Experience Supporting Clinical Next Generation Sequencing

Heidi L. Rehm, PhD, FACMG Director, Laboratory for Molecular Medicine, PCPGM

Assistant Professor of Pathology, BWH, MGH, HMS

Laboratory for Molecular Medicine at PCPGM

25% of testing is from

Partners’ patients

75% is from other US and

International patients

• CLIA-certified in 2003

• LMM offers >150 tests in cardiovascular disease, cancer,

hearing loss, pharmacogenetics and genetic syndromes

• Main focus of testing is large multi-gene panels using

sequencing technologies (NGS, Sanger)

• Other technologies include TaqMan, Luminex, allele-specific

PCR, MLPA, PNAs, STRs, droplet PCR

• WGS service will launch this week

Targeted panels are enlarging and more and more labs

are launching exome and genome services.

Genomic testing is improving diagnoses, enabling

informed treatments, and defining disease risks.

With gaining community acceptance, we must now

focus on optimizing support for integrating genomics

into the practice of medicine.

Clinical Genomics is Evolving

Step 1: Improve the analytical validity of NGS

Improve variant calling accuracy

Indels, CNVs, SVs, repeat expansions

Fill in the holes

Targeted NGS panels miss a variable amount of content

depending on genes

Exome sequencing misses 5-15% of coding sequence

International Collaboration for Clinical Genomics

Medical Exome Project: Partners/Emory/CHOP

0

50

100

150

co

ve

rage

exons

AVERAGE COVERAGE PER EXON

Cardiomyopathy Test (75 bp, single read, GAII)

Courtesy of Siva Gowrisankar

Relative Contribution of HCM Genes

MYBPC3 46.3%

MYH7 32.9%

TNNI3 5.8%

TNNT2 5.2%

TPM1 2.6%

MYL2 2.6%

GLA 1.7%

MYL3 1.5%

10 genes <0.5%

Need to address missing data for clinically important

genes.

Genes with high

contributions should

be fully covered

HCM Genes

0

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Failed Regions

Uncallable bases

Variant Confirmation

Am

plic

on

s

Sample number

Sanger Sequencing Follow-Up for OtoGenome

Average Coverage: 876 (range 275-1643)

Average Follow-up: 27 amplicons (out of 1439)

Failed Regions (7 entries, 9 amplicons)Gene % uncallableUncallable/Total basesCoordinatesNGS ROI intervalSanger ConfirmationMapping Messages

CTF1 100 55/55 16:30907950-30908004CTF1_EXON_01

CTF1 48 237/492 16:30913384-30913875CTF1_EXON_03

DES 2 14/608 2:220283170-220283777DES_EXON_01

DSC2 9 50/527 18:28681851-28682377DSC2_EXON_01

DSG2 15 Nov-75 18:29078200-29078274DSG2_EXON_01

RBM20 98 217/221 10:112404198-112404418RBM20_EXON_01

TPM1 73 119/162 15:63340760-63340921TPM1_EXON_02A_(1)

Uncallable bases Gene Exon cDNA PositionReference baseVariant baseCoverage A;B breakdownB frequencyCategory ClassificationPredicted ZygosityPredicted VariantPredicted AAStrand biasSanger ConfirmationCoordinatesMapping Messages

DSP Exon 24 c.8466T T . 0 0 0 Uncallable 0 Uncallable 6:7585961-7585961

DSP Exon 24 c.8470G G . 0 0 0 Uncallable 0 Uncallable 6:7585965-7585965

DSP Exon 24 c.8471G G . 0 0 0 Uncallable 0 Uncallable 6:7585966-7585966

LDB3 Exon 12 c.1294T T . 0 0 0 Uncallable 0 Uncallable 10:88476146-88476146

LMNA Exon 10 c.1706G G . 19 19;0 0 Uncallable -10.01 Uncallable 1:156107542-156107542

LMNA Exon 10 c.1707T T . 17 17;0 0 Uncallable -10.01 Uncallable 1:156107543-156107543

TNNT2 Intron 03 c.53-8C G . 0 0 0 Uncallable 0 Uncallable 1:201341177-201341177

TNNT2 Intron 03 c.53-7T A . 0 0 0 Uncallable 0 Uncallable 1:201341176-201341176

TTN Intron 45 c.10361-6TA . 0 0 0 Uncallable 0 Uncallable 2:179616772-179616772

TTN Intron 45 c.10361-5TA . 0 0 0 Uncallable 0 Uncallable 2:179616771-179616771

Follow-up (16 entries, 16 amplicons)Gene Exon cDNA PositionReference baseVariant baseCoverage A;B breakdownB frequencyCategory ClassificationPredicted ZygosityPredicted VariantPredicted AAStrand biasSanger ConfirmationCoordinatesMapping Messages

DSC2 Intron 07 c.942+12_942+13insTTAT TTAA 524 74;450 0.86 Variant (Het) c.942+12_942+13insTTAUnknown Variant 18:28666526-28666526

LAMA4 Exon 08 c.827C G T 573 0;573 1 Variant (Hom) c.827C>A p.Ala276Glu -7610.62 Variant 6:112508770-112508770

LDB3 Intron 07 c.756-12_756-11delTCTTC T 294 133;161 0.55 Variant (Het) c.756-12_756-11delTCUnknown Variant 10:88458996-88458998

LMNA Exon 10 c.*4C C G 35 11;24 0.69 Variant (Het) c.*4C>G -10.01 Variant 1:156107559-156107559

MYH6 Exon 07 c.622G C T 809 389;420 0.52 Variant (Unclassified)(Het) c.622G>A p.Asp208Asn-6747.06 Variant 14:23873940-23873940

PKP2 Exon 09 c.1955_1956insGAAGG GCTTC 822 577;245 0.3 Variant (Het) c.1955_1956insGAAGp.Ser652ArgfsX92Unknown Variant 12:32975416-32975416

PRKAG2 Exon 05 c.700_701insGG GC 74 66;8 0.11 Variant (Het) c.700_701insGp.Ala234GlyfsX39Unknown Variant 7:151329208-151329208

RBM20 Exon 09 c.2303G G C 430 0;430 1 Variant (Unclassified)(Hom) c.2303G>Cp.Trp768Ser -7751.62 Variant 10:112572458-112572458

RBM20 Intron 12 c.3452-9G G C 561 0;561 1 Variant (Unclassified)(Hom) c.3452-9G>C -7959.5 Variant 10:112590810-112590810

RYR2 Intron 15 c.1477-11_1477-10insTA AT 358 134;224 0.63 Variant (Het) c.1477-11_1477-10insTUnknown Variant 1:237619875-237619875

RYR2 Intron 29 c.3599-9delTAT A 707 393;314 0.44 Variant (Unclassified)(Het) c.3599-9delT Unknown Variant 1:237753074-237753075

RYR2 Intron 97 c.14091-11_14091-10insTA AT 417 291;126 0.3 Variant (Het) c.14091-11_14091-10insTUnknown Variant 1:237965133-237965133

TTN Exon 43 c.10049C G A 628 308;320 0.51 Variant (Het) c.10049C>Tp.Pro3350Leu-3786.36 Variant 2:179628969-179628969

TTN Exon 44B c.10213G C T 365 0;365 1 Variant (Unclassified)(Hom) c.10213G>Ap.Ala3405Thr-3228.31 Variant 2:179621477-179621477

TTN Intron 45 c.10361-5delTGA G 349 244;105 0.3 Variant (Het) c.10361-5delT Unknown Variant 2:179616770-179616771

TTN Exon 275 c.77167C G A 846 407;439 0.52 Variant (Het) c.77167C>Tp.Arg25723Cys-5407.69 Variant 2:179425988-179425988

Common SNPs (71 entries, 66 amplicons)Gene Exon cDNA PositionReference baseVariant baseCoverage A;B breakdownB frequencyCategory ClassificationPredicted ZygosityPredicted VariantPredicted AAStrand biasSanger ConfirmationCoordinatesMapping Messages

CASQ2 Intron 03 c.420+6T A G 626 0;626 1 Variant Benign (Hom) c.420+6T>C -5986.63 Variant 1:116283343-116283343

CASQ2 Exon 11 c.1185C G A 597 299;298 0.5 Variant Benign (Het) c.1185C>Tp.Asp395Asp-4456.04 Variant 1:116243877-116243877

DSC2 Exon 15A c.2326A T C 784 361;423 0.54 Variant Benign (Het) c.2326A>Gp.Ile776Val -7024.3 Variant 18:28649042-28649042

DSC2 Exon 15A c.2393G C T 713 393;320 0.45 Variant Benign (Het) c.2393G>Ap.Arg798Gln-5346.04 Variant 18:28648975-28648975

DSG2 Exon 08 c.861C C T 797 431;366 0.46 Variant Benign (Het) c.861C>T p.Asn287Asn-6084.92 Variant 18:29104698-29104698

DSG2 Exon 14 c.2318G G A 449 256;193 0.43 Variant Benign (Het) c.2318G>Ap.Arg773Lys -871.89 Variant 18:29122799-29122799

DSG2 Exon 15 c.3321T T C 693 368;325 0.47 Variant Benign (Het) c.3321T>Cp.Val1107Val-3592.72 Variant 18:29126670-29126670

DSP Exon 24 c.7122C C T 1000 520;480 0.48 Variant Benign (Het) c.7122C>Tp.Thr2374Thr-7131.27 Variant 6:7584617-7584617

DSP Exon 24 c.8175C C A 591 290;301 0.51 Variant Benign (Het) c.8175C>Ap.Arg2725Arg -3177.6 Variant 6:7585670-7585670

FILTER OUT

• No Sanger

• No further variant assessment

• Not included on patient report

SANGER SEQUENCE

PIPELINE OUTPUT List of exons that need sanger sequencing

Courtesy of Birgit Funke

A Genetic Sequencing Test is Not One Test

DNA

PCR

Hundreds of assays per sample

Sequencing

Hundreds of bases per exon

Failed exons/bases

One OtoGenome Test is actually ~380,000 tests with an infinite

number of possible results. After the NGS process, Sanger

follow-up begins:

VARIANT TYPE # FN SENSITIVITY 95%CI

All variants 400 2 99.50% 98.2-100

Substitutions 335 0 100.00% 98.9-100

Indels (all) 65 2 96.92% 98.8-99.2

>10 bp 14 1 92.86% 70.2-98.8

?10 bp 51 1 98.04% 89.9-99.7

5-10 bp 3 0 100.00% ND

3-5 bp 19 1 94.74% 76.4-99.1

1-2 bp 29 0 100.00% 88.3-100Pan Cardiomyopathy+ Hearing Loss Panel data FN = False Negative

VARIANT TYPE # FN SENSITIVITY 95%CI

All variants 400 2 99.50% 98.2-100

Substitutions 335 0 100.00% 98.9-100

Indels (all) 65 2 96.92% 98.8-99.2

>10 bp 14 1 92.86% 70.2-98.8

?10 bp 51 1 98.04% 89.9-99.7

5-10 bp 3 0 100.00% ND

3-5 bp 19 1 94.74% 76.4-99.1

1-2 bp 29 0 100.00% 88.3-100

VARIANT TYPE # FN SENSITIVITY 95%CI

All variants 400 2 99.50% 98.2-100

Substitutions 335 0 100.00% 98.9-100

Indels (all) 65 2 96.92% 98.8-99.2

>10 bp 14 1 92.86% 70.2-98.8

?10 bp 51 1 98.04% 89.9-99.7

5-10 bp 3 0 100.00% ND

3-5 bp 19 1 94.74% 76.4-99.1

1-2 bp 29 0 100.00% 88.3-100CNVs 16 0 100.00%

Modified from Birgit Funke

% FN SENS 95% CI % FP SPEC 95% CI

Substitutions * 0% 100% 99.3 - 100 0.08% 92.0% 99.6 - 99.9

In/dels 4.29% 95.70% 89.8 - 99.2 15.00% 85.0% 73.9 - 91.9

CNVs ? ?

Other structural ? ?

*non-repetitive regions

% FN SENS 95% CI % FP SPEC 95% CI

Substitutions * 0% 100% 99.3 - 100 0.08% 92.0% 99.6 - 99.9

In/dels 4.29% 95.70% 89.8 - 99.2 15.00% 85.0% 73.9 - 91.9

CNVs ? ?

Other structural ? ?

*non-repetitive regions

NGS Validation

99.9%

Relative sequence coverage is reproducible and comparable across exons and samples

Base c

overa

ge p

rofile

s fro

m 1

0 P

anC

ard

iom

yopath

y

tests

MYH7 exons

0-206

0-249

0-272

0-270

0-250

0-240

0-274

0-280

0-247

0-272

Trevor Pugh

Detection of full and partial gene deletions

through NGS: VisCap

Lo

g2

ratio

sa

mp

le/b

atc

h m

ed

ian

USH2A heterozygous

exon 10 deletion

All exons, sorted by genome position USH2A exons (3’→5’)

OTOF deletion

47 exons

Trevor Pugh

Need to balance FN and FP Rates

CNVs are confirmed by ddPCR

WGS Case 1: Non-syndromic Hearing Loss

• Mild-moderate “Cookie-bite” hearing loss

• Inheritance could be recessive or dominant with reduced penetrance

• Three affected children sequenced by WGS

• ~ 3.6 million variants identified

• No obvious candidates through initial analysis

Analyzed Hearing Loss Case by OtoGenome

STRC locus

All exons, sorted by genome position

Chromosome 15: STRC gene

Francey et al., 2011

high homology to pSTRC

STRC pSTRC

STRC pSTRC

Hom deletion of STRC

pSTRC

pSTRC

3 2 1 0 -1 -2 -3

2 1 1

2

4

16

47

0

5

10

15

20

25

30

35

40

45

50

0% 1-24% 25-49% 50-74% 75-89% 90-98% >98%

Exome Coverage of 73 Hearing Loss Genes

miRNAs

PTPRQ:

Reference

genome

assembly

problem

STRC:

Pseudogene

Common CNV

100 kb deletion

(43.89 Mb to 43.99 Mb)

WGS Case 1: Nonsyndromic Hearing Loss

Males with this deletion

will be infertile due to

deletion of the adjacent

CATSPER gene

Take Home Message

Targeted gene panels is still the recommended approach

when clinical sensitivity is high and until the cost of

exome/genome sequencing drops below targeted panels

such that one could start with exome/genome and then

reflex to targeted if negative.

Case 2: Distal Arthrogryposis Type 5

Disease is known to be AD and to occur de novo

Skeletal Spine stiffness, Hunched anteverted shoulders, Pectus excavatum, Limited forearm rotation and

wrist extension, Bilateral club feet, Congenital finger contractures, Long fingers, Absent phalangeal

creases, Poorly formed palmar creases, Camptodactyly, Dimples over large joints

Muscle Decreased muscle mass (especially in lower limbs), Firm muscles

Face Triangular face, Decreased facial expression

Ears Prominent ears

Eyes Ophthalmoplegia, Deep-set eyes, Epicanthal folds, Ptosis, Duane anomaly, Keratoglobus,

Keratoconus, Macular retinal folds, Strabismus, Astigmatism, Abnormal electroretinogram, Abnormal

retinal pigmentation

Clinical features:

Case 2: Distal Arthrogryposis Type 5

Previous DA phenotypes associated with MYBPC1,

MYH3, TPM2, MYH8, FBN2, TNNI2, TNNT3 were negative for variants

Two de novo mutations in exonic sequence:

ACSM4 – acyl-CoA synthetase medium-chain family member 4

5 nonsense variants identified in ESP; 1 with 6.4% MAF; 4 occur once

PIEZO2: mechanosensitive

ion channel component 2

Great candidate, but how to we prove causality for a

novel gene-disease association?

Project under discussion with Ada Hamosh to

deploy the PhenoDB system as a means to

connect labs with candidate gene identifications

A New Paradigm in Clinical Genomics

Patient/Provider

Researcher Clinical Lab

Research Discoveries

Clinical Lab Patient Care

Traditional Paradigm

New Paradigm

What is the biggest

bottleneck in clinical

sequencing?

Variant Assessment and Reporting

NVAs

Fellows/Residents

Draft Reports

Genetic Counselors

Signout

Geneticists

Clinical Correlation

Pathologists

Somatic Cancer

~15,000 variants

interpreted in patient

reports to date

Average

22 min

25 min

120 min

Variant Assessment Type

Variant with no data

Variant with dbSNP/ESP data only

Variant with publications

0

50

100

150

200

250

300

350

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

HCM Gene Mutations – 3000 cases tested

>500 clinically significant mutations identified

66% of clinically significant mutations are seen in

only one family

Number of probands

Nu

mb

er

of va

ria

nts

MYBPC3 E258K

MYBPC3 MYH7 R502W W792fs R663H

0

50

100

150

200

250

300

350

400

450

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85 87 89 91

Hearing Loss Gene Mutations – 2000 Cases Tested

Number of probands

Num

ber

of variants

GJB2

35delG

GJB2

V37I

GJB2

M34T

USH2A

2299delG

SLC26A4

81% (423/523) of clinically significant variants have been seen in only one family

100 Healthy Patients (10 PCPs)

100 HCM Patients (10 cardiologists)

Cardiomyopathy

Report

Cardiac Risk

Report

General Genome

Report

50 50

Project 2 Workflow

Whole Genome

Sequencing

MedSeq – WGS Pilot Clinical Trial

Standard of

Care

with

Family

History

50 50

Standard of

Care

with

Family

History Cardiac

Risk

Report

General

Genome

Report

Compare Outcomes Compare Outcomes

The General Genome Report

Variant Analysis for General Genome Report

3,227,455 Substitutions and 418,331 Indels

20,240 CDS/Splice Variants

25 “DM” Variants

HGMD

Genes

5 PGx Class 1

5 Variants

PharmGKB

2 Pathogenic 0 Likely Pathogenic 6 VUS 4 Likely Benign 13 Benign

<1%

616 Rare CDS/Splice Variants

87 Variants 102 LOF Variants

HGMD Genes

LOF

2 Pathogenic 9 VUS

11 Variants

~2 CNVs >1kb

0 Pathogenic

Fetus with US finding: ↑NT

PTPN11 p.Ile309Val Published as “pathogenic” for Noonan syndrome

Patient contacted author of paper who said he later found the variant in 7% of AJ controls; now feels the variant is benign

Courtesy Sherri Bale

Noonan Syndrome Case

?

LMM Case

To improve our knowledge of DNA

variation will require a massive

effort in data sharing

GeneInsight Laboratory Network

BWH

MGH

Intermount

ain Health

Litwin

Center

Michigan

More to

come

MGH

Pathology

LMM

Bioreference

Genetic

Data

Hub

Patient

Care

Providers

Illumina

CLIA Lab

ARUP

GeneInsight

Network

GeneInsight Clinics

GeneInsight Labs

Mt Sinai

NY

Genome

Center

Alert Delivery

3

4

This screenshot was taken from a demonstration system – the content of this screen should

not be used for any clinical purpose

Alerts are Common

~4% of case per year

received medium or high

alerts (.33% per month)

ICCG Project Goals To raise the quality of patient care by:

• Standardizing testing platforms and result interpretation related to structural and sequence-level variation

• Creating a centralized database of clinically relevant variant annotations to share data for clinical and research purposes

• Implementing a QC and expert consensus process for curating data submitted across laboratories and developing evidence based classifications

www.ncbi.nlm.nih.gov/clinvar

ClinVar

ClinVar Contributors (>40)

Group Submissions

OMIM 22954

Partners Healthcare/Harvard Medical School (LMM) 7127

International Standards For Cytogenomic Arrays (ISCA) 4557

GeneReviews 2264

ARUP 1415

LabCorp (Correlagen) 1391

Sharing Clinical Report Project – BRCA1/BRCA2 902

GeneDx 589

NHGRI (ClinSeq) 423

Leiden Muscular Dystrophy 114

Emory 43

Total = 41923

Sequencing Laboratories Which Have Agreed to Share Data

Ackerman Lab, Mayo Alfred I Dupont Hospital for Children All Children's Hospital St. Petersburg Ambry Laboratories ARUP Athena Diagnostics Baylor Medical Genetic Laboratories Boston Children's Hospital Boston University Children's Hospital of Philadelphia Children's Mercy Hospital, Kansas City Cincinnati Children's Hospital City of Hope Molecular Diagnostic Lab CureCMD Denver Genetic Laboratories Detroit Medical Center Emory University Fullerton Genetics Laboratory GeneDx Cleveland Clinic Greenwood Genetics Harvard-Partners Lab for Molec. Medicine Henry Ford Hospital Huntington Medical Research Institutes

Illumina Clinical Services Lab Indiana University/Perdue University InSiGHT LabCorp / Integrated Genetics / Correlagen Masonic Medical Research Laboratory Mayo Clinic Mt. Sinai School of Medicine Nationwide Children's Hospital Nemours Biomolecular Core, Jefferson Medical Oregon Health Sciences University Providence Sacred Heart Medical Center Quest Diagnostics SickKids Molecular Genetic Laboratory Transgenomics University of Chicago University of Michigan University of Nebraska Medical Center University of Oklahoma University of Penn University of Sydney University of Washington Women and Children's Hospital Wayne State University School of Medicine Yale University

SCRP: The Sharing Clinical Report Project: BRCA1/2

Robert Nussbaum, Dawn Lee, Heidi Rehm, George Riley, Peter Kolchinsky, Breast Cancer Clinics

445

19

255

62

119 Pathogenic

ProbablyPathogenic

VUS

Likely Benign

Benign

1981 variants (962 unique) from 25 clinics

Large variant

datasets

Intra-laboratory

Evidence-based review

Practice guidelines

Expert Curation

Single-Source Curation

Uncurated

Multi-Source Curation

Guideline

Inter-laboratory

dbSNP/dbVar

ClinVar

877

39004

1

ISCA

Maintaining Quality and Supporting Multiple Uses in ClinVar

N = 4,432 genes – not yet curated, still contain “false positives”

HGMD

OMIM

ClinVar

847

484

432

1897

108

91

31 Cosmic

11

CNV genes

7

CHOP

98 Animal

Models

278

Orpha

net

282

GeneTests

56

Defining the Medical Exome

Lead Groups: Partners, Emory, CHOP

1. Technical challenges • High GC • Pseudogenes/homologies • Repeat expansions • Common sites of structural variation

2. Variant types (denote common vs rare types) • Sequence variants (substitutions, small indels)

• Loss-of-function • Gain-of-function

• CNV – haploinsufficient • CNV – triplosensitive • Other structural changes (translocations, inversions, etc) • Imprinted loci • Repeat expansions

3. Medically relevant transcripts 4. Gene regions of pathogenic relevance 5. Patterns of inheritance (dominant, recessive, X-linked, mitochondrial, de novo, etc) 6. Phenotypes and evidence base for phenotype associations 7. Available approaches to define variant pathogenicity (assays, tools, etc) 8. Clinical utility measures 9. Clinical decision support opportunities

Community Efforts to Curate Genes and Loci in the Medical Exome/Genome

Annual Meeting

May 9-10, 2013

Bethesda, MD

Visit www.iccg.org for more details

Acknowledgements

NextGen Team

Birgit Funke

Lisa Farwell

Beth Duffy

Shangtao

Trevor Pugh

Siva Gowrisankar

Alison Brown

Natalie Boutin

Kevin Embree

Ana Holzbach

Nanda Kishore

Tom McLaughlin

Christine Poandl

Neeta Rathi

Brent Richter

Matilde Vickers

WGS Team

Matt Lebo

Robert Green – MedSeq

Sandy Aronson

Eugene Clark

Siva Gowrisankar

Amy Hernandez

Mike Murray

Shamil Sunyaev

GeneInsight Team

Sandy Aronson

Eugene Clark

Larry Babb

Frank Russell

Matt Varugheese

Tom Venman

Matt Lebo

Diana Toledo

Ashesh Patel

Fei Wang

Bob Hurley

Maureen Denning

Mike Oats

Gary Dombrowski

Juhan Sonin

Shane Thomas

Sharma Addepalli

Trung Do

Mike Band

Nancy Lugn

LMM Staff

ICCG/U41

Christa Martin

David Ledbetter

Robert Nussbaum

Joyce Mitchell

Andy Faucett

Erin Riggs

Erin Kaminsky

Sherri Bale

Madhuri Hegde

Patrick Willems

David Miller

Donna Maglott

Deanna Church

Justin Paschall

And many more……