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

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

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

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

underdose less effective overdose complications overdose complicationsunderdose Narrow therapeutic range drugs

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

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

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

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

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

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

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: * % * % Asian: *2 - 0% * % African-American: * % * %

Effect of CYP2C9 Genotype on Anticoagulation-Related Outcomes (Higashi et al., JAMA 2002) WARFARIN MAINTENANCE DOSE N 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

Analysis of Independent Predictors of Warfarin Dose Variable Change in Warfarin Dose P value Target INR, per 0.5 increase21%< BMI, per SD14%< Ethnicity (African-American, [Asian])13%, [ 10-15%] Age, per decade13% < Gender, Female12%< Drugs (Amiodarone)24% CYP2C9*2, per allele19%< CYP2C9*3, per allele 30%< 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?

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

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

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)

Warfarin maintenance dose (mg/day) 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?

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

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.)

SNP Selection: VKORC1 tagSNPs

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

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

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

CCGATCTCTG-H1 CCGAGCTCTG-H2 TAGGTCCGCA-H8 TACGTTCGCG-H9 (381, 3673, 6484, 6853, 7566) 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

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

* † † * * 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

European - mean ~ 5 mg/d African-American - higher ~ mg/d Asian - lower ~ 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

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

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

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

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

VKORC1 SNP alters liver-specific binding site

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

Associated SNPs in European-Americans

Long Range LD in Europeans 120 kb

137 kb

LD Region Narrowed in African-Americans 50 kb

39 kb

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

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

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

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.

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