Integrating Large Scale Sequencing
into Clinical Practice
Heidi L. Rehm, PhD, FACMGHeidi L. Rehm, PhD, FACMG
Director, Lab...
Laboratory for Molecular Medicine at PCPGM
15% of testing is from
Partners’ patients
85% is from other US and
Internationa...
Ryan Shay
Antonio Puerta
DeCarlo Polk
Joe KennedyJason
Collier
HCM SCD in Athletes
d. SCD, 7y
38y
Case Presentation
Cardiac gross exam: asymmetric septal LVH
Cardiac tissue histology: myofiber disarray
Aut...
Legend:
MI, 49y MI, 70y
39y
d. SCD, 7y6y 3y
38y
SCD = Sudden Cardiac Death
Case Presentation with Family History
40y
6 y
7...
HCM Family 90
Legend:
= Affected individuals
d. SCD, 49y d. SCD, 70y
39y
d. SCD, 7y
6y6y 3y
38y
LVH
Arrhythmia
SCD = Sudde...
HCM Family 90
Legend:
= Affected Individuals
+ = E187Q positive genotype
- = E187Q negative genotype
d. SCD, 49y d. SCD, 7...
2004: HCM Panel A (5 genes) $3000
HCM Panel B (3
genes) $1150
PRKAG2 and LAMP2 $1500
GLA $600
2007: HCM CardioChip (11 gen...
Targeted panels are enlarging and more and
more labs are launching exome and genome
services.
Genomic testing is improving...
Need improved analytical validity of NGS
Improve variant calling accuracy
Indels, CNVs, SVs, repeats
Fill in the holes
Tar...
AGMG NGS Guideline
ACMG (www.acmg.net) > Publications > Laboratory Standards and Guidelines > NGS
A Genetic Sequencing Test is Not One Test
DNA
PCR
Hundreds of assays per sample
Sequencing
Hundreds of bases per exon
Fail...
Detection of full and partial gene deletions
through targeted NGS: VisCap
Log2ratiosample/batchmedian USH2A heterozygous
e...
WGS Case 1: Nonsyndromic Hearing Loss
• Sept 2010: 2 yr old girl born presents to Genetics
• History of congenital bilater...
WGS Case 1: Nonsyndromic Hearing Loss
• July 2011: Patient enrolled into consented research study
• Obtained blood samples...
Variant Analysis Step 1
• ~4 million sequence variants per child
• ~1,250,000 shared variants among the three siblings
• F...
Variant Analysis Step 2
Genome sequence shared with external company
Proposed otopetrin (OTOP1) as causative gene:
Require...
Copyright 2013 – Partners HealthCare Incorporated – All Rights Reserved
Validation of WGS Alignment and Variant Calling
Va...
Copyright 2013 – Partners HealthCare Incorporated – All Rights Reserved
Validation of WGS Alignment and Variant Calling
Va...
WGS Case 1: Nonsyndromic Hearing Loss
• January 2012: Mother called to say that the 4 oldest children with hearing
loss we...
Variant Analysis Step 3
• Consider dominant inheritance with reduced penetrance
• Examined known hearing loss genes (~70) ...
Variant Analysis Step 4: Segregation
• Cost to develop Sanger assays for all 32 variants and test
for presence in 11 famil...
D3S1278 to D3S2453 = chr3:115,124,154-136,278,257 (3q13.31-22.3, 21 Mb)
Courtesy of Jun Shen
Some genes fail analysis by genome/exome sequencing
STRC
Exome Coverage of 73 Hearing Loss Genes
Analyzed case by new OtoGenome Test
STRC pSTRC
STRC pSTRC
Hom deletion
of STRC
pSTRC
pSTRCSTRC Gene
100 kb deletion
(43.89 Mb to 43.99 Mb)
STRC
Pseudogene
STRC
100,000 Base Deletion Identified
WGS Case 1: Nonsyndromic? Hearing Loss
• Called family to return cause of hearing loss
• Mother relayed that headaches in ...
WGS Case 1: Deafness Infertility Syndrome
Males with this deletion will be infertile due to deletion of the adjacent
CATSP...
WGS Case 2: Distal Arthrogryposis Type 5
Disease is known to be AD and to occur de novo
No known genes for DA5
Skeletal Sp...
Case 2: Distal Arthrogryposis Type 5
Two de novo mutations in exonic sequence:
ACSM4 – acyl-CoA synthetase medium-chain fa...
Matchmaker System for Exomes/Genomes
o Collaboration with Ada Hamosh (OMIM/Hopkins)
o Enhance PhenoDB to become a matchmak...
A New Paradigm in Clinical Genomics
Traditional Paradigm
New Paradigm
What is the biggest
bottleneck in clinical
sequencing?
Variant Assessment and Reporting
Somatic Cancer
~15,000 variants
interpreted in patient
reports to date
Average
22 min
25 ...
HCM Gene Mutations – 3000 cases tested
>500 clinically significant mutations identified
66% of clinically significant muta...
Hearing Loss Gene Mutations – 2000 Cases Tested
Number of probands
Numberofvariants
GJB2
35delG
GJB2
V37I
GJB2
M34T
USH2A
...
Variant Analysis for General Genome Report
3,227,455 Substitutions and 418,331 Indels3,227,455 Substitutions and 418,331 I...
Fetus with US finding: ↑NT
PTPN11 p.Ile309Val
Published as “pathogenic” for
Noonan syndrome
Patient contacted author of pa...
To improve our knowledge of DNA
variation will require a massive
effort in data sharing
U41 Genomic Resource Grant:
A Unified Clinical Genomics
Database
To raise the quality of patient care by:
• Standardizing ...
Sequencing Laboratories Which Have Agreed to Share Data
www.ncbi.nlm.nih.gov/clinvar
NIH NCBI ClinVar
ClinVar Contributors (>40)
Group Submissions
OMIM 22954
Partners Healthcare’s Laboratory for Molecular Medicine 7127
Inter...
LMM’s Disease Area
Submissions to ClinVar
Phenotypes Probands Genes Unique Variants
Cardiomyopathy 5485 51 3929
Somatic Ca...
Documenting arguments will improve the
evidence-based assessment of variants
Current Limitations of ClinVar
Many labs have limited and/or non-
standardized infrastructure to house their
data which li...
GeneInsight LabGeneInsight Lab
Reporting EngineReporting Engine
Laboratory KnowledgebaseLaboratory Knowledgebase
Case Repo...
Current Limitations of ClinVar
o Genomic data and other case-level data is difficult to
share in meaningful ways in the pu...
GeneInsight Data Sharing Networks
Networked
Standalone
Databases
Bioreference Lab-MGH Pathology
GeneInsight Clinical Lab N...
GeneInsight Lab – View of Networked Data
GeneInsight LabGeneInsight Lab
Reporting EngineReporting Engine
Laboratory
Knowledgebase
Laboratory
Knowledgebase
Case Rep...
Keeping up with Genomics
~4% of case per year
received medium or high
alerts (.33% per month)
GeneInsight Alert Delivery
This screenshot was taken from a demonstration system – the content of this screen should
not b...
Acknowledgements
The General Genome Report
Aug2013 Heidi Rehm integrating large scale sequencing into clinical practice
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Aug2013 Heidi Rehm integrating large scale sequencing into clinical practice

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  • 1946 Cases Tested, 1239 Variants Observed 523 variants reported as Pathogenic, Likely Pathogenic or VUS 716 variants reported as Benign or Likely Benign 1368 variants reported in literature or in databases not yet observed 882 GJB2 606 SLC26A4 323 Usher
  • PharmGKB Levels of Evidence: PharmGKB curators use specific criteria to assess the PharmGKB “variant annotations” and determine the level of evidence. Level 1 annotations require at least one study with at least 1000 cases and 1000 controls of the same ethnicity with statistically significant p-values <0.05. Level 2 associations are those found in at least one population of >100 with a statistically significant p-value of <0.05, and may or may not be replicated. Level 3 associations with lower levels of evidence are those that do not meet the above criteria for population size or p-value, or may be in vitro studies.
  • Transcript of "Aug2013 Heidi Rehm integrating large scale sequencing into clinical practice"

    1. 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. 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. 3. Ryan Shay Antonio Puerta DeCarlo Polk Joe KennedyJason Collier HCM SCD in Athletes
    4. 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. 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. 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. 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. 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. 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. 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. 11. AGMG NGS Guideline ACMG (www.acmg.net) > Publications > Laboratory Standards and Guidelines > NGS
    12. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 23. D3S1278 to D3S2453 = chr3:115,124,154-136,278,257 (3q13.31-22.3, 21 Mb) Courtesy of Jun Shen
    24. 24. Some genes fail analysis by genome/exome sequencing STRC Exome Coverage of 73 Hearing Loss Genes
    25. 25. Analyzed case by new OtoGenome Test STRC pSTRC STRC pSTRC Hom deletion of STRC pSTRC pSTRCSTRC Gene
    26. 26. 100 kb deletion (43.89 Mb to 43.99 Mb) STRC Pseudogene STRC 100,000 Base Deletion Identified
    27. 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. 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. 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. 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
    31. 31. 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)
    32. 32. A New Paradigm in Clinical Genomics Traditional Paradigm New Paradigm
    33. 33. What is the biggest bottleneck in clinical sequencing?
    34. 34. 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
    35. 35. 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
    36. 36. 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
    37. 37. 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
    38. 38. 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
    39. 39. To improve our knowledge of DNA variation will require a massive effort in data sharing
    40. 40. 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.
    41. 41. Sequencing Laboratories Which Have Agreed to Share Data
    42. 42. www.ncbi.nlm.nih.gov/clinvar NIH NCBI ClinVar
    43. 43. 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
    44. 44. 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
    45. 45. Documenting arguments will improve the evidence-based assessment of variants
    46. 46. 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
    47. 47. 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
    48. 48. 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
    49. 49. 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
    50. 50. GeneInsight Lab – View of Networked Data
    51. 51. 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
    52. 52. Keeping up with Genomics ~4% of case per year received medium or high alerts (.33% per month)
    53. 53. GeneInsight Alert Delivery This screenshot was taken from a demonstration system – the content of this screen should not be used for any clinical purpose
    54. 54. Acknowledgements
    55. 55. The General Genome Report
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