This document discusses integrating large scale genomic sequencing into clinical practice. It describes how the Laboratory for Molecular Medicine at Partners Healthcare has transitioned from testing a few genes to now offering over 150 tests using next generation sequencing technologies. It also discusses the evolution of testing panels, moving from targeted panels to exome and genome sequencing. Validation of sequencing methods and challenges of variant interpretation are discussed.

1. Integrating Large Scale Sequencing
into Clinical Practice
Heidi L. Rehm, PhD, FACMGHeidi L. Rehm, PhD, FACMG
Director, Laboratory for Molecular Medicine, PCPGM
Assistant Professor of Pathology, BWH, MGH, HMS

2. Laboratory for Molecular Medicine at PCPGM
15% of testing is from
Partners’ patients
85% 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
• Whole genome sequencing diagnostic service

3. Ryan Shay
Antonio Puerta
DeCarlo Polk
Joe KennedyJason
Collier
HCM SCD in Athletes

4. d. SCD, 7y
38y
Case Presentation
Cardiac gross exam: asymmetric septal LVH
Cardiac tissue histology: myofiber disarray
Autopsy
40y
6 y
Normal Deceased
Normal
Deceased

5. Legend:
MI, 49y MI, 70y
39y
d. SCD, 7y6y 3y
38y
SCD = Sudden Cardiac Death
Case Presentation with Family History
40y
6 y
71y 68y
36y

6. HCM Family 90
Legend:
= Affected individuals
d. SCD, 49y d. SCD, 70y
39y
d. SCD, 7y
6y6y 3y
38y
LVH
Arrhythmia
SCD = Sudden Cardiac Death
LVH = Left Ventricular Hypertrophy
40y
Normal
Echo
Normal Echo
Normal
Echo
Normal
Echo
40y
71y 68y
36y

7. HCM Family 90
Legend:
= Affected Individuals
+ = E187Q positive genotype
- = E187Q negative genotype
d. SCD, 49y d. SCD, 70y
39y
d. SCD, 7y 7y
Normal ECHO
6y 3y
SCD = Sudden Cardiac Death
LVH = Left Ventricular Hypertrophy
+
+- -
- 38y
LVH
Arrhythmia
E187Q
TPM1

8. 2004: HCM Panel A (5 genes) $3000
HCM Panel B (3
genes) $1150
PRKAG2 and LAMP2 $1500
GLA $600
2007: HCM CardioChip (11 genes) $3000
2011: Pan Cardiomyopathy Test (51 genes)
HCM (18
genes) $3200
(All Genes
$3950)
Evolution of Cardiomyopathy Testing at Partners
11 genes
$6250

9. 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.
When do we shift to “genomes for all”?
Genetic Testing is Evolving

10. Need improved analytical validity of NGS
Improve variant calling accuracy
Indels, CNVs, SVs, repeats
Fill in the holes
Targeted NGS panels miss
a variable amount of content
depending on genes
Exome sequencing misses
5-15% of coding sequence
0
50
100
150 NGS Coverage
Courtesy of Birgit Funke

11. AGMG NGS Guideline
ACMG (www.acmg.net) > Publications > Laboratory Standards and Guidelines > NGS

12. 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:

13. Detection of full and partial gene deletions
through targeted NGS: VisCap
Log2ratiosample/batchmedian USH2A heterozygous
exon 10 deletion
All exons, sorted by genome position USH2A exons (3’→5’)
OTOF deletion
47 exons
Trevor Pugh
However, deletion analysis is not robust in exome and
genome sequencing

14. WGS Case 1: Nonsyndromic Hearing Loss
• Sept 2010: 2 yr old girl born presents to Genetics
• History of congenital bilateral sensorineural hearing loss
• Mild-moderate “cookie-bite” shaped audiogram
• No other complaints
• Genetic testing for hearing loss (Cx26 and OtoChip): Negative
However, >100 loci, many without identified genes, are associated to hereditary hearing loss

15. WGS Case 1: Nonsyndromic Hearing Loss
• July 2011: Patient enrolled into consented research study
• Obtained blood samples on all 11 family members
• Whole genome sequencing ordered on 3 siblings
WGS on 3 children

16. Variant Analysis Step 1
• ~4 million sequence variants per child
• ~1,250,000 shared variants among the three siblings
• First considered a recessive model (hom or 2 het variants)
• No cause identified

17. Variant Analysis Step 2
Genome sequence shared with external company
Proposed otopetrin (OTOP1) as causative gene:
Required for normal formation of otoconia in the inner ear.
Two mutations identified:
p.Arg232Trp
4 bp insertion leading to frameshift
Upon manual examination of read data, frameshift variant was deemed
a false positive

18. Copyright 2013 – Partners HealthCare Incorporated – All Rights Reserved
Validation of WGS Alignment and Variant Calling
Variant type
1000G
Variants Sensitivity
With concordant
zygosity
SNVs 2762933 2735592 (99.0%) 2730826 (98.8%)
Indels 327474 299300 (91.4%) 285401 (87.2%)
Total 3090407 3034892 (98.2%) 3016227 (97.6%)
Concordance with 1000 Genomes Data
Courtesy of Matt Lebo

19. Copyright 2013 – Partners HealthCare Incorporated – All Rights Reserved
Validation of WGS Alignment and Variant Calling
Variant Type FP (before thresholds) FP (after thresholds)*
SNVs 20 1
Indels 1 0
Variant Type # FN Sensitivity 95% CI
SNVs 410 0 100% 99.1%-100%
Indels 15 0 100% 79.6%-100%
Specificity
Sensitivity
425 Sanger confirmed variants in 195 genes across 700kb of sequence
*Fisher Strand (FS) < 30 and Quality by Depth (QD) >= 4
Courtesy of Matt Lebo

20. WGS Case 1: Nonsyndromic Hearing Loss
• January 2012: Mother called to say that the 4 oldest children with hearing
loss were now complaining of headaches – could this be related?
• Also relayed that a paternal grandfather was reported to have hearing loss
detected at age 19 - described inability to hear fire alarm but the clicking of
an automatic seat belt annoyed him

21. Variant Analysis Step 3
• Consider dominant inheritance with reduced penetrance
• Examined known hearing loss genes (~70) for any single variant
• 32 rare/novel variants found across 31 different genes
• All missense variants with no strong data to support a deleterious
impact

22. Variant Analysis Step 4: Segregation
• Cost to develop Sanger assays for all 32 variants and test
for presence in 11 family members = $5428
• Examined cost to perform linkage analysis by SNP array:
$386.00 per sample x 11 samples = $4246
• Requested additional sample from grandfather

23. D3S1278 to D3S2453 = chr3:115,124,154-136,278,257 (3q13.31-22.3, 21 Mb)
Courtesy of Jun Shen

24. Some genes fail analysis by genome/exome sequencing
STRC
Exome Coverage of 73 Hearing Loss Genes

25. Analyzed case by new OtoGenome Test
STRC pSTRC
STRC pSTRC
Hom deletion
of STRC
pSTRC
pSTRCSTRC Gene

26. 100 kb deletion
(43.89 Mb to 43.99 Mb)
STRC
Pseudogene
STRC
100,000 Base Deletion Identified

27. WGS Case 1: Nonsyndromic? Hearing Loss
• Called family to return cause of hearing loss
• Mother relayed that headaches in 4 affected children had
resolved once it was discovered that all 4 needed glasses
• Question: Could STRC mutations cause vision problems?

28. WGS Case 1: Deafness Infertility Syndrome
Males with this deletion will be infertile due to deletion of the adjacent
CATSPER gene
Males can father children through intracytoplasmic sperm injection (ICSI)

29. WGS Case 2: Distal Arthrogryposis Type 5
Disease is known to be AD and to occur de novo
No known genes for DA5
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 from of Michael Murray, MD

30. Case 2: Distal Arthrogryposis Type 5
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
Great candidate, but how to we prove causality for a
novel gene-disease association?
Collaboration with Shamil Sunyeav

32. Matchmaker System for Exomes/Genomes
o Collaboration with Ada Hamosh (OMIM/Hopkins)
o Enhance PhenoDB to become a matchmaker system for
unsolved exomes and genomes
o Entry of structured phenotypes
o Ability to upload candidate genes/variants and vcf files
o Matches require data entry and dual notification of both
parties
o Build algorithm to prioritize matches based upon
phenotype match strength and phenotype/gene/variant
prior likelihood parameters
o Goal to also interface with other systems (e.g. Decipher,
LOVD, etc)

33. A New Paradigm in Clinical Genomics
Traditional Paradigm
New Paradigm

34. What is the biggest
bottleneck in clinical
sequencing?

35. Variant Assessment and Reporting
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

36. 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
Numberofvariants
MYBPC3
E258K
MYBPC3MYH7
R502WW792fsR663H

37. Hearing Loss Gene Mutations – 2000 Cases Tested
Number of probands
Numberofvariants
GJB2
35delG
GJB2
V37I
GJB2
M34T
USH2A
2299delG
SLC26A4
81% (423/523) of clinically significant variants have been seen in only one family

38. Variant Analysis for General Genome Report
3,227,455 Substitutions and 418,331 Indels3,227,455 Substitutions and 418,331 Indels
20,240 CDS/Splice Variants20,240 CDS/Splice Variants
25 “Pathogenic”
Variants
25 “Pathogenic”
Variants
Published as
Disease-Causing
Genes
5 PGx Class 1
5 Variants5 Variants
PharmGKB
2 Pathogenic
0 Likely Pathogenic
6 VUS
4 Likely Benign
13 Benign
<1%
616 Rare CDS/Splice Variants616 Rare CDS/Splice Variants
LOF in Disease
Associated Genes
2 Pathogenic
9 VUS
11 Variants11 Variants
~2 CNVs >1kb~2 CNVs >1kb
0 Pathogenic
Review evidence for
gene-disease
association and LOF role
Review evidence for
variant pathogenicity

39. 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

40. To improve our knowledge of DNA
variation will require a massive
effort in data sharing

41. U41 Genomic Resource Grant:
A Unified Clinical Genomics
Database
To raise the quality of patient care by:
• Standardizing the annotation and interpretation of
genomic variants
• Sharing variant and case level data through a
centralized database for clinical and research use
• Implementing an evidence-based expert consensus
process for curating variant interpretations
We will work jointly with the recipients of the Clinically Relevant
Variant Resource awards.

42. Sequencing Laboratories Which Have Agreed to Share Data

43. www.ncbi.nlm.nih.gov/clinvar
NIH NCBI ClinVar

44. ClinVar Contributors (>40)
Group Submissions
OMIM 22954
Partners Healthcare’s Laboratory for Molecular Medicine 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

45. LMM’s Disease Area
Submissions to ClinVar
Phenotypes Probands Genes Unique Variants
Cardiomyopathy 5485 51 3929
Somatic Cancer 3421 21 178
RASopathies 2781 12 376
Hearing Loss and Related Syndromes 2048 65 2218
Connective Tissue Disorders 915 3 227
Hereditary Cancer 665 9 81
Congenital Heart Defects 91 4 43
Ectodermal Dysplasia 81 1 36
Other 867

46. Documenting arguments will improve the
evidence-based assessment of variants

47. Current Limitations of ClinVar
Many labs have limited and/or non-
standardized infrastructure to house their
data which limits the quantity, quality and
efficiency of data sharing

48. GeneInsight LabGeneInsight Lab
Reporting EngineReporting Engine
Laboratory KnowledgebaseLaboratory Knowledgebase
Case RepositoryCase Repository
The GeneInsight Suite
• In clinical use since 2005; over 28,000 cases reported
• Commercialized and used by >10 clinical labs/research
consortia to date

49. Current Limitations of ClinVar
o Genomic data and other case-level data is difficult to
share in meaningful ways in the public domain
My lab has 25,000 cases with phenotypic data that I am willing
but unable to share through ClinVar right now
o We are gathering stakeholders at NIH in September
to discuss policies surrounding case-level data
submission and access for NCBI databases including
ClinVar and dbGaP

50. GeneInsight Data Sharing Networks
Networked
Standalone
Databases
Bioreference Lab-MGH Pathology
GeneInsight Clinical Lab Network
New York Genome Center
Lung Cancer Consortium
Genome Canada Grant

51. GeneInsight Lab – View of Networked Data

52. GeneInsight LabGeneInsight Lab
Reporting EngineReporting Engine
Laboratory
Knowledgebase
Laboratory
Knowledgebase
Case RepositoryCase Repository
GeneInsight ClinicGeneInsight Clinic
Patient Genomic
Profile Repository
Patient Genomic
Profile Repository
Alerting EngineAlerting Engine
EHR ConnectivityEHR Connectivity
Supports Molecular
Diagnostic Labs
Supports Molecular
Diagnostic Labs
Supports Clinicians Who
Order Genetic Tests
Supports Clinicians Who
Order Genetic Tests
The GeneInsight Suite

53. Keeping up with Genomics
~4% of case per year
received medium or high
alerts (.33% per month)

54. GeneInsight Alert Delivery
This screenshot was taken from a demonstration system – the content of this screen should
not be used for any clinical purpose

55. Acknowledgements

56. The General Genome Report