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

2. Pharmacogenomics as a Model for Association Studies Prospective testing reduce variability and identify outliers. 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

3. Warfarin Pharmacogenetics 1.Background Vitamin K cycle Pharmacokinetics/Pharmacodynamics Discovery of VKORC1 2.VKORC1 - SNP Discovery 3.VKORC1 - SNP Selection (tagSNPs) 4.Clinical Association Study VKORC1 and Wafarin Dose 5.VKORC1 - SNP Replication/Function

4. 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 Very effective rat poison! WARF+coumarin

5. underdose less effective overdose complications overdose complicationsunderdose 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. Add warfarin dose distribution Patient/Clinical/Environmental Factors Ave: 5.2 mg/d n = 186 European-American 30x dose variability 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 Ca 2+ binding, clot formation Vitamin K administration is the antidote for warfarin toxicity

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

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

12. Warfarin Metabolism (Pharmacokinetics) Major pathway for termination of pharmacologic effect Major pathway for termination of pharmacologic effect is through metabolism of S-warfarin in the liver by CYP2C9 CYP2C9 SNPs alter warfarin metabolism: 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 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. Effect of CYP2C9 Genotype on Anticoagulation-Related Outcomes (Higashi et al., JAMA 2002) WARFARIN MAINTENANCE DOSE N 127 28 4 18 3 5 mg warfarin/day - Variant alleles have significant clinical impact - Still large variability in warfarin dose (15-fold) in *1/*1 “controls”? TIME TO STABLE ANTICOAGULATION CYP2C9-WT ~90 days *2 or *3 carriers take longer to reach stable anticoagulation CYP2C9-Variant ~180 days

14. Analysis of Independent Predictors of Warfarin Dose Variable Change in Warfarin Dose P value Target INR, per 0.5 increase21%<0.0005 BMI, per SD14%<0.0001 Ethnicity (African-American, [Asian])13%, [ 10-15%] 0.003 Age, per decade13% <0.0001 Gender, Female12%<0.0001 Drugs (Amiodarone)24% 0.007 CYP2C9*2, per allele19%<0.0001 CYP2C9*3, per allele 30%<0.0001 Adapted from Gage et al., Thromb Haemost, 2004 ~ 30% of the variability in warfarin dose is explained by these factors What other candidate genes are influencing warfarin dosing?

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

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

17. Warfarin Resistance VKORC1 Polymorphisms Rare non-synonymous mutations in VKORC1 causative for warfarin resistance (15-35 mg/d) NO NO non-synonymous mutations found in ‘control’ chromosomes (n = ~400) Rost, et. al. Nature (2004)

18. Warfarin maintenance dose (mg/day) Inter-Individual Variability in Warfarin Dose: Genetic Liabilities SENSITIVITY CYP2C9 coding SNPs - *3/*3 RESISTANCE VKORC1 nonsynonymous coding SNPs 0.5515 Frequency Common VKORC1 non-coding SNPs?

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. 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 Do common SNPs associate with warfarin dose? SNP Discovery: Resequencing Results None of the previously identified VKORC1 warfarin-resistance SNPs were present (Rost, et al.)

21. SNP Selection: VKORC1 tagSNPs

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

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

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

25. CCGATCTCTG-H1 CCGAGCTCTG-H2 TAGGTCCGCA-H8 TACGTTCGCG-H9 (381, 3673, 6484, 6853, 7566) 5808 9041 861 B A 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 Explore the evolutionary relationship across haplotypes TCGGTCCGCA-H7 Multi-SNP testing: Haplotypes

26. VKORC1 clade diplotypes show a strong association with warfarin dose Low High A/A A/B B/B * † † * * All patients2C9 WT patients2C9 VAR patients AAABBB AA AB BB AAABBB (n = 181)(n = 124)(n = 57) Independent of INR levels across all groups

27. * † † * * All patients2C9 WT patients2C9 VAR patients AAABBB AAABBB AAABBB Univ. of Washington n = 185 All patients2C9 WT patients2C9 VAR patients AAABBB AAABBB AAABBB † † * † * 21% variance in dose explained Washington University n = 386 Brian Gage Howard McCleod Charles Eby

28. 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 Population differences in warfarin dose

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

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

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

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

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