genomics pathway prenatal lecture 20161102.pptx

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Gene)cs and Genomics of Disease Pathway Lecture @Washington Univ. in St. Louis

11/02/2016

Speakers…

� Kenan Omurtag, MD Assistant Professor, Obstetrics and Gynecology Division of Reproductive Endocrinology and Infertility

� Yoshiko Mito, PhD, FACMG Associate Director of Cytogenomics and Molecular Pathology/ Assistant Professor of Pathology and Immunology

� Amie Stanley, MS, CGC Department of Obstetrics and Gynecology, Division of Ultrasound Genetics

Dr. Kenan Omurtag

Preimplanta+on Gene+c Diagnosis/Screening

The Basics of real-‐+me PCR, array CGH, and Next-‐Genera+on Sequencing

The Basics of real-‐)me PCR, array CGH, and Next-‐Genera)on Sequencing

Bayer & Fisher

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Genetic Lesion

FISH aCGH rtPCR NGS

Whole-chromosome aneuploidy

+/- (limited by #

fluorochromes)

+ + +

Monogenic Disorders

- - - -

Familially Inherited

+ + + +

De novo Mutations

+ + + +

Whole-genome

amplification required

- + - +

rtPCR aCGH NGS

Sample [Polar Body,Blastomere or]

Trophectoderm biopsy



Genetic material analyzed

RNA DNA DNA/RNA

Amount of material required

Smallest Larger Smaller (1ng)

DNA-to-data time 4 weeks to make probe

7 days for results

24 hours 24h or less

Cost $5000 $5000 $5000

Technologies Progenesis, ThermoFisher

GeneDx, Igenomix

Illumina, 454, SOLiD, Ion Torrent

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Chromosomal Analysis (karyotype)

Chromosomal Aberration (trisomy 21) Chromosomal Aberrations in fetuses

Numerical 96% 85% 60%

Structural 　　　　　　　

balanced 0% 10% 30%

Unbalanced 4% 5% 10%

Frequency

1st trimester miscarriages　　

Fetuses of mother >35yo

General population

Total 1/2 1/50 1/160

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Frequency of chromosomal abnormalities

Livebirths: - 0.6% �  Congenital Anomalies with intellectual disability (ID) - 23% �  Congenital Heart Disease -13% �  Institutionalized individual with ID -12%

Infertility: �  Couples with recurrent spontaneous abortions - 5% Prenatal / Perinatal: �  Stillbirths and perinatal deaths - 6% �  First trimester spontaneous abortions - 60%

Chromosome abnormality in newborns

�  Balanced translocation – 1 in 500 �  Unbalanced translocation – 1 in 2,000 �  Pericentric inversion – 1 in 100 �  Trisomy 21 – 1 in 700 �  Trisomy 18 – 1 in 6,000 �  Trisomy 13 – 1 in 10,000 �  47,XXY– 1 in 1,000 males �  47,XYY– 1 in 1,000 males �  47,XXX– 1 in 1,000 females �  45,X – 1 in 5,000 females

Meiotic Errors lead to chromosomal aberrations

GeneticsLab

Invasive prenatal testing (amniocentesis and CVS)

>=15 wks >=10 wks

Byer/Shinberg/Galliano 1999 DOHS

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Risks associated with Prenatal Diagnostic Testing

�  Pregnancy loss (CVS: <1.0%; Amniocentesis:<0.5%) �  Fetal morbidity

�  Premature preterm rupture of membranes (PPROM) �  1.0% (within 48 hr)

�  Limb reduction defects �  5.2 to 5.7/10,000 vs 4.8 to 5.97 gen. pop

�  Hemangiomas �  3 fold increase

Wilkins-Haug LE ACMG 2013

Chromosome analysis of Amnio/CVS

Chromosome analysis of Amnio/CVS

� Chromosomes 13, 18, 21, X and Y

Trisomy 21

FISH testing for common aneuploidy

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Screening Maternal age at delivery Maternal Serum Screening �  1st trimester

�  PaPP-A, hCG �  Nuchal translucency

�  2nd trimester (quad test) �  MSAFP, hCG, estriol, inhibin

�  Integrated screening �  Combined screening Trisomy 21

�  Call 2-5% of individuals screened as “increased risk” �  80-90% of all trisomy 21 �  Chance of true positive is 2-4%.

Wilkins-Haug LE ACMG 2013 Wilkins-Haug LE ACMG 2013

>

80-90% detection rate in 2-5% individuals screened

Critical developments towards the Non-Invasive Prenatal Screening (NIPS)

Circulating cell-free “fetal” DNA (ccffDNA) �  Predominantly originated from placental cells’ apoptosis �  Detected at 5-7 weeks and thereafter �  Rapid turnover (cleared within hours) �  ccffDNA/RNA can be isolated with high fidelity �  ccffDNA constitutes ~10% of total cfDNA in maternal serum �  Amount of cfDNA correlates with gestational age (positive correlation), BMI

(inverse correlation), and multiple-gestation pregnancy, race, parity, smoking, maternal age, mode of conception, placental volume.

Next generation sequencing �  Enabled to produce many sequence reads at lower cost

Bianchi, 2006

NIPS by NextGen Sequencing detects fetal chromosomal aneuploidy

Swanson et al. Curr Genet Med Rep 2013

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Sensitivity: 100% Specificity: 98%

Chiu R W K et al. BMJ 2011

Performance of NIPS screening for high risk women Maternal Serum Screening vs Non-invasive Prenatal Screening

Maternal Serum Non-invasive Detection rate 80~ >90% 99% Screen positive 2-5% 0.2% Chance of true positive

2-4% >=98%

Limitation of NIPS

�  Clinically validated only for high-risk women �  Low fetal fraction (c.f. BMI) �  Multiple-gestation pregnancy �  False-positives: - Confined Placental Mosaicism - Vanished twin - Maternal Mosaicism �  Genetic counseling �  To be followed-up w/invasive prenatal diagnosis (confirmatory test)

�  ACOG recommends NIPT as a screening test for high-risk pregnant women

Multiple approaches of NIPS

Bianchi DW et al. 2014 Verinata Sequenom Ariosa Natera

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Morain S et al. NEJM 2013

Multiple approaches of NIPS

Morain S et al. NEJM 2013

Non-invasive fetal genome analysis

� Detection of microdeletion/duplications and other copy number variants.

� Detection of sequence variants, single-base or indels.

�  DNA segments with a variable copy number in comparison of two or more genomes

Lee and Scherer, 2010

Copy Number Variation/Variant (CNV)

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Copy Number Variation/Variant (CNV)

�  DNA segments with a variable copy number in comparison of two or more genomes

�  CNV vs indels: the definition of CNV is >1kb �  High proportion of the genome (~4.8-9.5%) is subject to CNV

“Variant” (common and benign?)

�  Historically, large genomic losses and gains were believed to be exceptionally rare, and always pathogenic.

�  Frequency undefined �  Continuous spectrum of phenotypic effects (from adaptive to embryonic

lethal) �  Many microdeletion/duplication syndromes have been identified.

Discovery of Copy Number Variations (2004)

�  Initial identification of copy number polymorphisms in 2004. �  ~250 loci examined in 20-55 individuals and revealed 76-102 CVPs. �  On average, a dozen CNPs per individual, of which more than half

involving genes, and average length of ~500kb

Genome-wide search for CNVs by array Comparative Genomic Hybridization (CGH)

http://www.microarray.lu

Patient’s sample vs “Normal” Control

Green > Red(Patient): deletion Green < Red(Patient): : duplication

BAC microarray >~300kb resolution

�  Oligo arrays and SNP arrays for better resolution (<~100kb)

�  Combined oligo/SNP arrays for genome-wide coverage and allelic information

Detection of Copy Number Variants by high-resolution oligo/SNP microarrays

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Copy Number Variation – updates (2015)

Zarrei et al, Nat Rev Genet 2015

�  24,000 CNVs (3,000 gains and 23,000 losses) �  Median size of ~1kB �  9.5% of the genome, ~273Mb, shows variation

Recurrent vs non-recurrent deletions/duplications

Non-recurrent

Breakpoints(BPs) not recurrent: - Wolf-Hirshhorn Syndrome (4p deletion) - Cri-du-chat syndrome (5p deletion) - 18p deletion syndrome

Recurrent

BP1 BP2

BP3 BP4

Mechanisms of CNV formation 1) Non-allelic homologous recombination (NAHR)

�  Segmental Duplication

-10-400kb DNA segments（>97% homologous）

- shares ~5% of the genome �  Most common mechanism for larger CNVs.　　

Weischenfeldt et al, Nat Rev Genet 2013 Luski et al. Trends in Genetics 2002; 18(2);74-82

deletion duplication

Mechanisms of CNV formation

�  1) Non-allelic homologous recombination (NAHR)

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Luski et al. Trends in Genetics 2002; 18(2);74-82

deletion duplication


�  1) Non-allelic homologous recombination (NAHR)

Luski et al. Trends in Genetics 2002; 18(2);74-82

NAHR leads to a variety of genomic rearrangements


2) Non-homologous End Joining (NHEJ)

Weischenfeldt et al, Nat Rev Genet 2013


3) Insertion of mobile element


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Mechanisms of CNV formation 4) Replication-based template switching (“Stalled or broken replication fork”) - Fork Stalling and Template Switching (FoSTeS) or - Microhomology-mediated break-induced replication(MMBIR)



5) Chromothripsis


Copy Number Variants and Pathogenisity ~ Microdeletion and Microduplication syndromes ~

�  A newborn baby presented with a high-pitched cry ("cat cry"), low birth weight, poor muscle tone, microcephaly.

�  Chromosome analysis (karyotype) is ordered. (resolution >5~10Mb)

�  Cri-du-Chat syndrome (5p deletion syndrome) https://www.mun.ca

Copy Number Variants and Pathogenisity ~ Microdeletion and Microduplication syndromes ~

�  A 3yo boy with Congenital Heart Defects (Tetralogy of Fallot) was evaluated at pediatric genetics clinic.

�  Fluorescent in situ hybridization (FISH) is ordered.

�  22q11.2 deletion syndrome (DiGeorge/Velocardiofacial Syndrome) (~3Mb)

HIRA(TUPLE1) 22q11.2 ARSA 22q13

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Genomic in Medicine 2007, by Thompson and Thompson

Syndrome Chromosomal region

Size Genes

William-Buren Syndrome

7q11.23 1.7 Mb ELN

Prader-Willi Syndrome/Angelman Syndrome

15q11-q13 4 Mb SNRPN, UBE3

Smith-Magenis Syndrome

17q11.2 5 Mb RAI1

DiGeorge/ Velocardiofacial Syndrome

22q11.2 3 Mb HIRA (TUPLE)

�  Large-scale genomic losses and gains were believed to be exceptionally rare, and always pathogenic.

Microdeletion and microduplication syndromes (2007)

Platform �  2.6 M copy number probes �  w/ 750k SNP probes Analysis software �  Signal intensities are extracted from a scanned image �  Software normalize the signal intensities to the control data

(“log2 ratio”) �  Signals are smoothed and converted to copy number states e.g. CN =2 (disomy) Segment Call �  Copy number loss(deletion), copy number gain(duplication) �  Region of homozygosity (ROH)

Oligo/SNP microarray analysis enabled detection of Copy Number Variants in a higher-resolution in a clinical setting

Representative Microarray Results Microarray analysis is a powerful tool for detection of Copy Number Variants

Log2 ratio

Copy Number

states

Loss Gain

segments

AOH

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Database of Genomic Variants (DGV)

* >2,000,000 CNVs are curated http://dgv.tcag.ca/dgv/app/home

DECIPHER: DatabasE of genomiC variation and Phenotype in Humans using Ensembl Resources

https://decipher.sanger.ac.uk/

Interpretation

�  1) Benign (Database of Genomic Variants) �  2) Likely benign �  3) Pathogenic (databases: ISCA and DECIPHER) �  4) Variants of uncertain significance (VOUS)

88,400 88,600 88,800 88,000 q21.21 q21.22

Gene A

(1)

DGV

(3) (2)

No genes

OMIM phenotype gene

(4)

Nature Genetics 2006; 38;1038-42

17q21.31 microdeletion syndrome

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Nature Genetics 2006; 38;1038-42

Microdeletions in patients show recurrent breakpoints Microdeletion and microduplication syndromes (2014)

Nevado et al. Genet Mol Bio, 2014

SNP probes can detect other chromosomal abnormalities

Allele Peaks (signal intensity of B) - (signal intensity of A)

segments

Loss Gain AOH

2 copies

A

A

B B

A B

A

B

3 SNP combinations AA, AB, BB

3 copies

A

A

B B

A B

A

B

A B B A

4 SNP combinations AAA, AAB, ABB, BBB

1 copy

A B B A

2 SNPs A or B

4 Allele Peaks

A

A

B B

A B

A

B

A B B A

SNP probes detects triploidy by abnormal allelic patterns

Copy Number State =2

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SNP probes detects UPDs by abnormal allelic patterns

Chromosome 11

4 Allele Peaks

Copy Number State =2

Paternal chr 11 Maternal chr 11

Mosaic Paternal isodisomy for 11p

Paternal chr 11 Maternal chr 11 Mitotic

recombination

Chromosomal Microarray Analysis is a powerful diagnostic approach

Pediatrics �  1st-tier clinical diagnostic testing for individuals with developmental

disabilities and congenital anomalies (including autism spectrum disorders) since 2010.

�  15-20% diagnostic yield �  Identified many microdeletion/duplication syndromes (copy number

alterations) �  Gene discovery �  Research component in clinical testing (by insurance)

Prenatal �  Hesitance due to the copy number variant of uncertain significance �  ACOG/SMFM recommendation came out late 2013

Copy number loss Copy number gain LCSH Prenatal 1 Mb 2 Mb 10 Mb

Postnatal 200 kb 500 kb 10 Mb

Chromosomal Microarray Testing in Maternal Fetal Medicine

-  Can detect smaller chromosomal aberrations than karyotyping

-  Unnecessary to culture cells – more likely to give results

ACOG/SMFM recommendations

1) A fetus w/major structural abnormalities by u/s : Microarray - arrayCGH detected additional clinically significant abnormalities in 6% 2) A structurally normal fetus: Microarray or karyotype - array CGH detected additional clinically significant abnormalities in 1.7% * VOUS reported in 3.4% (1.6% likely pathogenic; 1.8% likely benign) 3) Maternal age : should not restrict the use of microarray test 4) IUFD or stillbirth : Microarray -increased likelihood of obtaining results, improved detection 5) 1st/2nd trimester pregnancy losses : limited data on clinical utility 6) Comprehensive pretest/posttest genetic counseling with consent (regarding benefits, limitations, results, potential to identify findings of uncertain significance, non-paternity, consanguinity, and adult-onset diseases)

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Case #1 (amniotic fluid) 32yo G4P2SAB1TAB0 at 21w0d; fetus w/ abnormal ultrasound findings, Holoprosencephaly and TOF

Chromosome 18

arr[hg19] 18p11.32p11.21(136,226-12,305,082)x1

~12.169 Mb apparently terminal deletion on the short arm of chromosome 18 (p11.32p11.21)

CMA Case #2 (aminotic fluid) 27yo G1P0 @22w5d; for polydactyly, possible VSD, EIF, possible agenesis of the corpus callosum borderline enlarged lateral ventricles, SGA fetus.

Abnormal CMA findings:

*Clinical presentations overlaps w/1q43-q44 deletion syndrome:

polydactyly and other limb anomalies, craniofacial anomalies including corpus callosum agenesis, microcephaly, seizures, ID, and developmental delay

Chromosome Band Copy Number Change Size

1 q43q44 Loss ~11.363Mb

Summary of three cytogenetic techniques: Chromosomal Microarray vs FISH vs Chromosome

CMA FISH Chromosome Unbalanced abnormal ++

(high resolution) +/-

(if only targeted) +

(5~10Mb+) Balanced abnormal - *

(Robertsonian, familial

translocation)

+/- (metaphase FISH)

+

Mosaic +/- (FISH

confirmation)

++ (50-500 cells)

+ (20 mets)

Copy number-neutral AOH etc

+ - -

* Reflex Chromosome (karyotype) analysis is recommended.

Chrionic Villus sampling 40yo G5P1SAB3TAB0 @13w0d; Cystic Hygroma on u/s CVS CMA findings: Normal

Reflex Karyotype: 45,XX,der(13;14)(q10;q10) (Uniparental Disomy 14 (UPD14) testing recommended)

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Microarray Analysis

Suspected unbalanced translocations or structural abnormalities

Pathogenic CNV

If CNV too small for FISH, follow up w/ parental CMA or molecular studies

Reflex karyotype with metaphase FISH. Follow-up w/ parental testing (karyotype with metaphase FISH)

PGD* and/or prenatal CMA/FISH/Karyotype should be considered for future pregnancies (*PGD not currently offered at Wash U Cytogenomics lab)

Normal CMA

Can do reflex karyotype to detect balanced rearrangements

For failed CMA, FISH may be available

If FISH detectable, then do parental & proband's FISH

CNV of uncertain significance

Parental & Proband FISH

Chromosomal Microarray Analysis follow-up

Genomic analysis of recurrent miscarriages

Product of Conception �  50% show cytogenetically detectable aberrations �  Most of aberrations are numerical (trisomy, monosomy) �  Checking for an unbalanced translocation

Parents �  Follow-up chromosome analysis in search for balanced translocation

(Note: microarray analysis cannot detect balanced rearrangement)

First trimester spontaneous abortions

15% of all clinically recognized pregnancy end as SAB �  Normal chromosome - 40% �  Abnormal chromosome - 60% - Autosomal trisomy ~50% - Monosomy X ~25% - Triploid, Tetraploid ~20% - Structural abnormalities <5%

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Apparently “balanced” translocation carriers

Frequency

General population 0.2%

Recurrent pregnancy losses (0.5%) 5%

Consecutive pregnancy losses ~10%

Risk of carrying “balanced” translocation

�  PGD (preimplantation genetic diagnosis) - IVF (in vitro fertilization) - Genetic testing in single cell from 8-cell stage embryo

�  Invasive prenatal testing (amniocentesis or chorionic villus sampling)

From Gardner and Sutherland, Chromosome Abnormalities and Genetic Counseling

Translocation carriers produce balanced and unbalanced gametes

- A carrier is mother : 10-15% risk - A carrier is father : ~5% risk

2p terminal loss

CMA showed a terminal deletion and a terminal gain in POC

5p terminal gain

Normal

Balanced

Unbalanced 1

Chromosome 2 Chromosome 5

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Parental chromosome analysis à mother is a carrier father mother

Note: microarray analysis cannot detect balanced rearrangement

Metaphase FISH analysis on maternal specimen

Chr 5p Chr 5q

Normal chr 5

derivative chr 5 (5p loss)

derivative chr 2 (with 5p)

Amniotic fluid from 43yo G3P1 @16w1d GA; for AMA

Karyotype: 46,XX,t(13;14)(q12;q32)

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Amniotic fluid from 43yo G3P1 @16w1d GA; for AMA

Chromosome Band Copy Number Change Size

4 q27q28.1 Loss ~1.984Mb

14 q32.33 Loss ~1.117Mb

Abnormal CMA findings:

Karyotype: 46,XX,t(13;14)(q12;q32)

Lucigen NxSeq Long Mate Pair Library

Detection of balanced translocation using next-generation sequencing

Prevalence of genetic disorders

Types of genetic alterations Prevalence（/1000）

at birth 25yo population Genomic/ chromosomal disorders 6 1.8 3.8 Single-gene disorders 10 3.6 20 Complex diseases ~50 ~50 ~600

Genetics in Medicine (Thompson&Thompson)

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Genetic Diagnosis in Prenatal Care Timeline

Preconception

1st trimester

2nd trimester

Livebirth

Testing *Carrier screening *Infertility *Mendelian disorders *Preimplantation Genetic Diagnosis *Preimplantation Genetic Screening

*Aneuploidy screening *Chromosome analysis (karyotyping) *Other cytogenetic/molecular testings

Procedures

*Parental testing *IVF

*Ultrasound *Maternal serum screening *Non-Invasive Prenatal Screening *Chorionic villus sampling

10wks~

15wks~ *Amniocentesis

*miscarriages *intra-uterine fetal demise *stillbirth *live birth w/ genetic conditions

*Chromosome/Microarray/FISH analysis *Chromosome/Microarray/FISH analysis * Molecular testing

Genetic Diagnosis in Prenatal Care Pregnancy at risk of mendelian disorders �  Parental carrier screening (pre/post-conception) �  Parental sequence testing (familial history) �  Fetal diagnostic testing (known familial conditions or ultrasound findings)

General Population �  Aneuploidy testing - Invasive prenatal testing (Chorionic Villus Sampling and Amniocentesis) - Non-invasive prenatal screening (NIPS)

Recurrent Miscarriages/ Infertilities �  Chromosome analysis/microarray analysis on products of conception (POCs) �  Parental chromosome analysis

Molecular Cytogenetics: �  Fluorescent in situ hybridization (FISH) ◦  Numerical aberrations ◦  Microdeletions/duplications (<5 Mb) ◦  Cryptic rearrangements ◦  A targeted method

�  Chromosomal Microarray Analysis (CMA) ◦  a priori knowledge of chromosomal location is unnecessary ◦  Numerical and unbalanced structural aberrations ◦  Copy number changes ◦  Microdeletions/duplications (>~100kb) ◦  Allelic aberrations (consanguinity, uniparental disomy)

Cytogenetics: �  Chromosome analysis (karyotype) ◦  Numerical aberrations ◦  Structural aberrations (>5-10 Mb)

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Molecular Testing Molecular Testing: �  Mutation Analysis ◦  Sanger sequencing ◦  Single-base extension ◦  Next Generation Sequencing (whole-genome,

whole-exome, gene panel) ◦  Short tandem repeats (STR) ◦  Fragment analysis

�  Deletion/Duplication Analysis ◦  Quantitative PCR (qPCR) ◦  Multiplex Ligation-dependent probe amplification (MLPA)

Professional Societies and Regulatory Agencies

�  American Board of Medical Genetics and Genomics (American Board of Medical Specialties) �  American College of Medical Genetics �  American Society of Human Genetics �  Association for Molecular Pathology �  American Board of Genetic Counseling �  National Society of Genetic Counselors

�  CLIA(Clinical Laboratory Improvement Amendments) �  CAP (College of American Pathologists) �  FDA (Food and Drug Administration)

Acknowledgment

Dept. of Pathology and Immunology, Laboratory and Genomic Medicine �  Cytogenomics and Molecular Pathology �  Genomic and Pathology Services

genomics pathway prenatal lecture 20161102.pptx

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