Brynn Levy, M.Sc.(Med).,Ph.D., FACMGProfessor of Pathology & Cell Biology at CUMC

Director, Clinical Cytogenetics Laboratory

Co-Director, Division of Personalized Genomic Medicine Columbia University

Medical Center

Modern approach to laboratory prenatal workup

of congenital anomalies

Prenatal Diagnosis of Chromosome Abnormalities

• Chromosome abnormalities have been diagnosed

from prenatal specimens for almost 50 years

Likelihood of Fetal Aneuploidy is influenced by Presence of Fetal Structural Anomalies

• Well recognized ultrasound markers associated with the

common trisomies:

J Ultrasound Med. 2001 Jun;20(6):655-74.

Incidence & Spectrum of Karyotype Abnormalities in Prenatal Diagnosis

Karyotype

Result

CVS AmnioCVS

+

Amnio

Ultrasound

AnomalyN=411

All other

indicationsN=1,798

TotalN=2,209

Ultrasound

AnomalyN=652

All other

indicationsN=1,421

TotalN=2,073

Prenatal

totalN=4,282

Abnormal(Any Chromosome Abnormality)

49% 6% 14% 17% 3% 7% 11%

NORMAL 51% 94% 86% 83% 97% 93% 89%

Data from additional analysis of the 2012 NICHD microarray study dataset (Wapner et al. 2012)

Abnormal karyotype in:

• 29% of Prenatals (CVS + Amnio) referred for U/S anomalies

• 4.9% of prenatals (CVS + Amnio) referred for all other indications

Temporal Variability/Indication Variability: CVS vs Amnio

Incidence & Spectrum of Karyotype Abnormalities in Prenatal Diagnosis

Data from additional analysis of the 2012 NICHD microarray study dataset (Wapner et al. 2012)

Chromosome

Abnormality

CVS Amnio

CVS

+

AmnioUltrasound

Anomaly

N=411

All other

indications

N=1,798

Total

N=2,209

Ultrasound

Anomaly

N=652

All other

indications

N=1,421

Total

N=2,073

Overall total

N=4,282

Trisomy 21 21.17% 3% 6.38% 4.14% 1.41% 2.27% 4.39%

Trisomy 18 11.19% 0.61% 2.58% 5.37% 0.07% 1.74% 2.17%

Trisomy 13 4.38% 0.17% 0.95% 2.30% 0% 0.72% 0.84%

45,X 7.30% 0.06% 1.40% 1.23% 0% 0.39% 0.91%

47,XXY 0.97% 0.17% 0.32% 0.15% 0% 0.05% 0.19%

47,XXX 0.49% 0.06% 0.14% 0% 0.28% 0.19% 0.16%

47,XYY 0% 0.06% 0.05% 0.31% 0% 0.10% 0.07%

69,XXX / 69,XXY 1.7% 0.17% 0.45% 1.07% 0% 0.34% 0.40%

Struct Rearr: Unbalanced 1.22% 0.28% 0.45% 1.84% 0% 0.58% 0.51%

Struct Rearr: Balanced 0.24% 1.17% 1% 0.31% 1.13% 0.87% 0.93%

Other non-mosaic aneuploidy * 0% 0.17% 0.14% 0.15% 0% 0.05% 0.09%

Struct Rearr: Markers 0% 0% 0% 0.15% 0.14% 0.14% 0.07%

Benefits of Rapid Aneuploidy Screening

• Trisomies 13, 18 & 21 and Monosomy X are the most common aneuploidies related to maternal age or fetal abnormality

• Routine chromosome analysis take 7-10 days

• Rapid screening on uncultured cells can be done by:

❖ Interphase FISH

❖ MLPA

❖ qPCR

• Reduces emotional burden on the patient and/or physician in the face of an increased risk for chromosome abnormalities following an abnormal screening result

❖ Opportunity to reduce anxiety through earlier decision making

Incidence & Spectrum of Karyotype Abnormalities in Prenatal Diagnosis

Karyotype

Result

CVS AmnioCVS

+

Amnio

Ultrasound

AnomalyN=411

All other

indicationsN=1,798

TotalN=2,209

Ultrasound

AnomalyN=652

All other

indicationsN=1,421

TotalN=2,073

Overall totalN=4,282

Abnormal(Any Chromosome Abnormality)

49% 6% 14% 17% 3% 7% 11%

NORMAL 51% 94% 86% 83% 97% 93% 89%

Data from additional analysis of the 2012 NICHD microarray study dataset (Wapner et al. 2012)

What proportion of fetuses with a NORMAL KARYOTYPE

will have a genomic abnormality ??

Karyotype

Resolution:

>5-10 Million Base Pairs

(5-10 Mb)

Resolution:

<< 5 Million Base Pairs

(Kilobase Range…1kb-5Mb)

Chromosomal Microarray(CMA)

1,000 - 5,000 X Order of Diagnostic Magnitude

Clinically Relevant Information Seen by CMA and Reported to Patients in Cases with Normal Karyotype

Category Total Clinically Relevant

AMA

N=1966

34

(1.7%) 1.2 – 2.4~ 1:60

Positive Screen

N=72912

(1.7%) 0.9 – 2.9

US Anomaly

N=757

45

(6.0%) 4.5 – 7.9~ 1:17

95% CI

By Indications for Testing

Its not all Down SyndromeImportant Findings Not Seen on Karyotype

Micro-del/dup Syndromes Non-Syndromic Micro Del/Dups

DiGeorge22q11

Deletion3.5Mb 16p11.2 Autism 0.55Mb

Miller Dieker17p13.3

deletion1q21.1

ID,

microcephaly,

cardiac,

cataracts

0.8Mb

Prader Willi15q11-13

deletion4MB 16p13.11

Autism, ID, and

schizophrenia 0.8Mb

Smith

Magenis

17p11.2

deletion5Mb Postnatal Studies

Wolf

Hirshhorn

4p16.3

deletion1.9Mb 15-20% yield by CMA in children with

unexplained developmental delay/ID, and

congenital anomalies compared to ~3%

with karyotypeWilliams-

Beuren

7q11.23

deletion1.5Mb

Evaluation of Structural Anomaly Seen on Ultrasound

22q11 DeletionSpectrum of Clinical Features

Clinical Feature %

Learning Disabilities

none/mild

moderate/severe

62 %

30 %

Cardiac Defect 75 %

Genitourinary Defects 36 %

Palate Anomalies

Cleft Palate

Velopharyngeal insufficiency

76 %

9 %

67 %

Abnormal facial features Frequent

Growth Delay (<3rd %) 36 %

Psychosis /Behavior Problems 25 %

Hypoparathyroid 60 %

N =900

Prenatal Detection & Counseling for 22q11 deletion

It’s Not All 22q11.2

Fetuses with Structural Cardiac Defects & Normal Karyotype

Normal

Karyotype (N)

Number of Normal

Karyotype with

Micro Del/Dup (%)

Not Detected

by FISH (%)

All Cardiac

Defects210

30

(14.3%)67%

Isolated Cardiac

Defect59

8

(13.6%)63%

Structural Anomalies

Significant Microdeletions Associated with CHD

Region Name Phenotype

Del 1p36 1p Deletion Syndrome ID

Del 1q21.1 Mild MI

Del 4p16.3 Wolf-Hirschhorn Synd Microcephaly, severe ID, seizure

Del 5p15.2 Cri-du-chat Severe ID

Del 5q35.2 ASD and conduction Defect

Del 7q11.23 Williams-Bueren Synd Cognitive deficits, infantile hypocalcemia

Del 8p23.1 ID

Del 9q34

Del 11q23-qter Jacobsen Synd ID

Del 16p13.3 Rubinstein Taybi + CHD ID

Del 20p12.2 Alagille Synd Liver Disease

Del 22q11.2 DiGeorge Synd ID, Schizophrenia

Frequency of Ultrasound Detected Anomalies-Cytogenetics + Array

Anomaly

Systemn= 826

Abnormal

KaryotypeAbnormal Array Incremental Yield

Cardiac 232 78 (33.6%) 24 (10.3%) 15.58%

Face 108 42 (32.9%) 10 (9.3%) 15.15%

Thorax 50 10 (20.0%) 6 (12%) 15.00%

Renal 93 24 (25.8%) 8 (8.6%) 11.59%

CNS 200 51 ( 25.5%) 14 (7.0%) 9.40%

Skeletal 193 57 (29.5%) 12 (6.2%) 8.82%Donelly et al: Obs and Gynecol, 2014

Anomaly

System

n= 752 with

Normal KaryoCNV array

findings (n)

Additional

benefit (%)

SINGLE System 498 28 5.6%

MULTIPLE System 254 33 13.0%8.1%

Many New Micro-deletion/duplication Syndromes have No or Subtle U/S anomalies

Recurrent CNVs That Have The Potential To Cause Neurocognitive Impairment

Occurred in approximately

1:125 (0.8%)

cases sampled for AMA or positive

screening

Recurrent CNVs With Neurocognitive Phenotype Seen in NICHD Study in Pts with Normal Scan

Deletions N Nml U/S Phenotype

1q21.1 1 1 ID, microcephaly, cardiac and cataracts

7q11.23 1 0 Williams Beuren

15q11.2 2 2 Prader-Willi

15q13.2q13.3 1 1 ID and epilepsy

16p11.2 3 2 Autism

16p12.1 1 0 ID

16p13.11p12.3 3 1

16p13.11 5 3 Autism, ID and schizophrenia

17q12 6 1 Renal cysts, diabetes, autism and schizophrenia

22q11.2 11 3 DiGeorge/Velocardiofacial

Duplications N Nml U/S Phenotype

1q21.1 4 2 ID, microcephaly, cardiac and cataracts

15q11.2q13.1 1 1 Psychiatric Disease

15q11.2q13.1 1 1 Psychiatric Disease

16p13.11p12.3 2 1 Psychiatric Disease

16p13.11 4 3 Variable phenotype

17q12 3 2 Epilepsy

22q11.21 2 2 Variable Phenotype

Chromosomal Microarrays for Prenatal Cases

Consensus Statement

• American Congress of Obstetricians and Gynecologists (ACOG) and the Society for

Maternal Fetal Medicine recommend (SMFM) that all pregnant women should be offered

the option of diagnostic testing regardless of maternal age

• ACOG/SMFM recommend that CMA replace or supplement karyotype for prenatal

evaluation of fetuses with major structural anomalies

2%2%

?

Sales

AMA

Positive Screen

PathogenicSequenceVariation

The Frequency & Cause of Pathogenic Genomic Changes in Cases with a Normal Karyotype

Category Clinically Relevant CNV

AMA

N=1966

34

(1.7%)~ 1:60

Positive Screen

N=72912

(1.7%)

US Anomaly

N=75745

(6.0%)~ 1:17

6%

?

Sales

US Anomaly

PathogenicSequenceVariation

Exonic/whole

genome

Exonic/whole

genome

CNV

CNV

Studies of Whole Exome Sequencing

Undiagnosed DisordersStudy Journal N=4,676 Ascertainment % Resolved

Calvo 2012 Sci Transl Med 42 Mitochondrial 24%

Yang 2013 NEJM 250 80% Neuro 25%

DeLigt 2013 NEJM 100 Severe ID 16%

Srivastava 2014 Annals of Neuro 78 Neuro 41%

Yang 2014 JAMA 2,000 Mixture 25%

Lee 2014 JAMA 814 Mixture 26%

Soden 2014 Sci Transl Med 119 Neuro 45%

Wright 2015 Lancet 1113 Mixture 27%

Zhu 2015 GiM 119 Mixture 24%

Tarailo-Graovac 2016 NEJM 41 Neurometabalomic 68%

Fetal SeriesStudy Journal N Ascertainment % Resolved

Drury 2015 Prenatal Diagnosis 24 Fetal Anomaly 21%

Carss 2014 Hum Mol Genet 30 Fetal Anomaly 10%

Most Cases are WES, Postnatal and Selected

27%

Prenatal Sequencing Studies

• Recent meta-analysis by Best et.al (Prenat Diagn.

2017 Jun 27)

❖ 6.2% - 80% Diagnostic Rates across the 16 studies with

five or more fetuses (included conference abstracts)

❖ Primarily WES, <50% Trios

Prenatal Exome Sequencing Study at Columbia University Medical Center

All patients with Fetal Structural Anomalies

(Includes NT>3.5mm)

Informed Consent obtained

Parental Blood Available

Karyotype and CMA

Trio Whole Exome Sequencing

Clinically Significant Information Reported back to Patient

after confirmation in a CLIA Laboratory

Prenatal Exon Array Study at Columbia University Medical Center – Results

Exome Sequencing ResultsN=460 (65/460 14.1%)

Dominant(70.8%)

Autosomal Recessive(23.1%)

X-Linked Recessive

(6.2%)

de novo

40(87.0%)

Inherited

6(13.0%)

15(23.1%)

de novo

1(25%)

Inherited

3(75%)

Incidental finding reported

4

Total Screened 1,075

Excluded/Withdrawn 288*

Abnormal Karyotype or Array 127 (17.5%)

Eligible for Sequencing 660

Consent for Sequencing 429 (65%)

Over time, our pathogenic rate has

increased. Possible reasons:

• Early series was too small

• Ever expanding knowledge of

prenatal phenotypes has allowed

us to reclassify some variants (i.e.

SCN2A)

• New medical literature during

course of study associated

candidates genes with human

disease (i.e. ARMC9, RAC1,

LZTR1)

• Follow up postnatally allows us to

identify children growing into a

phenotype (i.e. RERE)

7

8

9

10

11

12

13

14

15

166 234 263 287 304 324 336 364 460

%

TRIOS ANALYZED

Exome Sequencing Pathogenic Hit Rate Over Time

7.8%

14.1%CompletedTrios

Pathogenic

Results

166 13 (7.8%)

234 22 (9.4%)

263 26 (9.9%)

287 31 (10.8%)

304 35 (11.5%)

324 38 (11.7%)

336 40 (11.9%)

364 50 (13.7%)

460 65 (14.1%)

SMFM Abstract 2017

Prospective Analysis of WES for Fetal Anomalies (N= 460 Cases)

N(Trios)

GeneticDiagnosis

Single Anomaly

319 30 (9.4%)

Multiple Anomalies

141 35 (25.0%)

Total 460 65 (14.1%)

• For single anomalies, specific organ systems likely have higher diagnostic yields

Prospective Analysis of WES for Fetal Anomalies

Frequency of Variants by Anomaly

SystemN

(Trios)Genetic

Diagnosis

Heart 142 11 (7.8%)

Nuchal 64 8 (13.0%)

CNS 102 26 (25.5%)

Skeletal 78 24 (30.8%)

IUGR 63 11 (17.5%)

Renal 61 11 (18.0%)

Effusion 33 10 (30.3%)

Cases With Multiple Anomalies Counted More Than Once

Exome Sequencing ResultsN=460 (65/460 14.1%)

Lessons Learned to Date

MATERNAL FETAL PRECISON MEDICINE

Columbia University

Medical Center

Case #1A Genetic Diagnosis can Provide Reassurance

Left Foot Right Foot

Differential Diagnosis for Polysyndactyly

• Aase Syndrome

• Diamond-blackfan Syndrome

• DOOR Syndrome

• Duane-radial Syndrome (DR Syndrome)

• Fanconi Anemia (Pancytopenia-dysmelia Syndrome)

• Fetal Hydantoin Syndrome (Dilantin Embryopathy)

• Goodman Syndrome

• Holt-Oram Syndrome

• Hypomelanosis Of Ito

• IVIC Syndrome

• Juberg-hayward Syndrome

• Lacrimo-auriculo-dento-digital Syndrome (LADD Syndrome) (Levy-hollister Syndrome)

• Mesomelic Dysplasia (Werner Type)

• Nager Syndrome

• Normal Variant : Isolated Anomaly

• Poland Syndrome (Pectoral Muscle Aplasia-syndactyly)

• Thalidomide Embryopathy

• Townes-brocks Syndrome

• Trichorhinophalangeal Dysplasia Type (Langer Gidieon Syndrome)

• Trisomy 13

• Trisomy 22

• VATER Association

Sequencing Analysis• Mutation in regulatory region of SHH gene

• Mutations in the Sonic hedgehog limb enhancer cause

limb malformations

• Consistent with a clinical diagnosis of Triphalangeal

thumb with Polysyndactyly

❖ Typically have no associated cognitive problems

❖ Usually do well with surgery

Sequencing Analysis

Case 2: Sequencing Analysis Reveals Incidental Finding of Medical Importance

• No pathogenic or suspicious sequencing variant identified

• Incidental finding of a pathogenic mutation causing Factor

XI deficiency

• Important medical factor that surgeons would need to know

before performing cardiac surgery when baby is born

Cardiac Defect

Single AV canalStomach

Single AV canal

Case #3The Importance of Reanalysis

• Background

❖ Vermian hypoplasia and Agenesis of the CC; IUGR <3%

❖ 46,XX and normal array

❖ European decent

❖ Patient elected to terminate pregnancy

❖ A de novo nonsyn missense mutation found in RAC1

c.170A>C exon 3 of 7

− This was flagged by our bioinformatics team, but no known

human disease reported in OMIM at that time

− RAC1 has important role in neurite outgrowth and axonal path

finding, as well as neuronal migration.

− Our variant was not present in ExAC

− Gene put on genematcher and PubMed alert set for new

publications

Case #3The Importance of Reanalysis

• A New Report about 1 year later found seven individuals

with de novo missense RAC1 mutations and variable

developmental delay with additional features

• Our variant was not included in the publication

Phenotypic Overlap + Inheritance Match + Disease Mechanism Match + OMIM Gene

• Phenotypic Overlap + Inheritance Match + Disease

mechanism Match + OMIM gene (# 617751)

❖ Prompting us to confirm and report to family

• When patient was called, she was already 8 months

pregnant.

❖ Already had a fetal MRI and U/S due to concern from prior

pregnancy.

❖ Relieved for low recurrence risk

− Planning to test her son who was born to r/o remote likelihood of germline mosaicism.

Case #3The Importance of Reanalysis

GeneNew

Publication

Child Grew into

Phenotype

RAC1

RERE

MYH10

ARMC9

LZTR1

RASA1

* RERE (OMIM#: 616975): Developmental delay, intellectual disability, and defects involving the brain, eye, ear, heart, and kidney.

Prenatal presentation: 3.5mm NT

*

Postnatal presentation: Hypotonia, dysmorphic, hearing loss

Have these Genomic Screening Advances Gone Far Enough?

Fetal WES Data and its Relationship to

Expanded Carrier Screening

Cell-free fetal DNA prenatal multigene

screening

Fetal WES Data and its Relationship to Expanded Carrier Screening

Ultrasound Finding

Recessive

Genes found

in CUMC

WES cohort

Arthrogryposis/clubfeet RAPSN

Small cerebellum. bilat cataracts OCRL

Encephalocele, renal, polydactyly CEP290

Agenesis of the Corpus LICAM

Cerebellar hypoplasia C5ORF42

Splaying of cerebellum/renal TMEM67

Hydranencephaly FLVCR2 (2 cases)

Encephalocele, renal, polydactyly CC2D2A

Agenesis of corpus, Ventriculomegaly GPSM2

ACC, Ventriculomegaly , clubfoot POMT2

Severe shortening limbs FGD1

Clubfoot, polyhydramnios KLH40

Hydrops PIEZO1 (2 cases)

Encephalocele ARMC9

Cardiomyopathy LZTR1

15 Genes (17 Cases)

Commercial expanded carrier screening still misses the majority of

conditions found in the structural anomaly cohort

• A case for further expanding carrier screening to whole exome level coverage?

Ultrasound Finding

Recessive

Genes found

in CUMC

WES cohort

Lab #1

141 genes

Arthrogryposis/clubfeet RAPSN N

Small cerebellum. bilat cataracts OCRL N

Encephalocele, renal, polydactyly CEP290 N

Agenesis of the Corpus LICAM N

Cerebellar hypoplasia C5ORF42 N

Splaying of cerebellum/renal TMEM67 N

Hydranencephaly FLVCR2 (2 cases) N

Encephalocele, renal, polydactyly CC2D2A N

Agenesis of corpus, Ventriculomegaly GPSM2 N

ACC, Ventriculomegaly , clubfoot POMT2 N

Severe shortening limbs FGD1 N

Clubfoot, polyhydramnios KLH40 N

Hydrops PIEZO1 (2 cases) N

Encephalocele ARMC9 N

Cardiomyopathy LZTR1 N

15 Genes (17 Cases)TOTAL 0/17

Percent % 0%

Ultrasound Finding

Recessive

Genes found

in CUMC

WES cohort

Lab #1 Lab #2

141 genes 158 genes

Arthrogryposis/clubfeet RAPSN N Y

Small cerebellum. bilat cataracts OCRL N Y

Encephalocele, renal, polydactyly CEP290 N N

Agenesis of the Corpus LICAM N N

Cerebellar hypoplasia C5ORF42 N N

Splaying of cerebellum/renal TMEM67 N N

Hydranencephaly FLVCR2 (2 cases) N N

Encephalocele, renal, polydactyly CC2D2A N N

Agenesis of corpus, Ventriculomegaly GPSM2 N N

ACC, Ventriculomegaly , clubfoot POMT2 N N

Severe shortening limbs FGD1 N N

Clubfoot, polyhydramnios KLH40 N N

Hydrops PIEZO1 (2 cases) N N

Encephalocele ARMC9 N N

Cardiomyopathy LZTR1 N N

15 Genes (17 Cases)TOTAL 0/17 2/17

Percent % 0% 11.8%

Ultrasound Finding

Recessive

Genes found

in CUMC

WES cohort

Lab #1 Lab #2 Lab #3

141 genes 158 genes 173 genes

Arthrogryposis/clubfeet RAPSN N Y N

Small cerebellum. bilat cataracts OCRL N Y N

Encephalocele, renal, polydactyly CEP290 N N N

Agenesis of the Corpus LICAM N N N

Cerebellar hypoplasia C5ORF42 N N N

Splaying of cerebellum/renal TMEM67 N N N

Hydranencephaly FLVCR2 (2 cases) N N N

Encephalocele, renal, polydactyly CC2D2A N N N

Agenesis of corpus, Ventriculomegaly GPSM2 N N N

ACC, Ventriculomegaly , clubfoot POMT2 N N N

Severe shortening limbs FGD1 N N N

Clubfoot, polyhydramnios KLH40 N N N

Hydrops PIEZO1 (2 cases) N N N

Encephalocele ARMC9 N N N

Cardiomyopathy LZTR1 N N N

15 Genes (17 Cases)TOTAL 0/17 2/17 0/17

Percent % 0% 11.8% 0%

Ultrasound Finding

Recessive

Genes found

in CUMC

WES cohort

Lab #1 Lab #2 Lab #3 Lab #4

141 genes 158 genes 173 genes 281 genes

Arthrogryposis/clubfeet RAPSN N Y N Y

Small cerebellum. bilat cataracts OCRL N Y N N

Encephalocele, renal, polydactyly CEP290 N N N Y

Agenesis of the Corpus LICAM N N N N

Cerebellar hypoplasia C5ORF42 N N N N

Splaying of cerebellum/renal TMEM67 N N N N

Hydranencephaly FLVCR2 (2 cases) N N N N

Encephalocele, renal, polydactyly CC2D2A N N N N

Agenesis of corpus, Ventriculomegaly GPSM2 N N N N

ACC, Ventriculomegaly , clubfoot POMT2 N N N N

Severe shortening limbs FGD1 N N N N

Clubfoot, polyhydramnios KLH40 N N N N

Hydrops PIEZO1 (2 cases) N N N N

Encephalocele ARMC9 N N N N

Cardiomyopathy LZTR1 N N N N

15 Genes (17 Cases)TOTAL 0/17 2/17 0/17 2/17

Percent % 0% 11.8% 0% 11.8%

Ultrasound Finding

Recessive

Genes found

in CUMC

WES cohort

Lab #1 Lab #2 Lab #3 Lab #4 Lab #5

141 genes 158 genes 173 genes 281 genes >300 genes

Arthrogryposis/clubfeet RAPSN N Y N Y Y

Small cerebellum. bilat cataracts OCRL N Y N N Y

Encephalocele, renal, polydactyly CEP290 N N N Y Y

Agenesis of the Corpus LICAM N N N N N

Cerebellar hypoplasia C5ORF42 N N N N N

Splaying of cerebellum/renal TMEM67 N N N N N

Hydranencephaly FLVCR2 (2 cases) N N N N N

Encephalocele, renal, polydactyly CC2D2A N N N N N

Agenesis of corpus, Ventriculomegaly GPSM2 N N N N N

ACC, Ventriculomegaly , clubfoot POMT2 N N N N N

Severe shortening limbs FGD1 N N N N N

Clubfoot, polyhydramnios KLH40 N N N N N

Hydrops PIEZO1 (2 cases) N N N N N

Encephalocele ARMC9 N N N N N

Cardiomyopathy LZTR1 N N N N N

15 Genes (17 Cases)TOTAL 0/17 2/17 0/17 2/17 3/17

Percent % 0% 11.8% 0% 11.8% 17.6%

Ultrasound Finding

Recessive

Genes found

in CUMC

WES cohort

Lab #1 Lab #2 Lab #3 Lab #4 Lab #5 Lab #6

141 genes 158 genes 173 genes 281 genes >300 genes 327 genes

Arthrogryposis/clubfeet RAPSN N Y N Y Y Y

Small cerebellum. bilat cataracts OCRL N Y N N Y Y

Encephalocele, renal, polydactyly CEP290 N N N Y Y Y

Agenesis of the Corpus LICAM N N N N N Y

Cerebellar hypoplasia C5ORF42 N N N N N N

Splaying of cerebellum/renal TMEM67 N N N N N N

Hydranencephaly FLVCR2 (2 cases) N N N N N N

Encephalocele, renal, polydactyly CC2D2A N N N N N N

Agenesis of corpus, Ventriculomegaly GPSM2 N N N N N N

ACC, Ventriculomegaly , clubfoot POMT2 N N N N N N

Severe shortening limbs FGD1 N N N N N N

Clubfoot, polyhydramnios KLH40 N N N N N N

Hydrops PIEZO1 (2 cases) N N N N N N

Encephalocele ARMC9 N N N N N N

Cardiomyopathy LZTR1 N N N N N N

15 Genes (17 Cases)TOTAL 0/17 2/17 0/17 2/17 3/17 4/17

Percent % 0% 11.8% 0% 11.8% 17.6% 24%

Ultrasound Finding

Recessive

Genes found

in CUMC

WES cohort

Lab #1 Lab #2 Lab #3 Lab #4 Lab #5 Lab #6 Lab #7

141 genes 158 genes 173 genes 281 genes >300 genes 327 genes 992 genes

Arthrogryposis/clubfeet RAPSN N Y N Y Y Y Y

Small cerebellum. bilat cataracts OCRL N Y N N Y Y Y

Encephalocele, renal, polydactyly CEP290 N N N Y Y Y Y

Agenesis of the Corpus LICAM N N N N N Y Y

Cerebellar hypoplasia C5ORF42 N N N N N N Y

Splaying of cerebellum/renal TMEM67 N N N N N N Y

Hydranencephaly FLVCR2 (2 cases) N N N N N N Y

Encephalocele, renal, polydactyly CC2D2A N N N N N N Y

Agenesis of corpus, Ventriculomegaly GPSM2 N N N N N N Y

ACC, Ventriculomegaly , clubfoot POMT2 N N N N N N Y

Severe shortening limbs FGD1 N N N N N N Y

Clubfoot, polyhydramnios KLH40 N N N N N N N

Hydrops PIEZO1 (2 cases) N N N N N N N

Encephalocele ARMC9 N N N N N N N

Cardiomyopathy LZTR1 N N N N N N N

15 Genes (17 Cases)TOTAL 0/17 2/17 0/17 2/17 3/17 4/17 12/17

Percent % 0% 11.8% 0% 11.8% 17.6% 24% 70.6%

Fetal WES Data and its Relationship to Cell free fetal DNA screening for de novo variants

Ultrasound Finding

Genes with de

novo findings in

our CUMC WES

cohortSevere shortening limbs COL1A1

Pleural effusion RIT1

Cystic hygroma, CHAOS FGFR2

Ependymal nodules TSC2

Skeletal dysplasia FGFR3 (3 cases)

Arm anomaly NIPBL

Unilat Renal Agenesis, hemivertebrae KMT2D

Megacystis ACTG2

Renal agenesis RET

Increased NT RERE

Hypoplastic Left Heart Syndrome NR2F2

Diaphragmatic hernia ZFPM2

ACC, ventriculomegaly MYH10

Intracranial hemorrhage COL4A1 (2 cases)

Dandy Walker RAC1

Cerebellar hypoplasia TUBA1A

Ventriculomegaly AIRID1A (identical twins)

Severe shortening of long bones club feet, micrognathia FLNB

Severe Limb Shortening COL2A1 (3 cases)

Arthrogryposis/polymicrogyria SCN2A (2 cases)

20 Genes (27 Cases)

Commercial cff-

DNA screening

for de novo

variants still

misses the

majority

of conditions

found in the

structural

anomaly cohort

A case for further

expanding cff-DNA

screening models to

larger gene

panel/whole exome

level?

Ultrasound Finding

Genes with de

novo findings in

our CUMC WES

cohort

Screened on cff-DNA

Severe shortening limbs COL1A1 Y

Pleural effusion RIT1 Y

Cystic hygroma, CHAOS FGFR2 Y

Ependymal nodules TSC2 Y

Skeletal dysplasia FGFR3 (3 cases) Y

Arm anomaly NIPBL Y

Unilat Renal Agenesis, hemivertebrae KMT2D N

Megacystis ACTG2 N

Renal agenesis RET N

Increased NT RERE N

Hypoplastic Left Heart Syndrome NR2F2 N

Diaphragmatic hernia ZFPM2 N

ACC, ventriculomegaly MYH10 N

Iacranial hemorrhage COL4A1 (2 cases) N

Dandy Walker RAC1 N

Cerebellar hypoplasia TUBA1A N

Ventriculomegaly AIRID1A (identical twins) N

Severe shortening of long bones club feet, micrognathia FLNB N

Severe Limb Shortening COL2A1 (3 cases) N

Arthrogryposis/polymicrogyria SCN2A (2 cases) N

20 Genes (27 Cases)TOTAL 8/27

Percent 29.6%

WES Data Provides Evidence for Additional Value in Expanded Molecular Screening Modalities

Advantages

• Cell free fetal DNA screening for de novo variants

could be done in first trimester, leading to earlier

diagnosis, discussion of options, and pregnancy

management planning

• Expanded carrier screening could be done even

in preconception, allowing more couples the

option for IVF with PGD and/or an earlier

diagnostic procedure

WES Data Provides Evidence for Additional Value in Expanded Molecular Screening Modalities

Barriers

• Scalability

❖ More infrastructure needed to process and curate large

numbers of samples

• Costs $$$$

❖ What is the cost of screening in relation to the economic

benefit - need to assess healthcare dollars

• Curation

❖ In the absence of a phenotype, some variants may be

classified as VOUS and not reported to patients

❖ Many screening labs do not currently report VOUS to

decrease confusion and anxiety

Karyotype17%

CMA6%

WES14%

?63%

Contribution of Genomic Technologies in elucidating the etiology of Fetal Anomalies in the 2nd Trimester

Contribution of Genomic Technologies in elucidating the etiology of Fetal Anomalies in the 2nd Trimester

• What does Exon Array Analysis (Tier 1) add?

17%

6%

13.70%

28%

33.30%

2%

Karyotype

CNV

Pathogenic Sequence Variation

Plausible

Unknown

Exon

Technological and Scientific Advances are Rarely Ready for Immediate Clinical Use Following Their Discovery

• Logistic Reasons

❖ High Costs

❖ Transitioning R&D protocols into standard clinical

laboratory protocols

− High throughput

− Equipment

• Clinical Reasons

❖ Relevance to public health

❖ Significance of new findings

− Lack of supporting literature

• Financial Reasons

❖ Reimbursement of testing

− Who pays for the new (expensive) tests?

Copy Number Assessment Needs to be added to NGS in a Prenatal Setting

• If NGS is to be a standalone test, it will at minimum

require detection of:

• Whole chromosome aneuploidies

❖ eg.Trisomy 21, Monosomy X

• Partial Aneuploidies

❖ eg. Marker Chromosomes, unbalanced rearrangements

(inversions & translocations)

• Microdeletions and Microduplications

❖ e.g. Di George syndrome, Miller Dieker syndrome

Conclusions

• Take the lessons we have learned / are still learning from

microarray and apply them to NGS SNV/CNV detection

• Ask the right questions

• Do the necessary validations

NGS is a powerful tool that has the ability to reveal the

basic building blocks of a person’s being.

In a research setting it is uncovering a great deal of

knowledge regarding the etiology of common, rare and

complex genetic diseases

There is still a formidable amount of knowledge that is

lacking regarding the consequences of rare variants

Clinical utility improves as this knowledge gap closes

Databases with prenatal phenotypes is necessary

Acknowledgements

Columbia University Medical CenterInstitute of Genomic Medicine (IGM)

• David B. Goldstein

• Louise Bier

• Colin Malone

• Slave Petrovski

• Nicholas Strong

• Anya Revah Politi

• Natalie C. Lippa

• Michelle E. Ernst

• Caroline Mebane

• Michelle Saliba

• Neha Dagaonkar

Maternal fetal Medicine (MFM)

• Ronald J. Wapner

• Jessica L. Giordano

• Erica Spiegel

• Melissa Stosic

• Stephanie Galloway

• Brooke Johannes

Personalized Genomic Medicine (PGM/PGL)

• Vimla Aggarwal

• Vaidehi Jobanputra

• Mahesh Mansukhani

• Odelia Nahum

• Emily Clancy

Pediatrics

• Kwame Yeboa