Juha Koskenvuo, MD, PhDLaboratory Director, Chief Medical OffficerBlueprint Genetics

Geenitiedon hyödyntäminenharvinaissairauksien diagnostiikassa ja hoidossa

Harvinaiset sairaudet - tutkimusyhteistyöstä apuagenomitiedon hyödyntämiseen ja lääkekehitykseenTurku 13.4.2016

Disease gene and disease mutationGene (discovery/initial characterization)

-Multiple non-related patients with the same phenotype -Allele frequency in reference population vs. disease prevalence

iPAH-prevalence 1:1,000,000 >< individual variant absent in ExAC-Adequate functional studies, animal models -Co-segregation (iPAH vs. hPAH, low penetrance)-De novo cases when sporadic disease in the family(In-silico predictions are not really important)

New mutation in established gene-Multiple non-related patients with the same variant and the same phenotype-Allele frequency (notice that non-coding variants are not present in ExAC)-Co-segregation-De novo-In-silico prediction

Disease causing gene/variantRESEARCH

-’Any’ gene with a rare variant-Predicted deleterious by in-silico tools -No matter if allele frequency in reference population 100x disease prevalence-Negative co-segregation; no matter??-ACMG guidelines (not applicable?)

CLINICAL DIAGNOSTICS-Variant graded pathogenic in research setting is usually VUS in clinical setting-In best case variant graded pathogenic in research setting is likely pathogenic

1. Pathogenic mutation

2. Likely pathogenic mutation

3. Variant of uncertain significance (VUS)

4. Likely benign variant

5. Benign variant

6. Known disease modifier

Novel variants in novel genes are VUSes before clear functional data + extensive co-segregation

Genetic diagnosis

Variant classification

• Sequencing quality

Strategies to comprehensive geneticdiagnostics

Whole genome sequencing

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Whole exome sequencing- All protein coding exons of >20 000 genes

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Targeted sequencing- Sequencing of known clinically relevant genes and regions

Whole genome sequencing

1. Incomplete coverage of inherited disease genes (10-19% not covered properly)2. Low reproducibility of detection of genetic variation with the highest potential

clinical effects 3. Uncertainty about clinically reportable findings

Targeted sequencing (OS-Seq) vs. exome sequencing

• 99,5% of bases in OS-Seq covered >50x• MYBPC3 gene covered 100% >60x, all exons but one covered >200x

Exomesequencing; MYBPC3 gene

OS-Seq

30x

400x

200x60x

• 50% of exons are <30x average coverage in 60 000 samples (Exome Aggregation Data, Broad Institute)

• Several exons in exome sequencing are poorly covered (<15x), highest approx. 90x• MYBPC3 is an important gene clinically• Low performance illustrates the problems associated with using exome sequencing in the clinic

- Median coverage 825x- Percentage of targeted bases with >15x read depth 100.0%

No gaps (golden standard)

Patient case (odyssey)

Patient•9-year old girl•HCM from birth•2x operated for LVOTO•Epilepsy•Normal neurodevelopment

NegativeTargeted sequencingMYH7, PNNNT2, TPN1, MYBBC3, TNNI3, MYL3, MYL2, ACTC, TTR, PNNC1, CAV3, LAMB2, GLA, PRKAG2, MTTG, MTTI and MTTK)

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Whole exome sequencing. . .

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Negative

. Finnish founder mutations –MYBPC3-Gln1061X and TPM1-Asp175Asn

Negative

Patient case (odyssey)

Whole exome sequencing. . .. .. . .. .

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• CNV• defect outside the protein coding region• interpretation failure•mitochondrial

The critical gene or region wasnot covered properly

or

Genetic diagnostic work-up

Targeted sequencing101 Gene Pan Cardiomyopathy Panel

Isolated and childhood onset syndromiccardiomyopathies

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HRAS c.173C>T (p.T58I)

Costello syndrome

Whole exome sequencing vs. OS-seq

Comparison with whole exome sequencing

OS-Seq

G

Exome

Hedges, ASHG 2014

Eur Heart J 2015

Targeted sequencing panel (OS-Seq) vs. exome sequencing

PHARMACOGENETICS PANELDiagnostic tool for the prediction of drug response

Covers variation in genes influencing drug efficacy and risk for adverse drug reactions• Drug metabolizing enzymes• Drug transporters• Drug targets

Gene selection criteria• Clinical guidelines published by the Clinical PharmacogeneticsImplementation Consortium (CPIC; www.pharmgkb.org/page/cpic)• Guidelines are peer-reviewed and published in Clinical Pharmacology & Therapeutics• Currently 17 guidelines covering 33 drugs and 13 genes

PHARMACOGENETICS PANELDrug Gene(s) CPIC guideline(s)CARDIOLOGYClopidogrel CYP2C19 Scott et al. 2013

Scott et al. 2011Simvastatin SLCO1B1 Ramsey et al. 2014

Wilke et al. 2012Warfarin CYP2C9, VKORC1 Johnson et al. 2011INFECTIOUS DISEASESAbacavir HLA-B Martin et al. 2014

Martin et al. 2012Atazanavir UGT1A1 Gammal et al. 2016Boceprevir IFNL3 Muir et al. 2014Peginterferon alfa-2a IFNL3 Muir et al. 2014Peginterferon alfa-2b IFNL3 Muir et al. 2014Ribavirin IFNL3 Muir et al. 2014Telaprevir IFNL3 Muir et al. 2014NEUROLOGYCarbamazepine HLA-B Leckband et al. 2013Clopidogrel CYP2C19 Scott et al. 2013

Scott et al. 2011Phenytoin CYP2C9, HLA-B Caudle et al. 2014TRANSPLANTATIONAzathioprine TPMT Relling et al. 2013

Relling et al. 2011Tacrolimus CYP3A5 Birdwell et al. 2015

Drug Gene(s) CPIC guideline(s)ONCOLOGYAllopurinol HLA-B Saito et al. 2016

Hershfield et al. 2013Capecitabine DPYD Caudle et al. 2013Fluorouracil DPYD Caudle et al. 2013Mercaptopurine TPMT Relling et al. 2013

Relling et al. 2011Rasburicase G6PD Relling et al. 2014Tegafur DPYD Caudle et al. 2013Thioguanine TPMT Relling et al. 2013

Relling et al. 2011PSYCHIATRYAmitriptyline CYP2C19 Hicks et al. 2013Citalopram CYP2C19 Hicks et al. 2015Clomipramine CYP2C19 Hicks et al. 2013Doxepin CYP2C19 Hicks et al. 2013Escitalopram CYP2C19 Hicks et al. 2015Imipramine CYP2C19 Hicks et al. 2013Sertraline CYP2C19 Hicks et al. 2015Trimipramine CYP2C19 Hicks et al. 2013PULMONOLOGYIvacaftor CFTR Clancy et al. 2014

When 10,000 patients were tested, 91% had at least one clinically actionable variant

IMPACT OF PHARMACOGENETIC TESTING

Van Driest et al. 2014 Clin Pharmacol TherDunnenberger et al. 2015 Annu Rev Pharmacol Toxicol

BENEFITS OF GENETIC TESTING1) Genetic diagnosis confirms clinical diagnosis2) Genetic diagnosis guides risk assessment and

treatment strategies3) Genetic counseling and estimates of risk of

recurrence4) Risk assessment for asymptomatic family

members

Dynamic statementVariant re-classification

Co-segregation, database, publicationsP to LP, LP to P – low significance VUS to LP or P or vice versa – high importance

Re-classification scheduleOnce a year?When variant is discovered again?Reporting to all patients with the same variant?

Contacting physician or patient??

Thank you