exomes and the clinical research conundrum slides_science... · webinar series exomes and the...

Webinar Series

Exomes and the Clinical Research ConundrumNew Approaches to Enhancing the Value of Genomic DataOctober 15, 2014

Facebook login

if you need help

shows speaker bios

download slides and more info

LinkedIn login

shows slide window

opens the Ask a Question box

Twitter login (#ScienceWebinar)

search Wikipedia

shows the video screen

Change the size of any window by dragging the lower right corner. Use controls in top right corner to close or maximize each window.

What each widget does:

Instructions for Viewers

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:


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



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


Cytogenomic Array [50-250kb]


FISH (40 to 250 kb per clone)

Phenotype doesn’t match known genetic disorder

Phenotype matchesknown genetic disorder


Broad Phenotypic Categories

Chromosomal Microarray: 3,450 samples at BCH

6%

13%

23%

42%

CongenitalAnomaliesDysmorphicFeaturesASD

Dev Delay/ID


Rationale for Genetic Testing

Identify cause of genetic disorder• End the “odyssey”• Measured as % of known causal variants

identified in all samples


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%


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



Exome SequencingDNA sequence (1-10 bp)


Phenotype matchesknown genetic disorder


Identification of causal variants through exome sequencing

13

October 17, 2013



• 250 pediatric probands who received whole-exome sequencing (WGS)

• 80% had neurologic phenotypes

14

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



• 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)

15

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.


Phenotype-Based Case Selection

“…upfront investment to precisely define patient phenotypes contributes to decreased time required for the

interpretation of results...”


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


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


WGS Results Filtered by Phenotype

Remove common variants (391,472)

Remove non-deleterious variants (709)

Remove variants unrelated to patient phenotype (25)



Exome/Genome Sequencing

Phenotype matches single gene disorder(s) for which clinical single gene testing not available



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


Non-Overlap of Gene Panels

22

MicrocephalyPanel Seizure

Panel

BrainMalformation

Panel





• 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


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





• 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


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


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


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


Selecting the best test

29

Presentation suggests a genetic disorder

Single gene likely Geneticallyheterogeneous

Non-specific Phenotype

+/- consanguinity

Single gene test Gene Panel

WES / WGS





Webinar Series

Sponsored by:


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.

32

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

38

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

39

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

41

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

47

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

48

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


Webinar Series

Sponsored by:





For related information on this webinar topic, go to:

Look out for more webinars in the series at:

webinar.sciencemag.org

To provide feedback on this webinar, please e‐mailyour comments to [email protected]

Webinar Series

Sponsored by:

www.lifetechnologies.com/ionexome



exomes and the clinical research conundrum slides_science... · webinar series exomes and the...

Documents