Transcript On-Treatment Reactivity and Variant Loci at 12-24 hours Post

First Pharmacogenomic Analysis
Using Whole Exome Sequencing to
Identify Novel Genetic Determinants of
Clopidogrel
Response Variability
GIFT-EXOME
ACC/i2 2012
Exome
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
Influence of CYP2C19*2 allele identified
through a candidate gene approach
Hulot et al, Blood 2006; 108:2244-47
429 Healthy Amish after
Clopidogrel 75 mg X 7d
P=1.5 X 10-13
The CYP2C19*2 genotype accounts for only 12% of the variation in clopidogrel response
Shuldiner AR et al JAMA. Aug 26 2009;302(8):849-857.
SCRIPPS CLINIC
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
Enables targeted
enrichment of mostly
protein coding functional
sequence
•>50 Mb (~1.5% genome)
•Exons in >21,000 genes
•>700 miRNA
•>300 non-coding RNAs
Counting A,T,G,&C is only the beginning….
Computational Pipeline to Discover DNA variation
Illumina HiSeq 2000
Align Reads Genome
BWA
Quality checks
Read Error Verification
Image analysis and
Base-calling
CASAVA
Re-align gaps in reads
GATK
Remove duplicate reads
GATK
Re-calibrate QVs reads
GATK
Identify Variants
GATK
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
Price MJ et al , JAMA 2011
GRAVITAS Study Design
Elective or Urgent PCI with DES*
VerifyNow P2Y12 Test 12-24 hours post-PCI
Yes
PRU ≥ 230
No
High On-treatment Reactivity
High-Dose Clopidogrel†
clopidogrel 600-mg, then
clopidogrel 150-mg/day
Normal On-treatment Reactivity
Random Selection
R
N = 1109
N=5429
N = 1105
Standard-Dose Clopidogrel†
clopidogrel 75-mg/day
N = 586
Standard-Dose
Clopidogrel†
clopidogrel 75-mg/day
Primary Efficacy Endpoint: CV Death, Non-Fatal MI, Stent Thrombosis at 6 mo
Key Safety Endpoint: GUSTO Moderate or Severe Bleeding at 6 mo
Pharmacodynamics: Repeat VerifyNow P2Y12 at 1 and 6 months
*Peri-PCI clopidogrel per protocol-mandated criteria to ensure steady-state at 12-24 hrs
†placebo-controlled
All patients received aspirin (81-162mg daily)
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 periprocedural clopidogrel regimen
• 30±7 days
• N=192 self-identified Caucasians selected for exome
analysis
Filtering after Variant Calling Improves
Data Quality
Sample
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
Stage
Primary Results: On-Treatment Reactivity
and Variant Loci at 12-24 hours Post-PCI
3 Distinct Loci Associated With PRU at 12-24 hours after PCI
TIAM2
-log (P) value
ATP2B2
CYP2C18 &
CYP2C19
Green - no adjustment
Blue - adjusted for age, sex, BMI, smoking, CrCl, CHF, DM, HTN, hypercholesterolemia
ATP2B2: Plasma membrane calciumtransporting 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 P2Y12dependent 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
On-Treatment Reactivity and Variant Loci at
30 days Post-PCI
Single gene locus associated with PRU at 30 days
-log (P) value
CYP2C18 &
CYP2C19
Green - no adjustment
Blue - 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 calling
Janel Lee, Tiereny Phillips – Exome enrichment
Erin Lee
Sarah Shaw Murray
Rebecca Tisch
Eric Topol