First Pharmacogenomic Analysis Using Whole Exome Sequencing to Identify Novel Genetic Determinants...
-
Upload
willa-newman -
Category
Documents
-
view
217 -
download
1
Transcript of First Pharmacogenomic Analysis Using Whole Exome Sequencing to Identify Novel Genetic Determinants...
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