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Webinar Series
Exomes and the Clinical Research ConundrumNew Approaches to Enhancing the Value of Genomic DataOctober 15, 2014
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Brought to you by the Science/AAAS Custom Publishing Office
Participating Experts
David T. Miller, M.D., Ph.D.Boston Children’s HospitalBoston, MA
Patrice Milos, Ph.D.Claritas GenomicsCambridge, MA
Webinar Series
Sponsored by:
Exomes and the Clinical Research ConundrumNew Approaches to Enhancing the Value of Genomic DataOctober 15, 2014
Boston Children’s HospitalBoston Children’s Hospital
Exomes and the Clinical Research Conundrum: New Approaches to Enhancing the
Value of Genomic Data
AAAS WebinarOctober 15, 2014
David T. Miller, MD, PhDClinical Geneticist, Division of Genetics & Genomics, Boston Children’s Hospital
Clinical Consultant and Medical Director, Claritas Genomics, Inc.
Boston Children’s Hospital
Disclosure
Clinical Consultant and Medical Director for Claritas Genomics, Inc., a majority owned subsidiary of Boston Children’s Hospital
Boston Children’s Hospital 5
Phenotype Guides Test Choice
6 y/o girl with developmental delays
Mild dysmorphic facial features
Boston Children’s Hospital 6
Phenotype Guides Test Choice
6 y/o girl with developmental delays
Mild dysmorphic facial features
A-V canal malformation and aortic coarctationDiagnosed at 10 days of age
• Cardiac surgery at 10 days of age and again at age 4 months.
Bifid uvula and modified barium swallow shows velopharyngeal insufficiency
Boston Children’s Hospital
Cytogenomic Array [50-250kb]
Phenotype Guides Test Choice
FISH (40 to 250 kb per clone)
Phenotype doesn’t match known genetic disorder
Phenotype matchesknown genetic disorder
Boston Children’s Hospital 8
Broad Phenotypic Categories
Chromosomal Microarray: 3,450 samples at BCH
6%
13%
23%
42%
CongenitalAnomaliesDysmorphicFeaturesASD
Dev Delay/ID
Boston Children’s Hospital
Rationale for Genetic Testing
Identify cause of genetic disorder• End the “odyssey”• Measured as % of known causal variants
identified in all samples
Boston Children’s Hospital
Cytogenomic Microarray Yield
Ravnan JB et al. (2006). J Med Genet 43:478-89. Baldwin, E et al. (2008). Genet Med 10(6):415-29.Sagoo, GS et al. (2009). Genet Med 11(3):139-46.
2%
5%
9%
>10%
Boston Children’s Hospital
Rationale for Genetic Testing
Identify cause of genetic disorder• End the “odyssey”• Measured as % of known causal variants
identified in all samplesChange in Management
• Opportunities for preventive care (surveillance)
• Better counseling about recurrence risks
Boston Children’s Hospital
Phenotype Guides Test Choice
Exome SequencingDNA sequence (1-10 bp)
Phenotype doesn’t match known genetic disorder
Phenotype matchesknown genetic disorder
Boston Children’s Hospital
Identification of causal variants through exome sequencing
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October 17, 2013
Boston Children’s Hospital
Identification of causal variants through exome sequencing
• 250 pediatric probands who received whole-exome sequencing (WGS)
• 80% had neurologic phenotypes
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Yang Y et al. (2013). N Engl J Med 369(16):1502-11.
Phenotypic Category Number
Neurologic disorder 60
Neurologic disorder w/other system 140
Specific neurologic disorder 13
Non-neurologic disorder 37
TOTAL 250
Boston Children’s Hospital
Identification of causal variants through exome sequencing
• Identified 86 mutated alleles that were highly likely to be causative in 62 of the 250 patients
• 25% diagnostic yield (95% CI, 20 to 31)
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de novo Mutation Novel Mutation
Autosomal dominant 25/30 (83%) 24/36 (67%)
Autosomal recessive 0/40 20/40 (50%)
X-linked 4/10 (40%) 4/10 (40%)
Yang Y et al. (2013). N Engl J Med 369(16):1502-11.
Boston Children’s Hospital
Phenotype-Based Case Selection
“…upfront investment to precisely define patient phenotypes contributes to decreased time required for the
interpretation of results...”
Boston Children’s Hospital
Case 1 - BCH WGS Pilot
Male patient with 3 older brothers (unaffected)Both parents unaffectedClinical Features
• Hypoparathyroidism with marked PTH deficiency and hypocalcemia/hyperphosphatemia--stable w/Tx
• Short stature (1%) relative to tall parents (MPH 75%)• Bilateral sensorineural hearing loss-- requiring hearing aids• Speech delay-- perhaps related to #3• Icthyosis in the newborn period which spontaneously
resolved
Boston Children’s Hospital
Negative Results from Prior Tests
Normal: • Karyotype• Chromosomal microarray• 22q11-10p13 deletion studies• Fragile X genetic test• CASR (calcium sensing receptor (gene sequencing)• Head and spinal MRI• Skeletal survey• Renal ultrasound• Cardiac echo• Eye exam except lacrimal duct obstruction
Boston Children’s Hospital
WGS Results Filtered by Phenotype
Remove common variants (391,472)
Remove non-deleterious variants (709)
Remove variants unrelated to patient phenotype (25)
Boston Children’s Hospital
Phenotype Guides Test Choice
Exome/Genome Sequencing
Phenotype matches single gene disorder(s) for which clinical single gene testing not available
Phenotype doesn’t match known genetic disorder
Boston Children’s Hospital
Case 2 – BCH WGS Pilot
Clinical Presentation:• Microcephaly• Brain malformation• Seizures• Dysmorphic features• Global developmental delay• Short stature
Prior Testing (normal results):• Chromosomal microarray• Microcephaly panel
Boston Children’s Hospital
Non-Overlap of Gene Panels
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MicrocephalyPanel Seizure
Panel
BrainMalformation
Panel
Boston Children’s Hospital
Phenotype Guides Test Choice
Exome/Genome Sequencing
Phenotype doesn’t match known genetic disorder
• Phenotype matches single gene disorder(s) for which clinical single gene testing not available
• Multiple phenotypes, each of which could be due to a number of different genes
Boston Children’s Hospital
Case 3 – BCH WGS Pilot3 ½ year old boy with
• infantile onset spastic quadriparesis, • global developmental delay (nonverbal), • nystagmus,• cortical visual impairment• failure to thrive
MRI brain shows patchy hypomelination and mild diffuse volume loss.
Dysmorphic features
Parents are first cousins
Boston Children’s Hospital
Phenotype Guides Test Choice
Exome/Genome Sequencing
Phenotype doesn’t match known genetic disorder
• Phenotype matches single gene disorder(s) for which clinical single gene testing not available
• Multiple phenotypes, each of which could be due to a number of different genes
• Possible recessive disorder due to consanguinity
Boston Children’s Hospital
Case Selection – FORGE
FORGE (Finding of Rare Disease Genes) Canada Consortium
Objective: identify genes associated with rare pediatric-onset single-gene disorders in Canada over a 2-year period (April 2011 to March 2013)
21 different genetics centers
The American Journal of Human Genetics, Volume 94, Issue 6, 2014, 809 - 817
Boston Children’s Hospital
Strategy Sample Characteristics Approach to Analysis
A multiple unrelated individuals or families affected by the same very rare but highly recognizable clinical condition
identify a common disease-associated gene or pathway shared between unrelated affected individuals
B consanguineous families map on the basis of homozygosity to exclude the majority of the genome
C single affected individuals with no family history
identify deleterious variants in genes with disease associations
FORGE Strategies for Gene Discovery
Boston Children’s Hospital
Outcomes of 264 Disorders Studied
A
A multiple unrelated individuals or families affected by the same very rare but highly recognizable clinical condition
B consanguineous familiesC single affected individuals with
no family history
B C
Unsolved Disorders
Disorders with Known Gene Identified
Disorders with Novel Gene Identified
Boston Children’s Hospital
Selecting the best test
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Presentation suggests a genetic disorder
Single gene likely Geneticallyheterogeneous
Non-specific Phenotype
+/- consanguinity
Single gene test Gene Panel
WES / WGS
Brought to you by the Science/AAAS Custom Publishing Office
Participating Experts
David T. Miller, M.D., Ph.D.Boston Children’s HospitalBoston, MA
Patrice Milos, Ph.D.Claritas GenomicsCambridge, MA
Webinar Series
Sponsored by:
Exomes and the Clinical Research ConundrumNew Approaches to Enhancing the Value of Genomic DataOctober 15, 2014
CLARITAS GENOMICS
Exomes and the Clinical Research Conundrum: New Approaches to Enhancing theImpact of Genomic Data
Patrice M. Milos, PhDChief Executive Officer
AAAS WEBINAR, WASHINGTON, DCOCTOBER 15, 2014
CLARITAS GENOMICS
Life Technologies Disclaimer
Life Technologies and its affiliates are not endorsing, recommending, or promoting any use or application of Life Technologies products presented by third parties during this seminar. Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so.
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CLARITAS GENOMICS33
The Conundrum: Right-Sizing the Genetic TestHistorically, and for monogenic disorders with clear phenotypic presentation
Single gene test – 1 gene – e.g. CF, DMD, Gaucher, etc.• Great when you know exactly what you’re looking for
• Can lead to serial testing and high costs
The “Medical Mystery” – phenotypic presentation doesn’t match known syndromes, presentation is complex
Whole Exome – 20,000 genes• When you have no idea what you’re looking for
• Too much information – hard to focus on relevant genes
More recently, for multigenic disorders with clear phenotypic presentations
Gene Panels – 10-100 genes• When you have a good idea of what you’re looking for
• But hard to keep current/complete because of rapid pace of discovery
CLARITAS GENOMICS34
The Conundrum: Right-Sizing the Genetic Test
Is there a better way?
CLARITAS GENOMICS35
Tackling the ExomeOpportunities
Comprehensive – single blood draw, multiple conditionsEnables discovery of new knowledge
ChallengesNo technology is perfect – some areas are missedMany variants are of unknown significance – science needs to catch up with technology
How to prepare the exome for broad clinical applicability in the future?
Perform an exome with phenotypic focus.
The ClariFocus Exome Family:Rapid return of relevant results
In-depth analysis of genes related to phenotype Enables flexible reanalysis
CLARITAS GENOMICS
Whole Exome (>30K Genes)
ClariFocus Exome Approach
CLARITAS GENOMICS
Ion AmpliSeq™ Exome (25K Genes)
ClariFocus Exome Approach
CLARITAS GENOMICS
Ion AmpliSeq™ Exome Workflow: Simple, Timely
DNA Sample
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12 Pool AmpliSeq Library Prep
5 Hour RunVCF GenerationTorrent Suite™ Software
Variant Annotation
AnnotationVariant Classification
DAY ONE
DAY TWO
Claritas Report
CLARITAS GENOMICS
Ion AmpliSeq™ Exome for Discovery Research
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Ion AmpliSeq™ Exome Mean Gb Mean Depth Variants Coverage at 20x
Coverage at 10x
Read length
2 samples per Ion Proton I chip
12.5 96.6x 49,931 86.7% 94.0% 163
• Providing 19,000 exomes for the VA Million Veteran Program
• Just sequenced our 5,000th exome• 192 libraries prepared per day• 120 exomes sequenced per day• No sample minimums• 6 week maximum TAT, with 3 week TAT available• $799 per sample
CLARITAS GENOMICS
Ion Proton™/AmpliSeq™ Solution Covers More of the Exome
40
100
80
60
40
20
0
Percent Ref SeqCDSCovered
0 50 100 150Coverage Depth
Proton AmpliSeq 1Proton AmpliSeq 2
HiSeq SureSelectV 1HiSeq SureSelectV 2
MiSeq SureSelectV
Sequencing Technology and Capture System
CLARITAS GENOMICS
Excellent Research Exome Amplicon Coverage
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At 2 samples per Ion Proton™ I chip:• >90% covered at 10x or greater• <2% not covered
CLARITAS GENOMICS
ClariFocus Exome Confirmation StrategyIon AmpliSeq™ Exome (25K Genes)
MiSeq® TruSight One Panel (4800 Genes)
Orthogonal approach combines technology platforms
Pass
ConfirmationAllows rapid confirmation of a subset of genes
Needs
additional work
CLARITAS GENOMICS43
Ion AmpliSeq™ Exome
TruSightOne Panel
680100%
8367%
18793%
SNPs+indelsPPV
Comparison to truth set of 614 genes within NIST NA12878 reference
Technology Platforms are Complementary
CLARITAS GENOMICS
ClariFocus Exome for Pediatric Neurology (614)
Pass
Needs
additional work
Confirmation
ClariFocus Exome ApproachBuilding Phenotypically Defined Regions Of Interest
CLARITAS GENOMICS45
Why Pediatric Neurology?
• Many neurological conditions are complex in presentation with no clear connection to underlying genotype – e.g. seizures
• Subjects with these conditions can be on an endless “odyssey”
• Represents a large financial expenditure for pediatric hospitals (each spending an average of $1M annually on these tests)
• Identification of causal variants with comprehensive tests (e.g. exome) is relatively high in neurology research
• Rapidly developing field of science; active research area
• As a result, demand for exome tests high for these subjects
CLARITAS GENOMICS46
Genetic testing is currently fragmented
Dysmorphology, Developmental delay Neurology Hematology Metabolic Cardio Skeletal Pharm Pulm ENT
Uniting Disparate Tests into One Product
CLARITAS GENOMICS
Area of Focus Chosen for Phenotypic Relevance
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OMIM Disease refseq HGNC Gene Symbol omim_id_gene
cytogenic_location
Charcot-Marie-Tooth disease,axonal,type 2N AARS AARS CMT2N 601065 16q22.1
GABA-transaminase deficiency ABAT ABAT GABAT 137150 16p13.2
{Colchicine resistance};{Inflammatory bowel disease 13} ABCB1 ABCB1 PGY1,MDR1,IBD13,CLCs
171050 7q21.12
Adrenoleukodystrophy;Adrenomyeloneuropathy,adult ABCD1 ABCD1 ALD,AMN 300371 Xq28
Chanarin-Dorfman syndrome ABHD5 ABHD5 CGI58,IECN2,NCIE2 604780 3p21.33
VLCAD deficiency ACADVL ACADVL VLCAD 609575 17p13.1
Peroxisomal acyl-CoA oxidase deficiency ACOX1 ACOX1 ACOX,SCOX 609751 17q25.1
Combined malonic and methylmalonic aciduria ACSF3 ACSF3 614245 16q24.3
Myopathy,actin,congenital,withcores;Myopathy,actin,congenital,with excess of thin myofilaments;Myopathy,congenital,with fiber-type disproportion 1;Nemaline myopathy 3,autosomal dominant or recessive
ACTA1 ACTA1 ASMA,NEM3,CFTD1 102610 1q42.13
Baraitser-Winter syndrome 1;Dystonia,juvenile-onset ACTB ACTB BRWS1 102630 7p22.1
List continues…
Region of focus well-verified for utility in pediatric neurology
CLARITAS GENOMICS48
ClariFocus Exome: Pediatric Neurology
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Disorder Category Genes
Neuromuscular disorders 297
Movement disorder 39
Epilepsy/Seizure 244
Brain Malformation 67
Hereditary peripheral neuropathy 84
Leukodystrophy/ Mitochondrial Encephalomyopathy
61
Comprehensive area of focus covers 6 disease subcategories in pediatric neurology
CLARITAS GENOMICS49
Product Summary
Justification
Key Features
Region of interest on an exome, covering pediatric inherited neurological disorders, with the exception of autism/ID/DD. Ordered as large set (614 genes). Gene list has been defined by phenotypically-delineated groupings (6 disease sub-categorizations in total).
Subjects with neurological phenotypes, cross-over phenotypes or complex presentations. Can substitute for clinical exome.
Combines attributes of other test providers in a single test. Use in place of exome. More efficient and lower cost than performing either an exomeor multiple panels and single gene tests each addressing one of the phenotypes. Allows quick gathering of comprehensive information and could be used in the future to confirm a suspected diagnosis. Useful for neurologists and geneticists alike.
Technical Approach Dual-platform preparations and data comparison. Ion Ampliseq™ Exome/Ion Proton™ System + complementary technology. Sanger confirmation under select conditions only.
ClariFocus Exome for Pediatric Neurology
CLARITAS GENOMICS
Spun Out of Boston Children’s Hospital Feb 2013
Commercial reference laboratory performing genetic testing services
• Best quality services, from pre-ordering to post-report • Testing for any condition• Single gene tests to microarrays to exomes• All technology platforms • Based on the expertise at pediatric hospitals• Research sequencing services to support discovery
CLARITAS GENOMICS51
Claritas Services: Clinical and Discovery ResearchSingle Gene Sequencing and Deletions/Duplication AnalysisArrays:
• Optimized for autism/ID/DD• MRD testing and pediatric oncology researchPanels: • Muscular Dystrophy• Nephrotic Syndrome ClariFocus Exomes:• Pediatric NeurologyComprehensive Exome Sequencing for Discovery
Assays arising from the clinic, developed for clinicians
CLIA facility, using all technology platforms
Goals: Quality, efficiency, analytic automation, scalability, affordability
Webinar attendees can receive first research sequencing project for $699/sample (code: AAAS)Contact: [email protected]
CLARITAS GENOMICS52
Summary• Clinical use of molecular diagnostic testing is in transition. New
technology is opening up approaches previously unavailable to both researchers and clinical practitioners.
• Exomes and genomes represent a huge potential opportunity to inform and drive the practice of individualized medicine in the future. However, the knowledge of disease genetics needs to catch up with pace of technology.
• Approaches that focus on a tight tie between genotype and phenotype and that connect research discovery to clinical knowledge will be the most efficient and successful to translate genomics into medical applicability in the future.
• Questions?
CLARITAS GENOMICS53
CONTACT US
Discovery Research Projects:[email protected]
Clinical testing services:[email protected]
To submit yourquestions, type them into the text box and
click
Participating Experts
Webinar Series
Sponsored by:
Brought to you by the Science/AAAS Custom Publishing Office
Exomes and the Clinical Research ConundrumNew Approaches to Enhancing the Value of Genomic DataOctober 15, 2014
David T. Miller, M.D., Ph.D.Boston Children’s HospitalBoston, MA
Patrice Milos, Ph.D.Claritas GenomicsCambridge, MA
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Brought to you by the Science/AAAS Custom Publishing Office
Exomes and the Clinical Research ConundrumNew Approaches to Enhancing the Value of Genomic DataOctober 15, 2014