stuart schwartz msgf presentation7!16!10
TRANSCRIPT
DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT
PHENOTYPEKARYOTYPE CORRELATION UTILIZING
MOLEUCLAR CYTOGENETICS
STUART SCHWARTZ PhD
LABORATORY CORPORATION OF
AMERICA RESEARCH TRIANGLE PARK NORTH CAROLINA
DISCLOSURE I am an employee of LabCorp (Laboratory
Corporation of America)
UNDERLYING QUESTIONS
bull What is a SNP bull How effective is the whole genome array analysis
to detect chromosomal imbalances not seen with standard cytogenetic methods
bull How small of an alteration can be routinely detected ndash easily and integrated into routine analysis
bull What do these small alteration mean phenotypically
OVERVIEW bull Introduction bull SNP Array - methodology bull SNP Array findings ~ 3000 abnormals bull Complexity bull Uniparental Disomy bull Consanguinity bull Prenatal Diagnosis and POCs bull Conclusions
OBJECTIVES bull Describe the types of abnormalities
detected by microarrays bull Review the implications of complexity
bull Translocations markers two hits bull Review the impact of UPD and
consanguinity bull Discuss the utilization of arrays for prenatal
diagnosis and POC analysis
DETECTION OF GENOMIC CHANGES
bull Unbanded Chromosomes 20 Mb bull Chromosomes - 550 Band Level 10 Mb bull High Resolution Chromosomes 3-5 Mb bull FISH 150 kb
ndash DIRECTED ANALYSIS bull Array Analysis 50 - 150 kb
ndash NOT DIRECTED ANALYSIS
GENOME ndash ARRAY TECHNOLOGY
bull Gene number ~25000 functional genes bull Gene density one per 45 kb
bull But very varied among chromosomes bull Gene size Average 20 kb
bull But enormous variation
bull Half of genes ndash unknown function bull Interpretation of findings
bull Can detect abnormalities ndash interpretation
More than 18 million markers across the entire genome for copy number analysis
906600 SNPs (Polymorphic probes for assessing genotype and copy number) 945826 Structural Probes (Non-polymorphic probes for assessing copy number)
Marker spacing = Average 12 Kb Median 07 Kb (694 bases) 12 average
GENOME-WIDE AFFYMETRIX SNP ARRAY 60
WHAT IS A SNP bull Single Nucleotide Polymorphism bull A SNP is a single base pair substitution of
one nucleotide for another bull This substitution must be found in the
population at a frequency greater than 10
bull Eg one individual has a CAACCT sequence and another has a CAGCCT
LabCorp
SNP DESIGN
TAGCCATCGGTA N T G
GTA C TCAATGATCAGCT
ATCGGTAGCCAT A
ATCGGTAGCCAT C
CAT G AGTTACTA
CAT G AGTTACTA
PM Allele
PM Allele
A
B 25mers
Patient DNA
Genomic Sequence
5acute 3acute SNP T G
SNP probe = 25 bases
A
B
Log 2
CN State
AA +1 AB 0 BB -1
NORMAL ALLELE DOSAGE
ALLELIC DIFFERENCE - DELETION
ALLELIC DIFFERENCE - GAIN
ABNORMALITY - CRITERIA
bull Deletion ndash gt200 kb in size ndash less than 1000 copy number variation (CNV) ndash greater than 50 SNPsCN probes within a 200 kb segment ndash at least one OMIM annotated gene or within a region of clear clinical
significance
bull Duplication ndash gt500 kb in size ndash at least one OMIM annotated gene
bull Known clinically significant gene region ndash Deletions and duplications are reported as small as 50 Kb
TYPES OF ABNORMALITIES
CATEGORIES OF ABERRATIONS
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
DISCLOSURE I am an employee of LabCorp (Laboratory
Corporation of America)
UNDERLYING QUESTIONS
bull What is a SNP bull How effective is the whole genome array analysis
to detect chromosomal imbalances not seen with standard cytogenetic methods
bull How small of an alteration can be routinely detected ndash easily and integrated into routine analysis
bull What do these small alteration mean phenotypically
OVERVIEW bull Introduction bull SNP Array - methodology bull SNP Array findings ~ 3000 abnormals bull Complexity bull Uniparental Disomy bull Consanguinity bull Prenatal Diagnosis and POCs bull Conclusions
OBJECTIVES bull Describe the types of abnormalities
detected by microarrays bull Review the implications of complexity
bull Translocations markers two hits bull Review the impact of UPD and
consanguinity bull Discuss the utilization of arrays for prenatal
diagnosis and POC analysis
DETECTION OF GENOMIC CHANGES
bull Unbanded Chromosomes 20 Mb bull Chromosomes - 550 Band Level 10 Mb bull High Resolution Chromosomes 3-5 Mb bull FISH 150 kb
ndash DIRECTED ANALYSIS bull Array Analysis 50 - 150 kb
ndash NOT DIRECTED ANALYSIS
GENOME ndash ARRAY TECHNOLOGY
bull Gene number ~25000 functional genes bull Gene density one per 45 kb
bull But very varied among chromosomes bull Gene size Average 20 kb
bull But enormous variation
bull Half of genes ndash unknown function bull Interpretation of findings
bull Can detect abnormalities ndash interpretation
More than 18 million markers across the entire genome for copy number analysis
906600 SNPs (Polymorphic probes for assessing genotype and copy number) 945826 Structural Probes (Non-polymorphic probes for assessing copy number)
Marker spacing = Average 12 Kb Median 07 Kb (694 bases) 12 average
GENOME-WIDE AFFYMETRIX SNP ARRAY 60
WHAT IS A SNP bull Single Nucleotide Polymorphism bull A SNP is a single base pair substitution of
one nucleotide for another bull This substitution must be found in the
population at a frequency greater than 10
bull Eg one individual has a CAACCT sequence and another has a CAGCCT
LabCorp
SNP DESIGN
TAGCCATCGGTA N T G
GTA C TCAATGATCAGCT
ATCGGTAGCCAT A
ATCGGTAGCCAT C
CAT G AGTTACTA
CAT G AGTTACTA
PM Allele
PM Allele
A
B 25mers
Patient DNA
Genomic Sequence
5acute 3acute SNP T G
SNP probe = 25 bases
A
B
Log 2
CN State
AA +1 AB 0 BB -1
NORMAL ALLELE DOSAGE
ALLELIC DIFFERENCE - DELETION
ALLELIC DIFFERENCE - GAIN
ABNORMALITY - CRITERIA
bull Deletion ndash gt200 kb in size ndash less than 1000 copy number variation (CNV) ndash greater than 50 SNPsCN probes within a 200 kb segment ndash at least one OMIM annotated gene or within a region of clear clinical
significance
bull Duplication ndash gt500 kb in size ndash at least one OMIM annotated gene
bull Known clinically significant gene region ndash Deletions and duplications are reported as small as 50 Kb
TYPES OF ABNORMALITIES
CATEGORIES OF ABERRATIONS
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
UNDERLYING QUESTIONS
bull What is a SNP bull How effective is the whole genome array analysis
to detect chromosomal imbalances not seen with standard cytogenetic methods
bull How small of an alteration can be routinely detected ndash easily and integrated into routine analysis
bull What do these small alteration mean phenotypically
OVERVIEW bull Introduction bull SNP Array - methodology bull SNP Array findings ~ 3000 abnormals bull Complexity bull Uniparental Disomy bull Consanguinity bull Prenatal Diagnosis and POCs bull Conclusions
OBJECTIVES bull Describe the types of abnormalities
detected by microarrays bull Review the implications of complexity
bull Translocations markers two hits bull Review the impact of UPD and
consanguinity bull Discuss the utilization of arrays for prenatal
diagnosis and POC analysis
DETECTION OF GENOMIC CHANGES
bull Unbanded Chromosomes 20 Mb bull Chromosomes - 550 Band Level 10 Mb bull High Resolution Chromosomes 3-5 Mb bull FISH 150 kb
ndash DIRECTED ANALYSIS bull Array Analysis 50 - 150 kb
ndash NOT DIRECTED ANALYSIS
GENOME ndash ARRAY TECHNOLOGY
bull Gene number ~25000 functional genes bull Gene density one per 45 kb
bull But very varied among chromosomes bull Gene size Average 20 kb
bull But enormous variation
bull Half of genes ndash unknown function bull Interpretation of findings
bull Can detect abnormalities ndash interpretation
More than 18 million markers across the entire genome for copy number analysis
906600 SNPs (Polymorphic probes for assessing genotype and copy number) 945826 Structural Probes (Non-polymorphic probes for assessing copy number)
Marker spacing = Average 12 Kb Median 07 Kb (694 bases) 12 average
GENOME-WIDE AFFYMETRIX SNP ARRAY 60
WHAT IS A SNP bull Single Nucleotide Polymorphism bull A SNP is a single base pair substitution of
one nucleotide for another bull This substitution must be found in the
population at a frequency greater than 10
bull Eg one individual has a CAACCT sequence and another has a CAGCCT
LabCorp
SNP DESIGN
TAGCCATCGGTA N T G
GTA C TCAATGATCAGCT
ATCGGTAGCCAT A
ATCGGTAGCCAT C
CAT G AGTTACTA
CAT G AGTTACTA
PM Allele
PM Allele
A
B 25mers
Patient DNA
Genomic Sequence
5acute 3acute SNP T G
SNP probe = 25 bases
A
B
Log 2
CN State
AA +1 AB 0 BB -1
NORMAL ALLELE DOSAGE
ALLELIC DIFFERENCE - DELETION
ALLELIC DIFFERENCE - GAIN
ABNORMALITY - CRITERIA
bull Deletion ndash gt200 kb in size ndash less than 1000 copy number variation (CNV) ndash greater than 50 SNPsCN probes within a 200 kb segment ndash at least one OMIM annotated gene or within a region of clear clinical
significance
bull Duplication ndash gt500 kb in size ndash at least one OMIM annotated gene
bull Known clinically significant gene region ndash Deletions and duplications are reported as small as 50 Kb
TYPES OF ABNORMALITIES
CATEGORIES OF ABERRATIONS
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
OVERVIEW bull Introduction bull SNP Array - methodology bull SNP Array findings ~ 3000 abnormals bull Complexity bull Uniparental Disomy bull Consanguinity bull Prenatal Diagnosis and POCs bull Conclusions
OBJECTIVES bull Describe the types of abnormalities
detected by microarrays bull Review the implications of complexity
bull Translocations markers two hits bull Review the impact of UPD and
consanguinity bull Discuss the utilization of arrays for prenatal
diagnosis and POC analysis
DETECTION OF GENOMIC CHANGES
bull Unbanded Chromosomes 20 Mb bull Chromosomes - 550 Band Level 10 Mb bull High Resolution Chromosomes 3-5 Mb bull FISH 150 kb
ndash DIRECTED ANALYSIS bull Array Analysis 50 - 150 kb
ndash NOT DIRECTED ANALYSIS
GENOME ndash ARRAY TECHNOLOGY
bull Gene number ~25000 functional genes bull Gene density one per 45 kb
bull But very varied among chromosomes bull Gene size Average 20 kb
bull But enormous variation
bull Half of genes ndash unknown function bull Interpretation of findings
bull Can detect abnormalities ndash interpretation
More than 18 million markers across the entire genome for copy number analysis
906600 SNPs (Polymorphic probes for assessing genotype and copy number) 945826 Structural Probes (Non-polymorphic probes for assessing copy number)
Marker spacing = Average 12 Kb Median 07 Kb (694 bases) 12 average
GENOME-WIDE AFFYMETRIX SNP ARRAY 60
WHAT IS A SNP bull Single Nucleotide Polymorphism bull A SNP is a single base pair substitution of
one nucleotide for another bull This substitution must be found in the
population at a frequency greater than 10
bull Eg one individual has a CAACCT sequence and another has a CAGCCT
LabCorp
SNP DESIGN
TAGCCATCGGTA N T G
GTA C TCAATGATCAGCT
ATCGGTAGCCAT A
ATCGGTAGCCAT C
CAT G AGTTACTA
CAT G AGTTACTA
PM Allele
PM Allele
A
B 25mers
Patient DNA
Genomic Sequence
5acute 3acute SNP T G
SNP probe = 25 bases
A
B
Log 2
CN State
AA +1 AB 0 BB -1
NORMAL ALLELE DOSAGE
ALLELIC DIFFERENCE - DELETION
ALLELIC DIFFERENCE - GAIN
ABNORMALITY - CRITERIA
bull Deletion ndash gt200 kb in size ndash less than 1000 copy number variation (CNV) ndash greater than 50 SNPsCN probes within a 200 kb segment ndash at least one OMIM annotated gene or within a region of clear clinical
significance
bull Duplication ndash gt500 kb in size ndash at least one OMIM annotated gene
bull Known clinically significant gene region ndash Deletions and duplications are reported as small as 50 Kb
TYPES OF ABNORMALITIES
CATEGORIES OF ABERRATIONS
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
OBJECTIVES bull Describe the types of abnormalities
detected by microarrays bull Review the implications of complexity
bull Translocations markers two hits bull Review the impact of UPD and
consanguinity bull Discuss the utilization of arrays for prenatal
diagnosis and POC analysis
DETECTION OF GENOMIC CHANGES
bull Unbanded Chromosomes 20 Mb bull Chromosomes - 550 Band Level 10 Mb bull High Resolution Chromosomes 3-5 Mb bull FISH 150 kb
ndash DIRECTED ANALYSIS bull Array Analysis 50 - 150 kb
ndash NOT DIRECTED ANALYSIS
GENOME ndash ARRAY TECHNOLOGY
bull Gene number ~25000 functional genes bull Gene density one per 45 kb
bull But very varied among chromosomes bull Gene size Average 20 kb
bull But enormous variation
bull Half of genes ndash unknown function bull Interpretation of findings
bull Can detect abnormalities ndash interpretation
More than 18 million markers across the entire genome for copy number analysis
906600 SNPs (Polymorphic probes for assessing genotype and copy number) 945826 Structural Probes (Non-polymorphic probes for assessing copy number)
Marker spacing = Average 12 Kb Median 07 Kb (694 bases) 12 average
GENOME-WIDE AFFYMETRIX SNP ARRAY 60
WHAT IS A SNP bull Single Nucleotide Polymorphism bull A SNP is a single base pair substitution of
one nucleotide for another bull This substitution must be found in the
population at a frequency greater than 10
bull Eg one individual has a CAACCT sequence and another has a CAGCCT
LabCorp
SNP DESIGN
TAGCCATCGGTA N T G
GTA C TCAATGATCAGCT
ATCGGTAGCCAT A
ATCGGTAGCCAT C
CAT G AGTTACTA
CAT G AGTTACTA
PM Allele
PM Allele
A
B 25mers
Patient DNA
Genomic Sequence
5acute 3acute SNP T G
SNP probe = 25 bases
A
B
Log 2
CN State
AA +1 AB 0 BB -1
NORMAL ALLELE DOSAGE
ALLELIC DIFFERENCE - DELETION
ALLELIC DIFFERENCE - GAIN
ABNORMALITY - CRITERIA
bull Deletion ndash gt200 kb in size ndash less than 1000 copy number variation (CNV) ndash greater than 50 SNPsCN probes within a 200 kb segment ndash at least one OMIM annotated gene or within a region of clear clinical
significance
bull Duplication ndash gt500 kb in size ndash at least one OMIM annotated gene
bull Known clinically significant gene region ndash Deletions and duplications are reported as small as 50 Kb
TYPES OF ABNORMALITIES
CATEGORIES OF ABERRATIONS
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
DETECTION OF GENOMIC CHANGES
bull Unbanded Chromosomes 20 Mb bull Chromosomes - 550 Band Level 10 Mb bull High Resolution Chromosomes 3-5 Mb bull FISH 150 kb
ndash DIRECTED ANALYSIS bull Array Analysis 50 - 150 kb
ndash NOT DIRECTED ANALYSIS
GENOME ndash ARRAY TECHNOLOGY
bull Gene number ~25000 functional genes bull Gene density one per 45 kb
bull But very varied among chromosomes bull Gene size Average 20 kb
bull But enormous variation
bull Half of genes ndash unknown function bull Interpretation of findings
bull Can detect abnormalities ndash interpretation
More than 18 million markers across the entire genome for copy number analysis
906600 SNPs (Polymorphic probes for assessing genotype and copy number) 945826 Structural Probes (Non-polymorphic probes for assessing copy number)
Marker spacing = Average 12 Kb Median 07 Kb (694 bases) 12 average
GENOME-WIDE AFFYMETRIX SNP ARRAY 60
WHAT IS A SNP bull Single Nucleotide Polymorphism bull A SNP is a single base pair substitution of
one nucleotide for another bull This substitution must be found in the
population at a frequency greater than 10
bull Eg one individual has a CAACCT sequence and another has a CAGCCT
LabCorp
SNP DESIGN
TAGCCATCGGTA N T G
GTA C TCAATGATCAGCT
ATCGGTAGCCAT A
ATCGGTAGCCAT C
CAT G AGTTACTA
CAT G AGTTACTA
PM Allele
PM Allele
A
B 25mers
Patient DNA
Genomic Sequence
5acute 3acute SNP T G
SNP probe = 25 bases
A
B
Log 2
CN State
AA +1 AB 0 BB -1
NORMAL ALLELE DOSAGE
ALLELIC DIFFERENCE - DELETION
ALLELIC DIFFERENCE - GAIN
ABNORMALITY - CRITERIA
bull Deletion ndash gt200 kb in size ndash less than 1000 copy number variation (CNV) ndash greater than 50 SNPsCN probes within a 200 kb segment ndash at least one OMIM annotated gene or within a region of clear clinical
significance
bull Duplication ndash gt500 kb in size ndash at least one OMIM annotated gene
bull Known clinically significant gene region ndash Deletions and duplications are reported as small as 50 Kb
TYPES OF ABNORMALITIES
CATEGORIES OF ABERRATIONS
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
GENOME ndash ARRAY TECHNOLOGY
bull Gene number ~25000 functional genes bull Gene density one per 45 kb
bull But very varied among chromosomes bull Gene size Average 20 kb
bull But enormous variation
bull Half of genes ndash unknown function bull Interpretation of findings
bull Can detect abnormalities ndash interpretation
More than 18 million markers across the entire genome for copy number analysis
906600 SNPs (Polymorphic probes for assessing genotype and copy number) 945826 Structural Probes (Non-polymorphic probes for assessing copy number)
Marker spacing = Average 12 Kb Median 07 Kb (694 bases) 12 average
GENOME-WIDE AFFYMETRIX SNP ARRAY 60
WHAT IS A SNP bull Single Nucleotide Polymorphism bull A SNP is a single base pair substitution of
one nucleotide for another bull This substitution must be found in the
population at a frequency greater than 10
bull Eg one individual has a CAACCT sequence and another has a CAGCCT
LabCorp
SNP DESIGN
TAGCCATCGGTA N T G
GTA C TCAATGATCAGCT
ATCGGTAGCCAT A
ATCGGTAGCCAT C
CAT G AGTTACTA
CAT G AGTTACTA
PM Allele
PM Allele
A
B 25mers
Patient DNA
Genomic Sequence
5acute 3acute SNP T G
SNP probe = 25 bases
A
B
Log 2
CN State
AA +1 AB 0 BB -1
NORMAL ALLELE DOSAGE
ALLELIC DIFFERENCE - DELETION
ALLELIC DIFFERENCE - GAIN
ABNORMALITY - CRITERIA
bull Deletion ndash gt200 kb in size ndash less than 1000 copy number variation (CNV) ndash greater than 50 SNPsCN probes within a 200 kb segment ndash at least one OMIM annotated gene or within a region of clear clinical
significance
bull Duplication ndash gt500 kb in size ndash at least one OMIM annotated gene
bull Known clinically significant gene region ndash Deletions and duplications are reported as small as 50 Kb
TYPES OF ABNORMALITIES
CATEGORIES OF ABERRATIONS
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
More than 18 million markers across the entire genome for copy number analysis
906600 SNPs (Polymorphic probes for assessing genotype and copy number) 945826 Structural Probes (Non-polymorphic probes for assessing copy number)
Marker spacing = Average 12 Kb Median 07 Kb (694 bases) 12 average
GENOME-WIDE AFFYMETRIX SNP ARRAY 60
WHAT IS A SNP bull Single Nucleotide Polymorphism bull A SNP is a single base pair substitution of
one nucleotide for another bull This substitution must be found in the
population at a frequency greater than 10
bull Eg one individual has a CAACCT sequence and another has a CAGCCT
LabCorp
SNP DESIGN
TAGCCATCGGTA N T G
GTA C TCAATGATCAGCT
ATCGGTAGCCAT A
ATCGGTAGCCAT C
CAT G AGTTACTA
CAT G AGTTACTA
PM Allele
PM Allele
A
B 25mers
Patient DNA
Genomic Sequence
5acute 3acute SNP T G
SNP probe = 25 bases
A
B
Log 2
CN State
AA +1 AB 0 BB -1
NORMAL ALLELE DOSAGE
ALLELIC DIFFERENCE - DELETION
ALLELIC DIFFERENCE - GAIN
ABNORMALITY - CRITERIA
bull Deletion ndash gt200 kb in size ndash less than 1000 copy number variation (CNV) ndash greater than 50 SNPsCN probes within a 200 kb segment ndash at least one OMIM annotated gene or within a region of clear clinical
significance
bull Duplication ndash gt500 kb in size ndash at least one OMIM annotated gene
bull Known clinically significant gene region ndash Deletions and duplications are reported as small as 50 Kb
TYPES OF ABNORMALITIES
CATEGORIES OF ABERRATIONS
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
WHAT IS A SNP bull Single Nucleotide Polymorphism bull A SNP is a single base pair substitution of
one nucleotide for another bull This substitution must be found in the
population at a frequency greater than 10
bull Eg one individual has a CAACCT sequence and another has a CAGCCT
LabCorp
SNP DESIGN
TAGCCATCGGTA N T G
GTA C TCAATGATCAGCT
ATCGGTAGCCAT A
ATCGGTAGCCAT C
CAT G AGTTACTA
CAT G AGTTACTA
PM Allele
PM Allele
A
B 25mers
Patient DNA
Genomic Sequence
5acute 3acute SNP T G
SNP probe = 25 bases
A
B
Log 2
CN State
AA +1 AB 0 BB -1
NORMAL ALLELE DOSAGE
ALLELIC DIFFERENCE - DELETION
ALLELIC DIFFERENCE - GAIN
ABNORMALITY - CRITERIA
bull Deletion ndash gt200 kb in size ndash less than 1000 copy number variation (CNV) ndash greater than 50 SNPsCN probes within a 200 kb segment ndash at least one OMIM annotated gene or within a region of clear clinical
significance
bull Duplication ndash gt500 kb in size ndash at least one OMIM annotated gene
bull Known clinically significant gene region ndash Deletions and duplications are reported as small as 50 Kb
TYPES OF ABNORMALITIES
CATEGORIES OF ABERRATIONS
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
SNP DESIGN
TAGCCATCGGTA N T G
GTA C TCAATGATCAGCT
ATCGGTAGCCAT A
ATCGGTAGCCAT C
CAT G AGTTACTA
CAT G AGTTACTA
PM Allele
PM Allele
A
B 25mers
Patient DNA
Genomic Sequence
5acute 3acute SNP T G
SNP probe = 25 bases
A
B
Log 2
CN State
AA +1 AB 0 BB -1
NORMAL ALLELE DOSAGE
ALLELIC DIFFERENCE - DELETION
ALLELIC DIFFERENCE - GAIN
ABNORMALITY - CRITERIA
bull Deletion ndash gt200 kb in size ndash less than 1000 copy number variation (CNV) ndash greater than 50 SNPsCN probes within a 200 kb segment ndash at least one OMIM annotated gene or within a region of clear clinical
significance
bull Duplication ndash gt500 kb in size ndash at least one OMIM annotated gene
bull Known clinically significant gene region ndash Deletions and duplications are reported as small as 50 Kb
TYPES OF ABNORMALITIES
CATEGORIES OF ABERRATIONS
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
Log 2
CN State
AA +1 AB 0 BB -1
NORMAL ALLELE DOSAGE
ALLELIC DIFFERENCE - DELETION
ALLELIC DIFFERENCE - GAIN
ABNORMALITY - CRITERIA
bull Deletion ndash gt200 kb in size ndash less than 1000 copy number variation (CNV) ndash greater than 50 SNPsCN probes within a 200 kb segment ndash at least one OMIM annotated gene or within a region of clear clinical
significance
bull Duplication ndash gt500 kb in size ndash at least one OMIM annotated gene
bull Known clinically significant gene region ndash Deletions and duplications are reported as small as 50 Kb
TYPES OF ABNORMALITIES
CATEGORIES OF ABERRATIONS
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
ALLELIC DIFFERENCE - DELETION
ALLELIC DIFFERENCE - GAIN
ABNORMALITY - CRITERIA
bull Deletion ndash gt200 kb in size ndash less than 1000 copy number variation (CNV) ndash greater than 50 SNPsCN probes within a 200 kb segment ndash at least one OMIM annotated gene or within a region of clear clinical
significance
bull Duplication ndash gt500 kb in size ndash at least one OMIM annotated gene
bull Known clinically significant gene region ndash Deletions and duplications are reported as small as 50 Kb
TYPES OF ABNORMALITIES
CATEGORIES OF ABERRATIONS
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
ALLELIC DIFFERENCE - GAIN
ABNORMALITY - CRITERIA
bull Deletion ndash gt200 kb in size ndash less than 1000 copy number variation (CNV) ndash greater than 50 SNPsCN probes within a 200 kb segment ndash at least one OMIM annotated gene or within a region of clear clinical
significance
bull Duplication ndash gt500 kb in size ndash at least one OMIM annotated gene
bull Known clinically significant gene region ndash Deletions and duplications are reported as small as 50 Kb
TYPES OF ABNORMALITIES
CATEGORIES OF ABERRATIONS
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
ABNORMALITY - CRITERIA
bull Deletion ndash gt200 kb in size ndash less than 1000 copy number variation (CNV) ndash greater than 50 SNPsCN probes within a 200 kb segment ndash at least one OMIM annotated gene or within a region of clear clinical
significance
bull Duplication ndash gt500 kb in size ndash at least one OMIM annotated gene
bull Known clinically significant gene region ndash Deletions and duplications are reported as small as 50 Kb
TYPES OF ABNORMALITIES
CATEGORIES OF ABERRATIONS
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
TYPES OF ABNORMALITIES
CATEGORIES OF ABERRATIONS
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
CATEGORIES OF ABERRATIONS
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
DELETED AND DUPLICATED SEGMENTS
Size Deleted Size Duplicated lt100kb 21 lt100kb 02 100-200kb 40 100-200kb 24 200 - 500kb 271 200 - 500kb 151 500kb ndash 1Mb 140 500kb ndash 1Mb 399 1Mb ndash 3Mb 286 1Mb ndash 3Mb 292 gt3Mb 243 gt3Mb 162
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
INHERITANCE
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletionknown pathogenic genes
[367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
bull 15Q133 DELETION bull 17Q2131 DELETION (MAPT) bull 1P36 DELETION bull 1Q21 MICRODELETION bull 1Q21 MICRODUPLICATION bull 22Q1123 DELETION bull 3Q29 DELETION bull 9P DELETION bull 9P DUPLICATION bull 9Q34 DELETION bull ANGELMAN bull AUTISM bull BPES bull BRANCHIOOTORENAL bull CONGENITAL DIAPHRAGMATIC bull CRI-DU-CHAT bull CHRONIC GRANULOMATOUS DISEASE bull DUCHENNE MUSCULAR DYSTROPHY bull HOLOPROSENCEPHALY bull ICHTHYOSIS bull MICROPTHALMIA
bull MOWAT-WILSON bull MULTIPLE EXOSTOSES bull NEUROFIBROMATOSIS bull NOONAN bull PELIZAEUS-MERZBACHER DISEASE bull PSEUDOVAGINAL PERINEOSCROTAL
HYPOSPADIAS bull PHELAN-MCDERMID bull POTOCKI-LUPSKI bull POTOCKI-SHAFFER bull PRADER-WILLI bull RENAL CYSTS AND DIABETES bull RETT bull SMITH-MAGENIS bull SOTOS bull SRY DELETION bull STICKLER bull VCF bull WARDENBURG-TYPE I bull WARDENBURG-TYPE IIA bull WILLIAMS bull WILLIAMS DUPLICATION bull WOLF-HIRSCHHORN
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
MICRODELETION SYNDROMES
bull Microdeletion syndromes well established ndash High resolution cytogenetics ndash FISH
bull New microdeletion syndromes identified by arrays ndash 17q2131 deletion
bull More older microdeletion syndromes identified by array ndash Genotype first
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
SUSCEPTIBILITY GENES bull Traditional view of genetics
ndash Dominant recessive multigenic bull Cytogenetics
ndash Haploinsufficient Over-expression bull New Category
ndash Susceptible raquo Important but not sufficient raquo Parents with aberrations may be mildly affected or
not affected
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
16p112 ABNORMALITIES bull 16p112 aberrations
bull Microdeletions bull Microduplications
bull Autism
bull Parents with aberrations may be normal bull Important but not sufficient
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
1q211 ABNORMALITIES bull 1q211 aberrations
bull Microdeletions and microduplications
bull Patients with 1q211 aberrations show variable phenotype bull Mild-moderate MR microcephaly cardiac anomalies
cataracts bull Parents with aberrations may be mildly affected bull Demonstrates difficulties with new
microdeletionduplication syndromes
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
QUESTIONABLE SUSCEPTIBILITY
bull Precise effect of absence of loss or gain of genes ndash questionable ndash Controversial at times ndash Duplications
raquo 15q133 16p1311
bull Genes identified by GWAS genes shown to have CNVs greater in autistic or other populations ndash PARK2 IMMP2L 15q112 deletion
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
COMPLEXITY OF ARRAY RESULTS
bull Overall ~28 of samples show complexity ndash Structural abnormalities ndash Two or more abnormalities in patient
raquo Derivative chromosomes raquo Recombinants raquo Contiguous duplicationdeletions raquo TWO UNRELATED ABNORMALITIES
ndash Will have an effect on phenotype
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
BALANCED REARRANGEMENTS
bull No loss or gain of genetic material ndash Inversions translocations amp insertions
bull Incidence 1 in 500 live births
ndash 2-3 fold more common in mental retardation populations
bull De novo prenatal cases ndash A major diagnostic dilemma ndash 8-10 risk of phenotypic abnormalities
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
CHROMOSOME 6 DELETION SECONDARY TO T(618)
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
Del
18
Del 18q122
18q211
Ins(11) 18q2133
Ins(11) 18q222-3
Break found by FISH Region not deleted from Array analysis Region deleted from Array analysis
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
RESULTS - REARRANGMENTS bull 100 de novo ldquobalancedrdquo rearrangements
ndash 56 with deletionduplication of material raquo 08 Mb to 15 Mb raquo 15 to 70 genes deleted
ndash 117 copy number changes identified ndash 16 of 17 studied without deletion - gene has been broken
raquo 1 neither broken or deleted
bull 9 familial ldquobalancedrdquo rearrangement ndash 0 with deletion of material ndash 8 where a gene has broken
raquo 2 cases of an inheritance of familial disorder raquo 6 cases where only the proband has the disease
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
RESULTS ndash ABNORMALITIES
bull 56 of de novo rearrangements with gain or loss of material
bull Considerable complexity bull Only 29 demonstrated loss at one breakpoint bull 10 with deletions at 2 breakpoints bull 61 involved more than two chromosomes and one deletion
bull Only 57 of deletionsduplications were adjacent to the breakpoint bull Many on same or other chromosome
bull 80 of copy number changes deletions 20 were duplications
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
MARKER - OVERVIEW bull 43 markers from 40 patients
bull SNP array analysis bull Cytogenetics and FISH
bull Multiple questions bull Identification bull Proper characterization bull Phenotype correlation bull Mechanism of formation
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
INV DUP (15)
4 COPIES
3 COPIES
2 COPIES
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
ACENTRIC MARKER
Partial Trisomy der(2)(q323-gtq34) Analphoid 2q
Size17533 Kb SNP1636 Genes 30 (14 of 30 genes in OMIM)
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
TWO markers derived from ONE chromosome in an individual
Pericentromeric G-band 2p112-q112 Size 130 Mb
Acentric G-band 2p241-p243 Size 66 Mb
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
TWO markers derived from TWO chromosomes in an individual
G-band 5p131 to 5q10 Size 619 Mb
G-band 15q10 to 15q133 Size 1077 Mb
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
MARKERS ndash UNUSUAL CHARACTERISTICS
G-Band 13Q313-gtQTER Size 2068 MB G-Band 19 (9 SEGMENTS) Size 689 MB
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
ACCESSORY MARKER RING CHROMOSOME 6 DISCONTINUOUS PORTIONS OF CHROMOSOME 15
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
Copy number state 4
Homozygosity Homozygosity HomoHeterozygosity
SUPERNUMERARY CHROMOSOME 8 AND UPD
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
bull A newborn was ascertained with a congenital heart defect and multiple congenital anomalies
bull SNP array analysis revealed ndash A small deletion (137 Mb) in 7q1123 consistent
with Williams syndrome ndash However a second abnormality a 139 Mb
duplication in 22q1121 was also detected ndash The second abnormality would not have been
detected with a directed FISH approach ndash The second abnormality is likely to expand the
phenotype of the proband
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
7q1123 microduplication
16p112 microdeletion
611 kb Deletion
Log 2
197 Mb Duplication
Log 2
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
PWSAS DELETION
ADDITIONAL DELETION NOT DELETED
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
TWO HIT HYPOTHESIS bull Girirjan et al (2010)
ndash Using 16p121 as a model have suggested that many susceptibility genes may act as a two hit hypothesis
ndash Approximately 24 of cases had a second hit raquo Patients more severely affected than parents
bull Overall ~ 28 of our patients with two abnormalities ndash Those with known susceptibility genes ~15
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
FAMILIAL ndash DE NOVO bull Overall fewer than expected abnormalities
are de novo bull Type of abnormality ndash parents studied
ndash More susceptibility genes than originally thought
ndash More susceptibility genes parents are studied than known pathogenic deletions
bull Deletion and complex abnormalities more likely to be de novo
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
FREQUENCY - DE NOVO SIZE OF ABNORMALITIY
SIZE DELETION DUPLICATION 100 ndash 200 kb 25 37 200 ndash 500 kb 31 85
500 kb ndash 1 Mb 113 157 1 ndash 3 Mb 323 123 gt 3 Mb 79 63
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
FAMILIAL ndash DE NOVO TYPE OF ABNORMALITIY
TYPE FAMILIAL DE NOVO Susceptibility 944 56 Susceptibility 848 152
Large 247 753 Pathogenic 229 771
Small 805 195
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
GENES ndash ARRAY [~3000 CASES]
bull Large changes ndash multiple genes [619] bull Microdeletion pathogenic genes [367] bull Susceptibility genes [411] bull Susceptibility genes [284] bull Unknown function [1329]
bull De novo [~311] bull Complex [372] bull Unknown [646 - ~21]
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
Array loss 958kb
Array loss 437Mb
Array gain 840kb
Array Loss 341kb Array gain 234kb
Array loss 275kb
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
CN=2
AA AB BB
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
Distribution of Longest Single Run of Homozygosity in 120 Consecutive Patients
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6 7 8 9 10 11 12 13
O
F P
ATI
EN
TS
Mb BLOCKS
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
Chromosome 10 97Mb Interval Total
IDENTITY BY DESCENT
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
0
100
200
300
400
500
600
700
800
900
1000
1 5 9 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 113
Tota
l Hom
ozyg
osity
gt10
Mb
Patient
IDENTITY BY DESCENT
Denied Consanguinity
2nd - 3rd Cousins
1st Cousins
First Degree Consanguinity
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
5
Proband
IQ=60
Autism DD
Speech Problems
Autism DD Speech Problems
Asperger syndrome
Asperger syndrome DD
MLD
All Non-dysmorphic IQ=70-90 but no significant genetic issues
5
PEDIGREE WITH HIGHEST LEVEL OF IBD= 953 MB LCSH
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
TYPICAL LCSH DISPLAY ASSOCIATED
WITH UPD
Red Brackets Regions of homozygosity Light Blue Brackets Regions of heterozygosity Dark Blue arrows Recombination sites
- -
Copy Number State = 20 UPD 15
Allelic Segregation
183 Mb 286 Mb
d15s217 d15s659
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
MATERNAL MEIOSIS 1 ERROR AND TRISOMY RESCUE
Confirmed hetero-isoUPD 7mat 299 and 8 Mb LCSH Intervals
Detected in AF after CVS trisomy 7 mosaicism
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
B
A
C
D
F
E
EXAMPLES OF LONG CONTIGUOUS STRETCHES OF HOMOZYGOSITY (LCSH)
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
Heterozygous Region (D11S1383) Homozygous region (D11S4463) Homozygous region (D11S4464)
D11S1383 D11S4463
D11S4463
90 DOSAGE CONVERSION TO SEGMENTAL UPD 11Q13-gtQTER
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
BECKWITH-WEIDEMANN SYNDROME Chromosome 11 SNP Array Results
MOSAIC ALLELE RATIOS IN SEGMENTAL UPD (dosage neutral)
CN=2
CN=2
AA
BB
AAAB
BBAB 0
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
UPD RELATED RISK 1 Imprinting syndromes
2 Recessive allele disorders- relative to the
lengthsite of the HZ run
3 Occult trisomy- early gestational effects of mosaicism pre-rescue
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
Cytogenetic Results Array Results Concordance
47XX+15 XX+15 + 47XY+16 XY+16 + 47XX+22 XX+22 + 47XX+9 XX+9 + 69XXX XXX Triploid +
47XY+18 XY+18 + 45XXder(1314)(q10q10) XX +
46XY XY + 46XY XY (60) + 46XY XY +
47XX+16[22]46XX[21] XX+16 (60) +
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
TRIPLOID RESULT
oTriploid results are diagnosed from the allele difference which shows 4 tracts for all autosomes with no 0 tract oThe software of all array types normalizes the log ratio and copy number state to 2 copy
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
CYTOGENETIC RESULT
ARRAY RESULT Cases
AneuploidyXX Pure Abnormal 16
AneuploidyXX Mixed Abnormal 3
Complete Aneuploidy Pure Abnormal 3
46XX (Fetal or MCC) Normal XX 7
46XY Normal XY 2
47XY+2[2]46XY Normal XY 1
46XXt(38)[3]46XX[17] 48XY+21+22 1
Tetraploid (XXYY) Normal Male 2
46XX (100 MCC) Mole 1
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
MOLAR GENOTYPES
Triploid normalization
~50 identity
100 identity
Normal
Normal
46XX (one sperm x 2)
46XY (two sperm)
69XXX
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
bull DNA isolated from residual tissue in long term storage ndash Array results obtained in 3334
bull NORMAL RESULTS = 17
ndash NL XX = 5 4 ldquoPurerdquo and 1 with MCC ndash NL XY = 12 8 ldquoPurerdquo and 4 with MCC
bull ABNORMAL RESULTS = 16
ndash PURE TRISOMY or 45X = 6 ndash PURE TRIPLOID = 2 (XXX and XXY) ndash PURE DELETION = 3 ndash COMPLETE MOLE = 1 (XY DISPERMY) ndash TRISOMY with MCC = 4
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
PRENATAL DIAGNOSIS - STUDIES
bull Validation of SNP array for prenatal in progress ndash Utilization of Affymetrix 60 array
raquo More conservative guidelines bull Deletions ndash 1MB Duplications 2 Mb bull More restrictive definitive gene list
ndash 138 prenatal cases studied raquo clinically significant abnormalities detected (~77)
bull Majority could not be detected by chromosomes
raquo UPD ndash 4 possible cases raquo Consanguinity ndash 6 cases
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
UTILITY OF SNP MICROARRAY ANALYSIS bull High density coverage throughout entire genome
bull Both known and regions of potential clinical significance targeted
bull Known regions targeted in high density bull More precise localization of abnormalities bull Ability to review archival data as new syndromes and
genes identified bull Delineation of abnormalities in ldquobalanced
rearrangementsrdquo and markers bull Routine detection of uniparental disomy bull Detection of identity by descent ndash recessive allele
risk
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
SNP ARRAY - LIMITATION bull Involves extra work
ndash Acquiring and using BACs ndash FISH ndash Problematic ndash Where can these probes come from
bull Variable phenotypic effects ndash 1q211 15q133 ndash This is a major problem that everyone faces ndash will
only be resolved with research and good data collection
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
CONCLUSIONS bull Have reviewed data of over 3000 abnormalities
detected by whole genome array bull Pathogenicity of genes can be delineated in ~80
of cases detected by array bull All but 4 of 15000 cases studied
bull Have delineated many new genesregions that contribute to phenotype
bull As more data is accumulated certainly more genes will be delineated and pathogenicity of more cases will be determined ndash lower unknown frequency
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
IMPLICATIONS - I Both retrospective and prospective cases
studied ndash ~155 of cases studied prospectively shown
not to be simple deletions or duplications ndash complex
ndash ~35 of cases studied retrospectively ndash complex
ndash Evidence for the need to study previously identified abnormalities with array analysis
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
IMPLICATIONS - II The majority of duplications (86) are
direct duplications not inverted tandem Most deletions do not appear to be terminal
(both retrospectively or prospectively ascertained)
A higher than expected number of individuals have two or more abnormalities ndash Accounts for phenotypic abnormalities
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
IMPLICATIONS - III Approximately 235 of abnormalities are
facilitated by LCRs (low copy repeats) Frequency of deletions and duplications are
similar ndash Fewer overall duplications formed by LCRs
raquo Phenotypically not ascertained
Most deletions are not facilitated by LCRs and are unique
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
IMPLICATIONS - IV New mechanisms responsible for
abnormalities ndash Facilitated by repeatsbut not LCRs ndash Discontinuous duplications or deletions
raquo Some facilitated by multiple sets of LCR ndash Duplication of chromosomal material from a
non-adjacent region in the precise area where a deletion has occurred
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
IMPLICATIONS - IV Multiple mechanism for ringmarker formation
ndash Breakpoint heterogeneity ndash Formation by multiple chromosome ndash Ring duplication rather than deletion ndash Formation associated with UPD ndash Facilitated by LCRs ndash Pericentric heterochromatin involved not alpha-
satellite DNA ndash Formation involves non-continuous chromosomal
segments
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
SNP ARRAY - IMPORTANCE Can detect extremely small abnormalities
anywhere in the genome Will allow for good breakpoint delineation
and determination of abnormalities ndash Importance in elucidation of mechanisms
Good whole genome coverage ndash Terminal vs interstitial abnormalities ndash LCR involvement
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
CONCLUSIONS
Much more complexity to chromosomal aberrations than originally thought
Structure of chromosomes examined and delineated ndash Fewer terminal deletions than previously
believed ndash Most duplications are tandem ndash LCRs involvement in 235 of deletions and
duplications ndash do not count for the formation of the majority of abnormalities
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
CONCLUSIONS
New mechanism of formation delineated ndash Only scratching the surface
Phenotypic findings
ndash Have always known considerable variability within cytogenetic syndromes
ndash Phenotypes may be altered by raquo Hidden complexity raquo Additional abnormalities
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
VERY LAST THOUGHTS bull Some abnormalities - difficult to interpret
bull Many factors need to consider bull Size doesnrsquot always matter
bull Interpretation will only be possible with the acquisition of good clinical information and family follow-up bull Parental phenotype and abnormality
bull Imperative for clinicians and laboratory personal to work together
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-
ACKNOWLEDGEMENTS bull LabCorp
ndash Peter Papenhausen ndash Jim Tepperberg ndash Marcia Eisenberg ndash Inder Gadi ndash Rachel Burnside ndash Vikram Jaswaney ndash Hiba Risheg ndash Romela Pasion
bull Referral physicians
bull Affymetrix ndash Roger Schaller ndash Richard Shippy
bull LabCorp ndash Brian Williford ndash Carolyn Bullen ndash Jessica Whaley-Davis ndash Daniel Fuentes ndash Renee Royster ndash Josh Kesler
- DELINEATION OF CHROMOSOME STRUCTURE AND RESULTANT PHENOTYPEKARYOTYPE CORRELATION UTILIZING MOLEUCLAR CYTOGENETICS
- DISCLOSURE
- UNDERLYING QUESTIONS
- OVERVIEW
- OBJECTIVES
- DETECTION OF GENOMIC CHANGES
- GENOME ndash ARRAY TECHNOLOGY
- GENOME-WIDE AFFYMETRIX SNP ARRAY 60
- WHAT IS A SNP
- Slide Number 10
- NORMAL ALLELE DOSAGE
- Allelic Difference - Deletion
- Allelic Difference - Gain
- ABNORMALITY - CRITERIA
- TYPES OF ABNORMALITIES
- CATEGORIES OF ABERRATIONS
- DELETED AND DUPLICATED SEGMENTS
- INHERITANCE
- GENES ndash ARRAY [~3000 CASES]
- EXAMPLES OF SYNDROMES IDENTIFIED BY ARRAY ANALYSIS
- MICRODELETION SYNDROMES
- SUSCEPTIBILITY GENES
- 16p112 ABNORMALITIES
- 1q211 ABNORMALITIES
- QUESTIONABLE SUSCEPTIBILITY
- COMPLEXITY OF ARRAY RESULTS
- BALANCED REARRANGEMENTS
- CHROMOSOME 6 DELETION SECONDARY TO T(618)
- Slide Number 29
- RESULTS - REARRANGMENTS
- RESULTS ndash ABNORMALITIES
- MARKER - OVERVIEW
- Slide Number 33
- Slide Number 34
- ACENTRIC MARKER
- TWO markers derived from ONE chromosome in an individual
- TWO markers derived from TWO chromosomes in an individual
- MARKERS ndash UNUSUAL CHARACTERISTICS
- Slide Number 39
- SUPERNUMERARY CHROMOSOME 8 AND UPD
- DELINEATION OF TWO SIGNIFICANT ABNORMALITIES
- CHROMOSOME 16 DELETION AND CHROMOSOME 7 GAIN
- Slide Number 43
- TWO HIT HYPOTHESIS
- FAMILIAL ndash DE NOVO
- FREQUENCY - DE NOVOSIZE OF ABNORMALITIY
- FAMILIAL ndash DE NOVOTYPE OF ABNORMALITIY
- GENES ndash ARRAY [~3000 CASES]
- Slide Number 49
- Slide Number 50
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- COPY NEUTRAL HOMOZYGOSITY RUNS gt1MB
- Slide Number 58
- Slide Number 59
- Slide Number 60
- Slide Number 61
- TYPICAL LCSH DISPLAY ASSOCIATED WITH UPD
- MATERNAL MEIOSIS 1 ERRORAND TRISOMY RESCUE
- Slide Number 64
- Slide Number 65
- Slide Number 66
- UPD RELATED RISK
- CYTOGENETIC amp ARRAY RESULTS - CULTURED CELLS
- TRISOMY 9 RESULT ndash ALLELE DIFFERENCE
- TRIPLOID RESULT
- CYTOGENETIC VS ARRAY COMPARISON OF DIRECT RAW TISSUE
- MOLAR GENOTYPES
- ARRAY ANALYSIS OF 34 DIRECT TISSUE DNA FROM FAILED CULTURE SAMPLES
- PRENATAL DIAGNOSIS - STUDIES
- UTILITY OF SNP MICROARRAY ANALYSIS
- SNP ARRAY - LIMITATION
- CONCLUSIONS
- IMPLICATIONS - I
- IMPLICATIONS - II
- IMPLICATIONS - III
- IMPLICATIONS - IV
- IMPLICATIONS - IV
- SNP ARRAY - importance
- CONCLUSIONS
- CONCLUSIONS
- VERY LAST THOUGHTS
- ACKNOWLEDGEMENTS
-