cytogenetic rearrangements mediated by genomic architecture in meiosis 22q11
TRANSCRIPT
Cytogenetic Cytogenetic Rearrangements Mediated Rearrangements Mediated by Genomic Architecture by Genomic Architecture
in Meiosisin Meiosis
Cytogenetic Cytogenetic Rearrangements Mediated Rearrangements Mediated by Genomic Architecture by Genomic Architecture
in Meiosisin Meiosis
22q1122q11
A Closer Look at A Closer Look at 22q11.222q11.2
A Closer Look at A Closer Look at 22q11.222q11.2
Most frequent Most frequent microdeletion syndromemicrodeletion syndrome
Most common recurrent Most common recurrent balanced translocationbalanced translocation
Bisatellited marker Bisatellited marker chromosome of CESchromosome of CES
Numerous other Numerous other translocations at 22q11translocations at 22q11
The 22q11.2 Deletion The 22q11.2 Deletion Syndrome Syndrome (VCFS/DGS)(VCFS/DGS)
The 22q11.2 Deletion The 22q11.2 Deletion Syndrome Syndrome (VCFS/DGS)(VCFS/DGS)
Estimated frequency 1/3000-4000 Estimated frequency 1/3000-4000 live birthslive births
Autosomal Dominant Autosomal Dominant DisorderDisorder
Cleft or palatal anomalies Cleft or palatal anomalies ((69%69%)) Congenital heart defect Congenital heart defect ((74%74%)) Learning disabilityLearning disability Speech and language abnormalitiesSpeech and language abnormalities Psychiatric abnormalitiesPsychiatric abnormalities Immune dysfunctionImmune dysfunction Characteristic facial featuresCharacteristic facial features
DGCRDGCR22q11.222q11.2
ConstitutionalConstitutional t(11;22)t(11;22) ConstitutionalConstitutional t(11;22)t(11;22) Only known, recurrent, non-Only known, recurrent, non-Robertsonian translocation in manRobertsonian translocation in man
Seen in several hundred familiesSeen in several hundred families
Balanced carriers are Balanced carriers are phenotypically normalphenotypically normal
Risk of Supernumerary Risk of Supernumerary der(22)t(11;22) due to der(22)t(11;22) due to chromosomal malsegregationchromosomal malsegregation
47, XX or XY, +der(22) t(11;22)47, XX or XY, +der(22) t(11;22) Multiple malformation syndrome Multiple malformation syndrome Emanuel syndromeEmanuel syndrome
D22S427
D22S36
D22S941
D22S788
HCF2
CHKAD26
D22S801
Cen Tel
D22S75
BAC/PAC/Cosmid ContigBAC/PAC/Cosmid Contig
c106e4 c103a2 N25 c102g9 c68a1 c87f9 c2c9 c45c9c87h3
BAC 444p24 BAC 562f10PAC 194m11
BAC 32i11PAC 408l11
BAC 135h6
BAC 379n11PAC 413m7BAC 445f23
PAC 134n5
BAC 677f7BAC 293j14
BAC 291k7BAC 48m11
PAC 423n14
1.2 Mb DGCR cosmid contig
PAC 393h21
*
cH K89
low copy repeats = LCRslow copy repeats = LCRs
ZNF74
AA DDBB CC
22q11 Deletion and 22q11 Deletion and Rearrangement EndpointsRearrangement Endpoints22q11 Deletion and 22q11 Deletion and
Rearrangement EndpointsRearrangement Endpoints
t(11;22)t(11;22)CESCES CESCES
N25/HIRAN25/HIRA
D22S427 D22S36 D22S75 UFD1L D22S788 ZNF74 HCF2 CHKAD26 D22S936 Ki1165 D22S801 c61e11 c102d1CDC45L
TBX1
IGLL IGLLA A' B C D E F
DA
BA
A C
A' D
22/300 (7.3%)
5/300 (1.7%)5/300 (1.7%)
259/300 (86.3%)
Yamagishi et al.
CH01-242 CH98-205
Unbalanced translocations McQuade et al. Kurahashi et al.
CH04-18
CH98-18
Rauch et al.
CH97-221
CH03-029
CH03-064
CH03-162
CH02-008
Patient "g"
O'Donnell et al.
9/300 (3%)
Low Copy Repeats (LCRs) Low Copy Repeats (LCRs) in 22q11in 22q11
Structure and CompositionStructure and Composition
Low Copy Repeats (LCRs) Low Copy Repeats (LCRs) in 22q11in 22q11
Structure and CompositionStructure and Composition Modular StructureModular Structure
Modules range in size from 10-65 kbModules range in size from 10-65 kb
Modules contain previously identified Modules contain previously identified markers, truncated genes and markers, truncated genes and pseudogenespseudogenes
Variation in the content, orientation Variation in the content, orientation and organization of modules between and organization of modules between the different copiesthe different copies
AA
DD
CC
Genomic Organization of the Genomic Organization of the LCRs in 22q11.2LCRs in 22q11.2Genomic Organization of the Genomic Organization of the LCRs in 22q11.2LCRs in 22q11.2
AA
DD
CC
BBBB
LCRs and DeletionLCRs and Deletion Mechanism MechanismLCRs and DeletionLCRs and Deletion Mechanism Mechanism Normal Recombination EventNormal Recombination Event
A D
A
B
B C D
C
A
CA
B
B
C D
D
Crossover at LCR-DCrossover at LCR-D
Hypothetical Duplication or Hypothetical Duplication or Deletion MechanismDeletion Mechanism
Hypothetical Duplication or Hypothetical Duplication or Deletion MechanismDeletion Mechanism
““Misalignment followed by Recombination”Misalignment followed by Recombination”
A B C D
A DB C
A case of a deletion or A case of a deletion or duplication syndromeduplication syndrome
A D
Crossover between A & DCrossover between A & D
A B B C DADC
Detection of Rearranged Fragments
Detection of Rearranged Fragments
P1 P3 C 1 C2 C3 C4P2
A-B DeletionA-B DeletionProbed with ZNF74Probed with ZNF74
650 kb
145 kb
..
NotI
NotI
NotI
D22S427
ZNF74
D22S788
NotI
650 kb
NotI
D22S427
ZNF74
A B
145 kb
PCR Analysis of LCRA-LCRB Junction Fragment
PCR Analysis of LCRA-LCRB Junction Fragment
1 2 3 6 1 2 3 6 1 2 344 55 44 55 44 55 6M M
NormalNormal
DeletedDeleted
BB
Not I SiteNot I Site
PCR MarkerPCR Marker
145 kb
Lane 4Lane 4
650 kb
Lane 5Lane 5
ZNF74
ZNF74
444p24Sp6444p24Sp637g5T3
37g5T3
A B
ZNF74 444p24Sp6 37g5T3
Parent of Origin Parent of Origin DeterminationsDeterminationsParent of Origin Parent of Origin DeterminationsDeterminations
Maternal DeletionsMaternal Deletions36 36
Paternal DeletionsPaternal Deletions 3333
Number of patients Number of patients examined = 69examined = 69
Examining the Examining the Mechanism of DeletionMechanism of Deletion
Examining the Examining the Mechanism of DeletionMechanism of Deletion
Haplotype reconstruction Haplotype reconstruction
3 generation families3 generation families
Markers flanking the Markers flanking the deletion regiondeletion region
Genescan analysis Genescan analysis
Results of Studies Results of Studies to Dateto DateResults of Studies Results of Studies to Dateto Date
20 informative families20 informative families
19 consistent with an interchromosomal 19 consistent with an interchromosomal crossover event - much greater than expected crossover event - much greater than expected for meiotic distancefor meiotic distance
1 consistent with an intrachromosomal event1 consistent with an intrachromosomal event normal 22 only has 2 interchromosomal events normal 22 only has 2 interchromosomal events in 15 examined - more consistent with in 15 examined - more consistent with meiotic mapping studiesmeiotic mapping studies
no evidence for phenotypic differences no evidence for phenotypic differences related to heart or palate based on parental related to heart or palate based on parental origin of deletion in any of the familiesorigin of deletion in any of the families
FISH to Detect an FISH to Detect an InversionInversionFISH to Detect an FISH to Detect an InversionInversion
3 Mb Deletion Region in 22q11.2
A DD22S427 CHKAD26
c106e4 87h3 CHKAD26
N25 D22S788 ZNF74
B
Summary of 22q11.2 Summary of 22q11.2 DeletionsDeletionsSummary of 22q11.2 Summary of 22q11.2 DeletionsDeletions
No major parent of origin effects - deletion No major parent of origin effects - deletion or phenotypeor phenotype
Majority are Majority are de novode novo 3 Mb deletions 3 Mb deletions
No genotype-phenotype correlations- No genotype-phenotype correlations- phenotype is quite variablephenotype is quite variable
Unusual number of interchromosomal exchangesUnusual number of interchromosomal exchanges unequal crossing over involving segmental unequal crossing over involving segmental duplicationsduplications
other meiotic studies indicate proximal crossovers other meiotic studies indicate proximal crossovers are infrequentare infrequent
Inversions do not predispose to the deletion Inversions do not predispose to the deletion or the phenotype in absence of a deletionor the phenotype in absence of a deletion
Isolation and analysis of Isolation and analysis of the the
Recurrent t(11;22)Recurrent t(11;22)(q23;q11), t(17;22) and (q23;q11), t(17;22) and
t(A;22)t(A;22)
Isolation and analysis of Isolation and analysis of the the
Recurrent t(11;22)Recurrent t(11;22)(q23;q11), t(17;22) and (q23;q11), t(17;22) and
t(A;22)t(A;22)
LCR-B on 22q and the LCR-B on 22q and the Constitutional t(11;22)Constitutional t(11;22)LCR-B on 22q and the LCR-B on 22q and the
Constitutional t(11;22)Constitutional t(11;22)
N N
AA BB CC DDcentromere
telomere
(AT)n (AT)n(AT)n ZNF74v
t(11;22)t(11;22)
b562f10b444p24
cHK89
duplication modules
90 kb gap90 kb gap
N41
PATTR at 11q23 and Stem PATTR at 11q23 and Stem Loop Cruciform StructuresLoop Cruciform StructuresPATTR at 11q23 and Stem PATTR at 11q23 and Stem Loop Cruciform StructuresLoop Cruciform Structures
Inverted Inverted Repeats Repeats
(palindrome(palindrome))
TA
TC
TG
A
TA
G
A
CA T
A
TCTGTATCTGT
A
CA
G
ATA
CA
G
A
T
TA
CA
G
A
A
TA
G
A
C
TA
TC
TG
AGA
CA
TA
GA
C
ATG
TC
T
A
TGTCTATGTC
T
TA
ATT C T G
A G A C A T A G A C
T A T C T G T A T C T GA
T A G A C AT TGTCT
ACAGATACAGA
ATGTCTATGTCT
TACAGA
A
TA
G
A
C
T
A
TC
TG
A
TC
TG
TA
G
A
CA
TG
T
C
T
TA
C
A
GA
TA
C
A
G
A
A
TGT
C
TA
TG
T
C
T
TA
C
A
GA
A
TA
G
A
C
TA
T
C
TG
ATT
G
A
T
C
TG
TA
TC
T
A
CA
T
T
C
T
A
T
C
T
A T
AG
A
CA
TAG
A
C
T
G
A
TA
G
ACA
TG
T
C
T
T
A
A
A
G
A
A
TG
T
C
TA
TG
T
C
T
A
A
AA
TA
A
T
T
T
T
T
T
A
A
A
G
G
G
T
T
C
Genomic Instability Mediates Genomic Instability Mediates the t(11;22)the t(11;22)Genomic Instability Mediates Genomic Instability Mediates the t(11;22)the t(11;22)
Chromosome 22Unclonable
BacteriaTranslocation
Human Genome
Human Genome
Deletion
Bacteria
Chromosome 11
Analysis of the t(11;22) Analysis of the t(11;22) Breakpoint (BP)Breakpoint (BP)Analysis of the t(11;22) Analysis of the t(11;22) Breakpoint (BP)Breakpoint (BP) > 42 independent BPs have been examined - PATRRs > 42 independent BPs have been examined - PATRRs at 11q23 and 22q11 BPs at 11q23 and 22q11 BPs
Breakpoints in all families are almost identical Breakpoints in all families are almost identical with very limited variationwith very limited variation
Families are of various ethnic and racial Families are of various ethnic and racial backgrounds backgrounds i.e., implicates a similar mechanism rather than a i.e., implicates a similar mechanism rather than a founder effectfounder effect
BPs occur at the tip of the palindrome with BPs occur at the tip of the palindrome with small, symmetrical deletionssmall, symmetrical deletions
suggests that there is likely a palindrome mediated suggests that there is likely a palindrome mediated mechanism responsible for the recurrent nature of the mechanism responsible for the recurrent nature of the translocationtranslocation
Analysis of Analysis of t(17;22)s in the t(17;22)s in the
NF1 GeneNF1 Gene
Analysis of Analysis of t(17;22)s in the t(17;22)s in the
NF1 GeneNF1 Gene
exon 31exon 31 exon 32exon 32centromerecentromere telomeretelomere
********************************************************************************
proximal proximal CTAGTTATTTGCACAGTCTCCTTCAAGGCATAATTATATA--------TATATATATATA 52CTAGTTATTTGCACAGTCTCCTTCAAGGCATAATTATATA--------TATATATATATA 52distal CTAGTTATTTGCACAGTCTCCTTCAAGGCATAATTATATACATATATATATATATATATA 60distal CTAGTTATTTGCACAGTCTCCTTCAAGGCATAATTATATACATATATATATATATATATA 60 **************************************** **************************************************** ************
proximalproximal TATGTATATAATTATATAGGATTATATGTAGGATTATATTTATATGTATATAATTATATAGGATTATATGTAGGATTATATTTA 9494distaldistal TATGTATATAATTATATAGGATTATATGTAGGATTATATTA- 101TATGTATATAATTATATAGGATTATATGTAGGATTATATTA- 101
209 bp209 bp
PATRRPATRR
195 195 bpbp
Unexpectedly High Rate of Unexpectedly High Rate of t(11;22)s in Sperm from t(11;22)s in Sperm from
Normal MalesNormal Males
Unexpectedly High Rate of Unexpectedly High Rate of t(11;22)s in Sperm from t(11;22)s in Sperm from
Normal MalesNormal Males PCR performed on sperm samples from four normal PCR performed on sperm samples from four normal males was positive for the t(11;22) PCR product males was positive for the t(11;22) PCR product
The estimated frequencies are : The estimated frequencies are : 1.24 - 9.46x101.24 - 9.46x10-5-5
Testicular DNA: Testicular DNA: positivepositive
Twenty normal lymphoblast or fibroblast DNAs: Twenty normal lymphoblast or fibroblast DNAs: negativenegative
Lymphoblasts from patients with Bloom syndrome Lymphoblasts from patients with Bloom syndrome or ataxia-telangiectasia:or ataxia-telangiectasia: negativenegative
PCR for t(17;22) in same sperm samples: PCR for t(17;22) in same sperm samples: negativenegative
Polymorphism of the PATRR affects translocation Polymorphism of the PATRR affects translocation frequencyfrequency
Polymorphisms of the 11q Polymorphisms of the 11q PATRRPATRRPolymorphisms of the 11q Polymorphisms of the 11q PATRRPATRR
Allele typeAllele type NumberNumber Frequency(%)Frequency(%) StructureStructure
Long (L)Long (L) 343343 87.187.1
Short (S)Short (S) S1S1 2020 (40.0)(40.0)
55 (10.0)(10.0)
S3S3 2424 (48.0)(48.0)
S4S4 11 (2.0)(2.0)
5050 12.712.7
11 0.30.3
TotalTotal 394394 100100
Extra-long (EL)Extra-long (EL)
SubtotalSubtotal
S2S2
Deletion/insertion type polymorphisms have been identifiedDeletion/insertion type polymorphisms have been identified
6.81 x 106.81 x 10-7-7
<1.69 x 10<1.69 x 10-7-7
1.07-2.27 x 101.07-2.27 x 10-5-5
<3.05 x 10<3.05 x 10-7-7
Translocation Translocation FrequencyFrequency
1.20-2.93 x 101.20-2.93 x 10-6-6
1.55-1.71 x 101.55-1.71 x 10-6-6
Kato et al., Science 311:971 2006Kato et al., Science 311:971 2006
AB
Does the 11q23 PATRR Adopt a Cruciform Does the 11q23 PATRR Adopt a Cruciform Configuration?Configuration?
Atomic Force MicroscopyAtomic Force Microscopy
Does the 11q23 PATRR Adopt a Cruciform Does the 11q23 PATRR Adopt a Cruciform Configuration?Configuration?
Atomic Force MicroscopyAtomic Force Microscopy
Other Translocations Other Translocations to 22q11.2to 22q11.2Other Translocations Other Translocations to 22q11.2to 22q11.2 Numerous translocations have been describedNumerous translocations have been described None of the others are recurrent None of the others are recurrent translocationstranslocations
Question: Are they located in LCR-B?Question: Are they located in LCR-B? Analysis to the sequence level to determineAnalysis to the sequence level to determine
Is a PATRR a consistent finding at the BP?Is a PATRR a consistent finding at the BP? Do the different partner chromosomes share Do the different partner chromosomes share sequence homologysequence homology with one anotherwith one another with 22q?with 22q?
Do they demonstrate any similarities to one Do they demonstrate any similarities to one another with respect to location or organization another with respect to location or organization of the BPsof the BPs
Stem Loop Structures of LCR-B Stem Loop Structures of LCR-B and its Partnersand its Partners
Stem Loop Structures of LCR-B Stem Loop Structures of LCR-B and its Partnersand its Partners
22q11.2 LCR-B22q11.2 LCR-B(594 bp)(594 bp)
100
500 400
200
BP Region
11q2311q23(445 bp)(445 bp)
BP Region100
400 300
17q1117q11(205 bp)(205 bp)
4080
120160200
4q35.14q35.1(1072 bp)(1072 bp)
100 2001000 900
300
600800
1000
600
100
500
300
900
0/1200 700
1100
200
400
800
Deduced 1p21.2 AT-rich Deduced 1p21.2 AT-rich BreakpointBreakpointSequence Sequence
[t(1;22) patient][t(1;22) patient]
600
100500300
900
800
0/1200
700
1000
1100
200
400
Reference 1p21.2Reference 1p21.2SequenceSequence
(RP4-600M23)(RP4-600M23)
Stem Loop Structure of Stem Loop Structure of t(1;22) BPt(1;22) BP
Stem Loop Structure of Stem Loop Structure of t(1;22) BPt(1;22) BP
Non-LCR-B Translocations Non-LCR-B Translocations No Palindromic Sequences - No No Palindromic Sequences - No
Stem LoopsStem Loops
Non-LCR-B Translocations Non-LCR-B Translocations No Palindromic Sequences - No No Palindromic Sequences - No
Stem LoopsStem Loopst(2;22))
2q142q14 22q11.222q11.2t(10;22)
10q2610q26 22q11.222q11.2 15q2615q26 22q12.122q12.1t(15;22)
t(21;22)21p1221p12 22q11.222q11.2 2q37.22q37.2 Xp21.1Xp21.1
t(X;2) t(X;4)4q35.24q35.2 Xp21.1Xp21.1
Analysis of Recombination Analysis of Recombination and Proximity in Meiosisand Proximity in MeiosisAnalysis of Recombination Analysis of Recombination and Proximity in Meiosisand Proximity in Meiosis
LEGENDBlue = CRESTRed = SCP3Green = MLH1
Fuschia = c87f9Gold - 442e11
Comparison Between Comparison Between Oocytes and Oocytes and
SpermatocytesSpermatocytes
Comparison Between Comparison Between Oocytes and Oocytes and
SpermatocytesSpermatocytes
00
200200
400400
600600
800800
10001000
12001200
14001400
16001600
18001800
11 33 55 77 99 1111 1313 1515 1717 1919 2121 2323 2525 2727 2929 3131 3333 3535 3737 3939 4141 4343 4545 4747 4949
DistanceDistance
Oocyte or Spermatocyte #Oocyte or Spermatocyte #
11q-22q
6q-22q
11q-22q
6q-22q
spermatocytesspermatocytes oocytesoocytes
Are the Breakpoints at Are the Breakpoints at Recombination “Hot-Recombination “Hot-
Spots?”Spots?”
Are the Breakpoints at Are the Breakpoints at Recombination “Hot-Recombination “Hot-
Spots?”Spots?”
SummarySummarySummarySummary
The “genomic instability” of 22q11.2 is The “genomic instability” of 22q11.2 is presumed to be based on the presence of presumed to be based on the presence of multiple LCRs or segmental duplicationsmultiple LCRs or segmental duplications
Recurrent 22q11.2 deletion endpoints coincide Recurrent 22q11.2 deletion endpoints coincide with the low copy repeats (LCRs) on chromosome with the low copy repeats (LCRs) on chromosome 22 22
Inversions are not a common feature that Inversions are not a common feature that predisposes to the deletionpredisposes to the deletion
Interchromosomal recombination occurs in most Interchromosomal recombination occurs in most de novode novo deletions - not the favored location deletions - not the favored location for recombination eventsfor recombination events
Summary Summary ContinuedContinuedSummary Summary ContinuedContinued LCR-B is the location of the t(11;22) breakpoint as LCR-B is the location of the t(11;22) breakpoint as
well as many other translocations involving 22q11, well as many other translocations involving 22q11, but not all of thembut not all of them
A palindrome mediated mechanism is responsible for A palindrome mediated mechanism is responsible for the translocations occurring in LCR-Bthe translocations occurring in LCR-B
Other 22q11 translocations appear to be more randomOther 22q11 translocations appear to be more random
LCR-B translocation BPs exhibit a propensity to LCR-B translocation BPs exhibit a propensity to form secondary structure apparently related to form secondary structure apparently related to their rate of recurrence and perhaps polymorphismstheir rate of recurrence and perhaps polymorphisms
Constitutional translocations seem to occur Constitutional translocations seem to occur between sequences of similar melting temperature between sequences of similar melting temperature and/or secondary structure.and/or secondary structure.
AcknowledgemeAcknowledgementsntsAcknowledgemeAcknowledgementsnts
Anthony Gotter Anthony Gotter
Sulagna SaittaSulagna Saitta
Tamim Shaikh Tamim Shaikh
Hiroki KurahashiHiroki Kurahashi
Stacy HarrisStacy Harris
April HackerApril Hacker
Jill Yersak Jill Yersak
Mandy BookMandy Book
Danielle ConfortoDanielle Conforto
Debbie DriscollDebbie Driscoll
Elaine ZackaiElaine Zackai
Donna McDonald-Donna McDonald-McGinnMcGinn
Mellissa TonnesonMellissa Tonneson
Livija CelleLivija Celle
Jason CatanzaroJason Catanzaro
Jeanne MansonJeanne Manson
Marcia BudarfMarcia Budarf
MaimonCohMaimonCohenen
Terry Terry AshleyAshley
Ann GaethAnn Gaeth
Harker Harker RhodesRhodes
Tatsushi Tatsushi TodaToda
Takema Takema KatoKato
LocaLocall
Outside Outside InstitutionsInstitutions