First Pharmacogenomic Analysis Using Whole Exome Sequencing to

Identify Novel Genetic Determinants of Clopidogrel

Response Variability

GIFT-EXOMEACC/i2 2012

Disclosures

Consulting honoraria: Bristol-Myers Squibb/sanofi-aventis, Accumetrics, DSI/Lilly & Co., Merck, Janssen, AstraZeneca, The Medicines Company, Medicure

Equity Interest: Iverson Genetics

Speaker Honoraria: DSI/Lilly, AstraZeneca

Research Support: Bristol Meyers Squibb/sanofi-aventis, Quest Diagnostics, Accumetrics, Molecular Response

GIFT was supported through an grant from BMS/sanofi aventis, and exome analysis was made possible by a grant from Molecular Response and in-kind support from Agilent technologies

GRAVITAS was sponsored by Accumetrics

Exome

Influence of CYP2C19*2 allele identified through a candidate gene approach

Hulot et al, Blood 2006; 108:2244-47

SCRIPPS CLINIC

429 Healthy Amish after Clopidogrel 75 mg X 7d P=1.5 X 10-13

Shuldiner AR et al JAMA. Aug 26 2009;302(8):849-857.

The CYP2C19*2 genotype accounts for only 12% of the variation in clopidogrel response

What Is Whole Exome Analysis, and Why Do it?

• Sequencing of the entire protein coding regions of the human genome

• Exploratory approach, non hypothesis-driven (don’t need to know the mechanism of effect).

• Identifies both SNPs and insertion/deletions (indels)

• Unlike GWAS, can identify actual, causative variant associated with disease, rather than a SNP in linkage dysequilibrium (i.e., in keeping with bad company)

• More likely to detect mutations with a greater impact on disease

• Enriched portion of the genome that can be used to search for variants with large effect sizes

Enrichment of the human exome using biotinylated RNA probes

•>50 Mb (~1.5% genome)•Exons in >21,000 genes•>700 miRNA •>300 non-coding RNAs

Enables targeted enrichment of mostly protein coding functional sequence

Counting A,T,G,&C is only the beginning…. Computational Pipeline to Discover DNA variation

Align Reads GenomeBWA

Re-align gaps in readsGATK

Remove duplicate readsGATK

Quality checksRead Error Verification

Re-calibrate QVs readsGATK

Image analysis and Base-calling

CASAVA

Illumina HiSeq 2000

Identify VariantsGATK

Counting A,T,G,&C is only the beginning…. Computational Pipeline to Discover DNA variation

32-600 processor units employed for 7-10 days at a time to compute DNA variants on 192 exome samples.

Total serial compute time: >400 days

Standard-Dose Clopidogrel†

clopidogrel 75-mg/dayStandard-Dose Clopidogrel†

clopidogrel 75-mg/day

High-Dose Clopidogrel†

clopidogrel 600-mg, thenclopidogrel 150-mg/day

PRU ≥ 230

High On-treatment Reactivity

Yes No

N = 1109 N = 586

Normal On-treatment Reactivity

Random SelectionR

N = 1105

Primary Efficacy Endpoint: CV Death, Non-Fatal MI, Stent Thrombosis at 6 moKey Safety Endpoint: GUSTO Moderate or Severe Bleeding at 6 mo

Pharmacodynamics: Repeat VerifyNow P2Y12 at 1 and 6 months

†placebo-controlled All patients received aspirin (81-162mg daily)

*Peri-PCI clopidogrel per protocol-mandated criteria to ensure steady-state at 12-24 hrs

Price MJ et al , JAMA 2011

Elective or Urgent PCI with DES*

N=5429 VerifyNow P2Y12 Test 12-24 hours post-PCI

GRAVITAS Study Design

Sample Acquisition

• Samples (N=1,152) obtained at platelet function screening or during follow-up from patients participating in GRAVITAS at 42 participating sites

• OTR assessed using VerifyNow P2Y12 test per GRAVITAS protocol

• Baseline: 12-24 hours post-PCI, after standard peri-procedural clopidogrel regimen

• 30±7 days

• N=192 self-identified Caucasians selected for exome analysis

Filtering after Variant Calling Improves Data Quality

StageSample Number

# of SNPs detected

Indels detected

Raw GATK 189 6,191,317 518,757

Remove variants with QUAL<1000 and/or MQ<50

189 468,846 73,882

Remove genotypes with <4 reads

189 468,846 73,882

Remove variants with <75% samples reporting and/or MAF = 0

189 266,660 37,192

Remove outliers in gender, heterozygosity rate, and/or population statification

147 262,292 36,831

Primary Results: On-Treatment Reactivity and Variant Loci at 12-24 hours Post-PCI

CYP2C18 &CYP2C19ATP2B2 TIAM2

-log

(P

) va

lue

Green - no adjustmentBlue - adjusted for age, sex, BMI, smoking, CrCl, CHF, DM, HTN, hypercholesterolemia

3 Distinct Loci Associated With PRU at 12-24 hours after PCI

ATP2B2: Plasma membrane calcium-transporting ATPase 2

• Plays critical role in maintaining intracellular calcium homeostasis (exports calcium ions out of cell), thereby influencing platelet activation and in turn aggregation

• SNP variants associated with reactivity are within introns at border areas of exons

• Overall allelic frequency approximately 27% in general population

TIAM2: T-cell Lymphoma Invasion And Metastasis 2

• Rac1-specific guanine nucleotide exchange factor

• Principle mediator of Rac1 activation, which is essential for platelet lamellipodia formation, granule secretion, clot retraction, and PLCγ2 activation.

• Rac1 activation is potentiated by P2Y12 signaling, and affects P2Y12-dependent Rap1 activation

Stefanini et al, ATVB. 2012;32:434-44

TIAM2 Variants: Clinical Effects

• >10 SNPs in TIAM2 weakly associated with PRU

• Most significant SNP:

• Arg to Cys (CGC to TGC): non-synonymous, “damaging” substitution according to computational analysis (SIFT)

• Associated with lower levels of on-treatment reactivity

• Phenotype is consistent with predicted TIAM2 loss-of-function variant (i.e., decreased Rac1 activation)

• Allelic frequency approx 13% in general pop’n

CYP2C18 &CYP2C19

-log

(P

) va

lue

On-Treatment Reactivity and Variant Loci at 30 days Post-PCI

Single gene locus associated with PRU at 30 days

Green - no adjustmentBlue - adjusted for age, sex, BMI, smoking, CrCl, CHF, DM, HTN, hypercholesterolemia

Summary

• Exome analysis identified 2 novel loci that appear to be associated with early on-treatment reactivity.

• Findings preliminary, but identification of 2 genes critical to platelet function among the 21,000 sequenced genes lends credibility to the validity of the result

• Singular influence of CYP2C18/9 locus at 30 days of maintenance clopidogrel after PCI

• Unlikely that other protein-coding variant has large effect on response variability at this timepoint

Next Steps

• Validating variants in >1,000 subjects via genotyping

• Increase exome sequencing sample size

• Functional modeling

Conclusion

• Novel variants in genes downstream of clopidogrel metabolism appear to influence early on-treatment reactivity

• Over longer-term follow-up, CYP2C18/19 locus is the primary protein-coding determinant of clopidogrel response variability

• Our findings demonstrate the feasibility and potential of exploratory pharmacogenomics using exome sequencing to identify unanticipated mechanisms of drug response

The STSI Team

Andrew Carson PhD: Computational biology

Samuel Levy PhD, Director, Genomic Sciences, STSI

Guangfa Zhang – Image analysis and base callingJanel Lee, Tiereny Phillips – Exome enrichmentErin LeeSarah Shaw MurrayRebecca TischEric Topol