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A Pharmacogenomic Approach to Understanding the Warfarin Drug Response

A Pharmacogenomic Approach to Understanding the Warfarin Drug Response. Mark J. Rieder, PhD. Pharmacogenomics as a Model for Association Studies. Clear genotype-phenotype link intervention variable response Pharmacokinetics - 5x variation Quantitative intervention and response

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A Pharmacogenomic Approach to Understanding the Warfarin Drug Response

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  1. A Pharmacogenomic Approach to Understanding the Warfarin Drug Response Mark J. Rieder, PhD

  2. Pharmacogenomics as a Model for Association Studies Clear genotype-phenotype link intervention variable response Pharmacokinetics - 5x variation Quantitative intervention and response drug dose, response time, metabolism rate, etc. Target/metabolism of drug generally known gene target that can be tested directly with response Prospective testing reduce variability and identify outliers.

  3. Warfarin Pharmacogenetics Background Vitamin K cycle Pharmacokinetics/Pharmacodynamics Discovery of VKORC1 VKORC1 - SNP Discovery VKORC1 - SNP Selection (tagSNPs) Clinical Association Study VKORC1 and Wafarin Dose VKORC1 - SNP Replication/Function

  4. WARF+coumarin Very effective rat poison! Warfarin Background • Commonly prescribed oral anti-coagulant and acts as an inhibitor of the vitamin K cycle • In 2003, 21.2 million prescriptions were written for • warfarin (Coumadin) • Prescribed following MI, atrial fibrillation, stroke, • venous thrombosis, prosthetic heart valve replacement, • and following major surgery • Difficult to determine effective dosage • Narrow therapeutic range • Large inter-individual variation

  5. underdose underdose overdose overdose complications complications less effective less effective Narrow therapeutic range drugs

  6. Warfarin Complications (-) Major bleeding episodes in 1-2% of all patients (--) Death in as many as 0.1-0.7% (++) Prevents 20 strokes for each bleeding event • Probably underused because of the fear of bleeding/overdose

  7. Monitoring Warfarin Dosing • Measure prothrombin time(PT) - time to clot • Normal times are 10-13 sec • Also measured as INR (International Normalized • Ratio). Range 1.0 - 1.4 (normalized thromboplastin) • On warfarin therapy a patient is kept at about • 2-3x normal (PT = 20-39 seconds)

  8. Ave: 5.2 mg/d n = 186 European-American Add warfarin dose distribution 30x dose variability Patient/Clinical/Environmental Factors • Pharmacokinetic/Pharmacodynamic - Genetic

  9. Vitamin K Cycle • Vitamin K synthesized by plants and bacteria • e.g. leafy green vegetables and intestinal flora • Vitamin K - discovered from defects in blood “koagulation” • Vitamin K - required coenzyme for -carboxylation of glutamic acid (Glu) conversion to -carboxyglutamic acid (Gla) • Glu --> Gla modification needed for Ca2+ binding, clot formation • Vitamin K administration is the antidote for warfarin toxicity

  10. Warfarin is a competitive antagonist of Vitamin K Scully, M. The Biochemist, 2002

  11. Warfarin CYP2C9 Epoxide Reductase Inactivation Pharmacokinetic  -Carboxylase (GGCX) Warfarin inhibits the vitamin K cycle Vitamin K-dependent clotting factors (FII, FVII, FIX, FX, Protein C/S/Z)

  12. Warfarin Metabolism (Pharmacokinetics) • Major pathway for termination of pharmacologic effect • is through metabolism of S-warfarin in the liver by CYP2C9 • CYP2C9 SNPs alter warfarin metabolism: • CYP2C9*1 (WT) - normal • CYP2C9*2 (Arg144Cys) - low/intermediate • CYP2C9*3 (Ile359Leu) - low • CYP2C9 alleles occur at a significant minor allele frequency • European: *2 - 10.7% *3 - 8.5 % • Asian: *2 - 0% *3 - 1-2% • African-American: *2 - 2.9% *3 - 0.8%

  13. TIME TO STABLE ANTICOAGULATION CYP2C9-WT ~90 days CYP2C9-Variant ~180 days *2 or *3 carriers take longer to reach stable anticoagulation N 127 28 4 18 3 5 Effect of CYP2C9 Genotype on Anticoagulation-Related Outcomes (Higashi et al., JAMA 2002) WARFARIN MAINTENANCE DOSE mg warfarin/day - Variant alleles have significant clinical impact - Still large variability in warfarin dose (15-fold) in *1/*1 “controls”?

  14. Analysis of Independent Predictors of Warfarin Dose Adapted from Gage et al., Thromb Haemost, 2004 Variable Change in Warfarin Dose P value Target INR, per 0.5 increase 21% <0.0005 BMI, per SD 14% <0.0001 Ethnicity(African-American, [Asian])13%, [ 10-15%] 0.003 Age, per decade 13% <0.0001 Gender, Female 12% <0.0001 Drugs (Amiodarone) 24% 0.007 CYP2C9*2, per allele19% <0.0001 CYP2C9*3, per allele30% <0.0001 ~ 30% of the variability in warfarin dose is explained by these factors What other candidate genes are influencing warfarin dosing?

  15. Warfarin Epoxide Reductase  -Carboxylase (GGCX) Warfarin acts as a vitamin K antagonist Pharmacodynamic CYP2C9 Inactivation Vitamin K-dependent clotting factors (FII, FVII, FIX, FX, Protein C/S/Z)

  16. Epoxide Reductase (VKORC1) New Target Protein for Warfarin  -Carboxylase (GGCX) Clotting Factors (FII, FVII, FIX, FX, Protein C/S/Z) Rost et al. & Li, et al., Nature (2004) 5 kb - chr 16

  17. Warfarin Resistance VKORC1 Polymorphisms Rost, et. al. Nature (2004) • Rare non-synonymous mutations in VKORC1 causative for warfarin resistance (15-35 mg/d) • NOnon-synonymous mutations found in ‘control’ chromosomes (n = ~400)

  18. 0.5 5 15 Inter-Individual Variability in Warfarin Dose: Genetic Liabilities SENSITIVITY CYP2C9 coding SNPs - *3/*3 RESISTANCE VKORC1 nonsynonymous coding SNPs Frequency Common VKORC1 non-coding SNPs? Warfarin maintenance dose (mg/day)

  19. SNP Discovery: Resequencing VKORC1 • PCR amplicons --> Resequencing of the complete genomic region • 5 Kb upstream and each of the 3 exons and intronic segments; ~11 Kb • Warfarin treated clinical patients (UWMC): 186 European • Other populations: 96 European, 96 African-Am., 120 Asian

  20. SNP Discovery: Resequencing Results VKORC1 - PGA samples (European, n = 23) Total: 13 SNPs identified 10 common/3 rare (<5% MAF) VKORC1 - Clinical Samples (European patients n = 186) Total: 28 SNPs identified 10 common/18 rare (<5% MAF) 15 - intronic/regulatory 7 - promoter SNPs 2 - 3’ UTR SNPs 3 - synonymous SNPs 1 - nonsynonymous - single heterozygous indiv. - highest warfarin dose = 15.5 mg/d None of the previously identified VKORC1 warfarin-resistance SNPs were present (Rost, et al.) Do common SNPs associate with warfarin dose?

  21. SNP Selection: VKORC1 tagSNPs

  22. e.g. Bin 1 - SNP 381 Bin 1 - p < 0.001 C/C C/T T/T SNP Testing: VKORC1 tagSNPs Five Bins to Test 381, 3673, 6484, 6853, 7566 2653, 6009 861 5808 9041 Bin 2 - p < 0.02 Bin 3 - p < 0.01 Bin 4 - p < 0.001 Bin 5 - p < 0.001 SNP x SNP interactions - haplotype analysis?

  23. 186 warfarin patients (European) PHASE v2.1 Multi-SNP testing: Haplotypes Five tagSNPs (10 total SNPs) 9 haplotypes/5 common (>5%)

  24. Adjusted for all significant covariates: age, sex, amiodarone, CYP2C9 genotype VKORC1 Haplotypes Associate with Dose 25% variance in dose explained

  25. Multi-SNP testing: Haplotypes Explore the evolutionary relationship across haplotypes 5808 (381, 3673, 6484, 6853, 7566) CCGATCTCTG-H1 A CCGAGCTCTG-H2 861 TCGGTCCGCA-H7 TAGGTCCGCA-H8 B 9041 TACGTTCGCG-H9 VKORC1 haplotypes cluster into divergent clades Patients can be assigned a clade diplotype: e.g. Patient 1 - H1/H2 = A/A Patient 2 - H1/H7 = A/B Patient 3 - H7/H9 = B/B

  26. † † * * * A/A A/B AA AB BB AA AB BB AA BB AB All patients 2C9 WT patients 2C9 VAR patients B/B • Independent of INR levels across all groups (n = 181) (n = 124) (n = 57) VKORC1 clade diplotypes show a strong association with warfarin dose Low High

  27. † † * * * Washington University n = 386 † † † * Brian Gage Howard McCleod Charles Eby * AA AA AB AB BB BB AA AA AB AB BB BB All patients All patients 2C9 WT patients 2C9 WT patients 2C9 VAR patients 2C9 VAR patients AA AA AB AB BB BB Univ. of Washington n = 185 21% variance in dose explained

  28. Population differences in warfarin dose • European - mean ~ 5 mg/d • African-American - higher ~ 6.0-7.0 mg/d • Asian - lower ~ 3.0-3.5 mg/d • Hypothesis:VKORC1 haplotypes contribute to racial • variability in warfarin dosing. • “Control” populations: 120 Europeans • 96 African-Americans • 120 Asian

  29. A (37%) B (58%) European (CEPH) Clade Distribution VKORC1 Haplotype Frequency Differs Between Populations Explore the evolutionary relationship across populations B (11%) A (14%) Other (39%) A (89%) B (47%) Asian Clade Distribution Low dose phenotype African-American Clade Distribution High dose phenotype Clade A = Low Clade B = High

  30. VKORC1 Predicts Warfarin Dose in Asians Joyce You 72 Asian - Hong Kong

  31. SNP Function: VKORC1 Expression mechanism Several SNPs are present in evolutionarily conserved non-coding regions - mRNA expression in human liver tissue

  32. SNP Function: VKORC1 Expression Expression in human liver tissue (n = 53) shows a graded change in expression.

  33. VKORC1 SNP alters liver-specific binding site

  34. Perlegen Large-scale SNP Dataset 1.58 millions SNPs genotyped 71 individuals from 3 American populations European, African and Asian ancestry

  35. Associated SNPs in European-Americans

  36. Long Range LD in Europeans 120 kb

  37. 137 kb

  38. LD Region Narrowed in African-Americans 50 kb

  39. 39 kb

  40. VKORC1 Confirmed in Other Studies (6484) (9041) D’Andrea, et al., Blood (2005) 21% - CYP2C9 - *2/*3 13% - VKORC1 - 6484 n = 147

  41. Bodin, et al. Blood 2005 n = 222 VKORC1 Affects Rapid Vit K Cycle Response 1 oral dose of acenocoumarol 37% reduction in F7 30% change in INR

  42. Future Studies: Other Warfarin Candidate Genes 25 genes ~200 informative SNPs

  43. VKORC1 Pharmacogenetics Summary 1. VKORC1 haplotypes are the major contributor to warfarin dose variability (21-25%). Overall variance described by clinical and genetic factors is 50-60%. 2. VKORC1 haplotypes are correlated with mRNA expression in the liver. 3. Distribution of high and low dose haplotypes is different in European, African, and Asian populations and may account for observed differences in average warfarin dose. 4. Prospective genotyping may lead to more accurate warfarin dosing and have impacts on the overall clinical treatment time.

  44. Acknowledgements Allan Rettie, Medicinal Chemistry Alex Reiner Dave Veenstra Debbie Nickerson Dave Blough Ken Thummel Noel Hastings Maggie Ahearn Josh Smith Chris Baier Peggy Dyer-Robertson Washington University Brian Gage Howard McLeod Charles Eby Joyce You - Hong Kong

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