7 Rieder VKORC1 CaseStudy 2006 by jWH4u22

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									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
      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.
  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
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• In 2003, 21.2 million prescriptions were written for
   warfarin (Coumadin)                                 Very effective rat poison!
                                                         WARF+coumarin
• 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
    Narrow therapeutic range drugs
                 underdose                overdose
                 less effective           complications
            1                                             1


           0.8                                            0.8
Efficacy




                                                                Toxicity
           0.6                                            0.6


           0.4                                            0.4


           0.2                                            0.2


            0                                             0

                              Drug Dose
         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
        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)
                  50                           Ave: 5.2 mg/d
                                               n = 186
                  40                           European-American
No. of patients                                 30x dose variability
                  30

                  20
      Add warfarin dose distribution
                  10

                  0
                       0   2   4      6    8   10    12    14    16
                                   Warfarin Dose (mg/d)

   Patient/Clinical/Environmental Factors
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 Ca2+ binding, clot
formation

• Vitamin K administration is the antidote for warfarin toxicity
Warfarin is a competitive antagonist of Vitamin K




                           Scully, M. The Biochemist, 2002
Warfarin inhibits the vitamin K cycle
                                                 Warfarin
           Epoxide
          Reductase                                   CYP2C9

                                                 Inactivation
                                           Pharmacokinetic




           -Carboxylase
              (GGCX)




          Vitamin K-dependent clotting factors
          (FII, FVII, FIX, FX, Protein C/S/Z)
 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%
Effect of CYP2C9 Genotype on Anticoagulation-Related Outcomes
(Higashi et al., JAMA 2002)

       WARFARIN MAINTENANCE DOSE          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


    - Variant alleles have significant clinical impact
    - Still large variability in warfarin dose (15-fold) in *1/*1 “controls”?
 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 allele                       19%                       <0.0001
 CYP2C9*3, per allele                       30%                       <0.0001

      ~ 30% of the variability in warfarin dose is explained by these factors

What other candidate genes are influencing warfarin dosing?
Warfarin acts as a vitamin K antagonist
                                 Pharmacodynamic       Warfarin
                  Epoxide
                 Reductase                                   CYP2C9

                                                       Inactivation




                 -Carboxylase
                    (GGCX)




                          Vitamin K-dependent clotting factors
                          (FII, FVII, FIX, FX, Protein C/S/Z)
              New Target Protein for Warfarin


          Epoxide
         Reductase

       (VKORC1)




         -Carboxylase
            (GGCX)




                   Clotting Factors
                   (FII, FVII, FIX, FX, Protein C/S/Z)
                                                         Rost et al. & Li, et al., Nature (2004)
5 kb - chr 16
             Warfarin Resistance VKORC1 Polymorphisms




                                                        Rost, et. al. Nature (2004)

• Rare non-synonymous mutations in VKORC1 causative for warfarin resistance (15-35 mg/d)
• NO non-synonymous mutations found in ‘control’ chromosomes (n = ~400)
Inter-Individual Variability in Warfarin Dose: Genetic Liabilities

    Frequency




                 SENSITIVITY                      RESISTANCE
                                                    VKORC1
                CYP2C9 coding    Common          nonsynonymous
                 SNPs - *3/*3    VKORC1              coding
                                non-coding            SNPs

                                  SNPs?

                       0.5            5          15
                      Warfarin maintenance dose (mg/day)
  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
       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?
SNP Selection: VKORC1 tagSNPs
  SNP Testing: VKORC1 tagSNPs
                    Five Bins to Test
                    1.   381, 3673, 6484, 6853, 7566
                    2.   2653, 6009
                    3.   861
                    4.   5808
                                          e.g. Bin 1 - SNP 381
                    5.   9041


                    Bin 1 - p < 0.001
                    Bin 2 - p < 0.02
                    Bin 3 - p < 0.01
                    Bin 4 - p < 0.001
                    Bin 5 - p < 0.001
                                                C/C C/T T/T


SNP x SNP interactions - haplotype analysis?
Multi-SNP testing: Haplotypes
             Five tagSNPs (10 total SNPs)
                   186 warfarin patients (European)
                   PHASE v2.1




                9 haplotypes/5 common (>5%)
VKORC1 Haplotypes Associate with Dose




  Adjusted for all significant covariates: age, sex, amiodarone, CYP2C9 genotype


   25% variance in dose explained
       Multi-SNP testing: Haplotypes
Explore the evolutionary relationship across haplotypes


(381, 3673, 6484, 6853, 7566)           5808
                                                    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
VKORC1 clade diplotypes show a strong association with warfarin dose


                                                                                        Low

                                                                                        High

                           8
                                                                 †                      A/A
                                             †
                                                                                        A/B
    Warfarin Dose (mg/d)




                           6
                                                         *
                                      *                                                 B/B
                                                                           *
                           4


                           2


                           0
                               AA     AB     BB   AA   AB     BB     AA    AB    BB
                                 All patients     2C9 WT patients    2C9 VAR patients
                                 (n = 181)           (n = 124)           (n = 57)

          Independent of INR levels across all groups
Univ. of Washington                            8
                                                                                †
n = 185                                                       †




                        Warfarin Dose (mg/d)
                                               6
                                                                           *
                                                         *
                                                                                             *
                                               4


                                               2


                                               0
                                                   AA AB BB         AA   AB BB        AA    AB BB
                                                    All patients   2C9 WT patients   2C9 VAR patients
                                               8
Washington University                                                           †
n = 386                                                       †
                        Warfarin Dose (mg/d)




                                               6                                                 †
                                                                          *
Brian Gage                                               *
Howard McCleod                                 4

Charles Eby
                                               2
 21% variance in
 dose explained                                0
                                                   AA AB BB         AA   AB BB        AA    AB BB
                                                    All patients   2C9 WT patients   2C9 VAR patients
    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
VKORC1 Haplotype Frequency Differs Between Populations


   Explore the evolutionary relationship across populations

                               B                           A
                             (11%)                       (14%)
                                                 Other
                A
                                                 (39%)
       B      (37%)
     (58%)                       A                         B
                               (89%)                     (47%)


   European (CEPH)             Asian            African-American
   Clade Distribution     Clade Distribution    Clade Distribution
                        Low dose phenotype     High dose phenotype

Clade A = Low
Clade B = High
                   VKORC1 Predicts Warfarin Dose in Asians

                                   Hong Kong Samples

                       7.00
Warfarin Dose (mg/d)




                       6.00

                       5.00

                       4.00

                       3.00

                       2.00

                       1.00

                       0.00

                              TT             CT          CC

                                      6853 Genotype


                                          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
Perlegen Large-scale SNP Dataset




1.58 millions SNPs genotyped
71 individuals from 3 American populations
     European, African and Asian ancestry
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



(6484)



(9041)




                                  D’Andrea, et al., Blood (2005)
21% - CYP2C9 - *2/*3              n = 147
13% - VKORC1 - 6484
  VKORC1 Affects Rapid Vit K Cycle Response




                               Bodin, et al. Blood 2005
1 oral dose of acenocoumarol   n = 222
37% reduction in F7
30% change in INR
 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   Washington University
Alex Reiner                         Brian Gage
Dave Veenstra                       Howard McLeod
Debbie Nickerson                    Charles Eby
Dave Blough
Ken Thummel                         Joyce You - Hong Kong


Noel Hastings
Maggie Ahearn

Josh Smith
Chris Baier
Peggy Dyer-Robertson

								
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