Transcript ppt

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
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
• In 2003, 21.2 million prescriptions were written for
Very effective rat poison!
warfarin (Coumadin)
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
overdose
less effective
complications
1
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
0
Drug Dose
Toxicity
Efficacy
1
underdose
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) Normal times are 10-13 sec
time to clot
• 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
European-American
No. of patients
40
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
BMI, per SD
Ethnicity (African-American, [Asian])
21%
14%
13%, [ 10-15%]
<0.0005
<0.0001
0.003
Age, per decade
Gender, Female
Drugs (Amiodarone)
CYP2C9*2, per allele
CYP2C9*3, per allele
13%
12%
24%
19%
30%
<0.0001
<0.0001
0.007
<0.0001
<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
Epoxide
Reductase
Warfarin
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)
5 kb - chr 16
Rost et al. & Li, et al., Nature (2004)
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)
Frequency
Inter-Individual Variability in Warfarin Dose: Genetic Liabilities
SENSITIVITY
CYP2C9 coding
SNPs - *3/*3
Common
VKORC1
non-coding
SNPs?
RESISTANCE
VKORC1
nonsynonymous
coding
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.
2.
3.
4.
5.
381, 3673, 6484, 6853, 7566
2653, 6009
861
5808
e.g. Bin 1 - SNP 381
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
861
9041
CCGATCTCTG-H1
CCGAGCTCTG-H2
A
TCGGTCCGCA-H7
TAGGTCCGCA-H8
TACGTTCGCG-H9
B
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
Warfarin Dose (mg/d)
8
†
A/A
A/B
B/B
†
6
*
*
*
4
2
0
AA
BB
AB
All patients
AA
AB
BB
2C9 WT patients
AA
AB
BB
2C9 VAR patients
(n = 181)
(n = 124)
(n = 57)
Independent of INR levels across all groups
8
Warfarin Dose (mg/d)
Univ. of Washington
n = 185
†
†
6
*
*
*
4
2
0
AA AB BB
All patients
Brian Gage
Howard McCleod
Charles Eby
21% variance in
dose explained
8
Warfarin Dose (mg/d)
Washington University
n = 386
AA
AB BB
2C9 WT patients
†
†
6
*
AA
AB BB
2C9 VAR patients
*
†
4
2
0
AA AB BB
All patients
AA
AB BB
2C9 WT patients
AA
AB BB
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
(11%)
B
(58%)
A
(37%)
European (CEPH)
Clade Distribution
Clade A = Low
Clade B = High
Other
(39%)
A
(89%)
Asian
Clade Distribution
Low dose phenotype
A
(14%)
B
(47%)
African-American
Clade Distribution
High dose phenotype
VKORC1 Predicts Warfarin Dose in Asians
Hong Kong Samples
Warfarin Dose (mg/d)
7.00
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)
21% - CYP2C9 - *2/*3
13% - VKORC1 - 6484
D’Andrea, et al., Blood (2005)
n = 147
VKORC1 Affects Rapid Vit K Cycle Response
1 oral dose of acenocoumarol
37% reduction in F7
30% change in INR
Bodin, et al. Blood 2005
n = 222
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