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The influence of CYP2C9 and VKORC1 on warfarin dose, time to target INR, time to stabilization, and risk of over-anticoagulation (INR>4) was assessed after adjustment for clinical factors, intra-individual variation in environmental factors and unobserved heterogeneity.
Common CYP2C9 and VKORC1 polymorphisms were assessed in 302 European American and 273 African Americans on warfarin. Race-stratified multivariable analyses evaluated the influence of CYP2C9 and VKORC1 on warfarin response.
CYP2C9 and VKORC1 accounted for up to 30% variability in warfarin dose among European Americans and 10% among African Americans. Neither CYP2C9 nor VKORC1 influenced time to target INR or stabilization among patients of either race or risk of over-anticoagulation among African Americans. The risk of over-anticoagulation was higher among European Americans with variant VKORC11173C/T (p<0.01) and marginally significant among those with variant CYP2C9 (p=0.08) genotype.
Although CYP2C9 and VKORC1 genotyping can facilitate individualized initiation of warfarin dose in African and European Americans, the ability to predict risk of over-anticoagulation is inconsistent across race. Identification of other factors that can predict such risk consistently in a racially diverse group will facilitate individualized maintenance of warfarin therapy.
Although the efficacy of warfarin in the treatment and prevention of thromboembolic disorders (TEDs) is proven,[1, 2] it is underutilized with difficulties in management and risk of complications being the main deterrents.[3–6] Recognition of genetic regulation of warfarin response has stimulated efforts aimed at quantifying this influence, but past efforts have focused on a limited number of polymorphisms in populations of mainly European descent. [7–17]Race appears to influence warfarin dose requirements with African Americans requiring larger and Asians requiring lower doses compared to Europeans.[18–23] This variation in dose requirement by race may at least partially be explained by genetic differences. Cytochrome P4502C9 (CYP2C9)*2 and *3 variants are reported to have significant influence on warfarin dose among patients of European descent. [7–17] However among patients of African descent the CYP2C9-dose association has not been consistent.[24–26]African American populations contain known or putative poor-metabolizer alleles (CYP2C9*5, CYP2C9*6 and CYP2C9*11) which are rarely found in European Americans. However the influence of these variants has not been extensively documented. In contrast to the influence of CYP2C9, Vitamin K epoxide reductase (VKORC1)1173C/T variants have been shown to influence warfarin dose requirements among patients of both racial groups.
CYP2C9*2 and *3 VKORC11173C/T variants have been associated with an increased risk of over-anticoagulation among European Americans[7, 13, 25, 28–30] but not African Americans.[25, 28] It is recognized that while genes may influence inter-individual variation in warfarin response, for the intra-individual variation environmental factors may be more important.[2, 17] For example in any given patient, the International Normalized Ratio (INR) deviates from target (2.5, range 2–3) over time in response to many independent perturbations. Such intra-individual variation in INR due to unobserved factors introduces heterogeneity. Difficulties in capturing and accounting for unobserved heterogeneity may underestimate the environmental contribution (e.g. medication changes) and overestimate the genetic contribution in explaining variance in warfarin response and limit the ability to assess gene-environment interactions.
Herein we evaluate the influence of CYP2C9 (*2 (rs1799853), *3 (rs1057910), *5 (rs28371686), *6 (rs9332131), and *11(rs28371685)) and four VKORC1 single nucleotide polymorphisms [SNPs: 1173C/T (rs9934438), 3730G/A (rs7294), 2255C/T (rs2359612), 1542G/C (rs8050894)] on warfarin maintenance dose. We also assess the influence of CYP2C9 and VKORC1 polymorphisms on anticoagulation status and risk of over-anticoagulation after accounting for unobserved heterogeneity and clinical factors.
The Pharmacogenetic Optimization of Anticoagulation Therapy (POAT) is an ongoing prospective cohort study aimed at defining the influence of CYP2C9 polymorphisms on warfarin response over a 2-year follow-up period.[26, 31, 32] The study is being conducted at the University of Alabama at Birmingham (UAB) enrolling patients from the anticoagulation clinic at The Kirklin Clinics (TKC-AC) and the Jefferson Clinic P.C., Jefferson County Health System (CGH-JC) under the approval of the respective Institutional Review Boards. Both clinics follow a standardized approach to manage anticoagulation therapy.
Patients ≥20 years of age were identified at the initiation of warfarin therapy. Patients were considered eligible if the intended duration of anticoagulation therapy was at least 2 years, the target INR range was 2–3 and are managed at one of the two anticoagulation clinics.
A structured interview form was used at the time of enrollment to obtain a detailed medical lifestyle and concomitant medication history. Information on self-reported race, indication for therapy, demographics, height and weight, medications and co-morbid conditions was documented. Lifestyle and socioeconomic data included smoking, alcohol use, education, annual household income, medical insurance, physical activity, and dietary vitamin K intake. Medical history was then verified by medical records review.
All patients were followed at monthly intervals for up to two years from initiation of therapy. At each visit factors influencing warfarin response such as warfarin dose, INR, concurrent medications, dietary vitamin K (number of servings of foods rich in vitamin K consumed per week) alcohol intake (number of alcoholic drinks per week), compliance and level of physical activity were documented.[35–37] Information on concurrent medications was updated at every clinic visit by self-report and verified by medical record review. Concomitant use of drugs such as non-steroidal anti-inflammatory drugs or antiplatelet agents, or drugs that alter warfarin pharmacokinetics, including CYP2C9 inhibitors (e.g., amiodarone), CYP2C9 inducers (e.g., rifampin), or CYP2C9 substrates (e.g., losartan) was recorded.[38, 39]
DNA extraction and genotyping methodology for CYP2C9 and VKORC1 polymorphisms were detailed in recent manuscripts.[26, 31] Blood (8ml) was collected in a Qaigen PAX gene tube and DNA was extracted using the PAXgene blood DNA extraction kits. Briefly CYP2C9 genotyping was conducted using pyrosequencing methods and PCR-RFLP methodology. VKORC1 genotyping (rs9934438, rs7294, rs2359612 and rs8050894) was conducted using the Sequenom iPLEX technology at the Broad Institute.
Analysis of variance was used to assess group differences for continuous variables and χ2 test of independence for categorical variables. The assumption of Hardy Weinberg Equilibrium (HWE) was tested using the χ2 test of independence and exact statistics were obtained using a Markov Chain Monte Carlo algorithm.
Dose was defined in two ways: the average maintenance dose required to maintain anticoagulation for the duration of therapy and stable dose defined as the first dose that leads to a stable INR over three consecutive visits following initiation of the drug. These INR measurements encompassing a period of at least 2 weeks, with a maximum difference between the mean daily dosages of 10%. The distribution of dose was marginally skewed to the right therefore log transformation was done to attain normality. Race-stratified linear-regression analysis was conducted to assess the influence of CYP2C9, VKORC1 genotype on log-transformed dose after adjustment for age, gender, BMI, clinic, income, education, health insurance, smoking status, level of physical activity, alcohol intake, vitamin K intake, comorbid conditions (e.g. CHF, renal failure and cancer) and drug interactions (e.g. amiodarone, statins, NSAIDs, antiplatelet agents). We evaluated dose genotype associations using models that assessed the additive and dominant effects of VKORC1 and CYP2C9. Genotypes were included as covariates with 3-levels in the additive models and as covariates with 2 levels (wild-type versus variant) in the dominant models. The effect size associated with each predictor was calculated as the percentage of the variation in warfarin dose explained by the predictor, divided by the total variance in the regression model.
We also examined the time required to attain target INR (calculated as the difference in days from initiation of therapy to achieving the first INR in target range) and time to attain stable dose (calculated as the time from the initiation of therapy until attainment of stable dose) using the Cox Proportional Hazard (PH) models. To assess the risk of over-anticoagulation (INR>4) the hazard ratio (HR) and 95% CI were obtained using the counting process format in the PH model.[42, 43] This format allows individuals to contribute more than one event (INR>4). Valid confidence intervals were obtained by correction of dependence using robust variance estimation.
In any given patient, INR deviates from target (2.5, range 2–3) over time in response to many independent perturbations. Such intra-individual variation in INR due to unobserved factors introduces heterogeneity, herein termed unobserved heterogeneity.
We recognize that such unobserved heterogeneity may potentially influence the time to stabilization and over-anticoagulation. To capture its effect we computed a patient-specific variance growth rate (Vscore), a cumulative measure of time-weighted variance of the INR for each time interval (between visits) as proposed by Fihn et al, with minor modification. This measure adjusts for the influence of the number of visits and the interval between visits on INR variation for each patient. The use of the Vscore from the preceding interval accounts for the patient-specific unobserved heterogeneity in the analyses of time to stabilization and time to over-anticoagulation.
σ2 = variance growth rate (Vscore)
n = number of visits (n-1 will compute the Vscore up to the preceding visit)
τi = duration in weeks since preceding clinic visit (INR measurement)
INRi = International Normalized ratio at the ith visit (target INR was 2–3 for all participants)
Modified from Fihn et al 
All multivariable analyses included CYP2C9, VKORC1 genotype, age, race, gender, BMI, socio-demographic factors, clinic, indication and comorbid conditions. Changes in medications, Vscore, vitamin K and alcohol intake, and level of physical activity, warfarin dose and INRs were included as time-varying covariates. All analyses were performed using SAS version 9.1 (SAS Institute, Cary, NC) at a non-directional alpha level of 0.05.
Patients meeting eligibility criteria between August 2003 and April 2007 (n=621) were asked to participate in the study. Forty-three (6. 9%) patients declined participation. The cohort was comprised mainly of African Americans (47.2%) and European Americans (52.2%). As described in previous reports,[26, 31, 32] there were no significant differences in gender distribution, length of follow-up or BMI across race groups. European American patients were older, more physically active, and more likely to be light drinkers (1–7 drinks/week); enrolled from TKC-AC, have medical insurance, higher education and higher income (Table 1).
Stroke and venous thromboembolism were more common indications for therapy in African American patients, while atrial fibrillation and valvular heart disease were more common in European Americans. The prevalence of individual comorbid conditions differed across racial groups. More European American patients had undergone coronary artery bypass grafting (CABG) or percutaneous coronary angioplasty (PTCA), had hyperlipidemia and malignancies while the prevalence of end stage renal disease and renal insufficiency was higher among African Americans. Other than the higher use of antiplatelet agents at baseline, the use of non-steroidal analgesics, CYP2C9 inhibitors or substrates was not significantly different across racial groups African American patients had a greater intra-individual variation in INR, captured by the variable VScore (p=0.026, Table 2).
Genotype distributions for CYP2C9 and VKORC1 were in HWE among European Americans (all p-values >0.5) and African Americans (all p-values >0.25). Of the variant CYP2C9 alleles tested only CYP2C9*5, CYP2C9*6, and CYP2C9*11 were observed among African Americans while CYP2C9*2, and CYP2C9*3 were observed among European Americans and African Americans. European Americans had higher frequency of variant genotype for CYP2C9, 1173C/T, 2255C/T and 1542G/C, whereas African Americans had higher variant allele frequencies for 3730G/A (Table 3). Due to significant racial differences in covariates (Tables 1 and and2)2) and frequencies of CYP2C9 and VKORC1 variants (Table 3) all further analyses were stratified by race.
In univariate analysis, among European Americans, variant CYP2C9 and VKORC1 (1173C/T, 2255C/T and 1542G/C) genotypes were associated with lower (all p-values <0.01) and 3730G/A was associated with higher (p =0.008) warfarin dose. Among African Americans, variant VKORC1 (1173C/T, 2255C/T) genotypes were associated with lower dose (p <0.005) while variant CYP2C9 (p =0.12) and VKORC1 (1542G/C, p =0.15 and 3730G/A, p =0.2) genotypes did not have a significant influence. Univariate associations of CYP2C9 and VKORC1 variants with dose demonstrate the higher dose requirements among African Americans (Figure 1).
Among European Americans, prior reports indicate strong linkage disequilibrium between the four VKORC1 SNPs.[49, 50] Therefore multivariable analyses included the most informative VKORC1 SNP (1173C/T) along with CYP2C9 and clinical covariates. Since the LD structure for VKORC1 in African Americans is unknown, all four VKORC1 SNPs were included in the initial model. However the final model retained only VKORC11173C/T as a significant predictor.
African Americans required higher warfarin maintenance doses compared to European Americans (Figure 2) after adjusting clinical and genetic covariates. The variability in dose explained by the VKORC11173C/T and CYP2C9 was lower in African Americans compared to European Americans (p<0.0001). After adjusting for potential confounders, variant CYP2C9 and VKORC11173C/T genotypes explained 30% variance in warfarin dose (20% in the dominant model) among European Americans compared to 10% variance in does (7% in the dominant model) among African Americans (Table 4). The variance in dose accounted for by CYP2C9 and VKORC11173C/T genotypes was similar for both dose definitions; average maintenance dose and first stable dose. The interaction between CYP2C9 and VKOR1173C/T genotype was not significant for either race group. Age, gender, BMI, vitamin K intake, concomitant therapy with CYP2C9 inhibitors also had significant influence on warfarin dose among patients of both race groups (p<0.05).
We assessed the effects of VKORC11173C/T on time to target INR, stabilization or risk of over-anticoagulation after accounting for clinical covariates and unobserved heterogeneity (Table 5).
Neither CYP2C9 nor VKORC11173C/T influenced the time to attain target INR or time to stabilization among patients of either race. European Americans patients with variant VKORC11173C/T genotype showed a 2 to 4 fold higher risk of over-anticoagulation (Figure 3a). Those with variant CYP2C9 genotype were at a 2 to 3 fold higher risk; however, this finding was of marginal statistical significance (Table 5, Figure 3b). Neither VKORC11173C/T nor CYP2C9 was significantly associated with risk of over anticoagulation among African Americans (Table 5, Figure 3c and 3d).
The interaction of CYP2C9 and VKOR1173C/T genotypes was not significant for any endpoint. Time to stable dosing was not significantly influenced by Vscore prior to stabilization for patients of either race (p-values >0.1). Higher Vscore was associated with a significantly higher risk of over anticoagulation through out the duration of therapy (p<0.0001) among both racial groups. The risk was higher during the first 30 days of therapy (p=0.06 for European Americans, p=0.03 for African Americans) but not around the time when stabilization is first achieved (p=0.56 for European Americans, p=0.81 for African Americans).
This prospective cohort study demonstrates the contribution of CYP2C9 and VKORC1 variants in determining warfarin dose in a racially diverse cohort. VKORC11173C/T genotype explains a higher proportion of variance in warfarin dose compared CYP2C9 for both race groups.
By evaluating both additive and dominant effects of VKORC1 and CYP2C9 we demonstrate the variance in warfarin dose explained is higher when VKORC1 and CYP2C9 genotype covariates were modeled on an additive scale compared to dominant scale. This finding was consistent among both European Americans and African Americans. Among African Americans, the variance in warfarin dose explained by CYP2C9 and VKORC1 (dominant model) was consistent with prior reports.[25, 26, 28] However, contrary to prior reports and concordant with those of Momary et al, the additive model explained a higher percent variance and revealed statistically significant effects of CYP2C9 on warfarin dose in African Americans. Consistent with prior reports and others age, gender, BMI, vitamin K intake, concomitant therapy with CYP2C9 inhibitors also had significant influence on warfarin dose among patients of both race groups.[8, 10–12, 15, 16, 24, 50–56]
By defining dose in two ways; dose at first stabilization and average maintenance dose we demonstrated consistent influence of VKORC1 and CYP2C9 genotype regardless of definition. VKORC1 and CYP2C9 along with clinical covariates explained up to 55% of the variance in dose in European Americans (up to 40% among African Americans) with VKORC1 and CYP2C9 accounting for up to 30% (10% among African Americans). Among European Americans these findings are consistent with the prior reports.[10, 11, 15, 19–21, 23, 49, 50, 57, 58] However the latter estimates, derived from mainly retrospective studies in homogeneous populations, may not hold in racially diverse populations as demonstrated by Schellman et al. In concordance with their findings, the variability in dose explained by the VKORC11173C/T and CYP2C9 was lower in African Americans compared to European Americans.
Consistent with Schellman et al and Kealy et al, our findings demonstrate the lack of influence of VKORC1 and CYP2C9 on time to stabilization in patients of either race. However these findings are discordant with those reported by Schalekamp et al.[7, 13, 29, 30] This discordance can potentially be explained by several factors including differences in the coumarin anticoagulant, dose initiation strategies, and important protocol differences such as exclusion of interacting drugs.
As reported by Schellman et al, the VKORC11173’T’ allele was significantly associated with an increased risk of over-anticoagulation among European Americans but not African Americans. The CYP2C9 variant was marginally associated with an increased risk of over-anticoagulation among European Americans, which is consistent with previous studies. Although we do not know the reason for the racial differences, we can speculate on the influence and interplay of various factors:
Among European Americans, the risk ratios in our study were lower than those reported by Kealy et al and Schellamn et al.[25, 28] This can perhaps be explained by the inclusion of a measure of unobserved heterogeneity in the analyses. Inability to account for such heterogeneity has been recognized as a limitation by several investigators.[9, 17, 28] Our results provide evidence that the higher risk of over-anticoagulation associated with variant VKORC11173C/T among European Americans is independent of such heterogeneity.
We recently reported a significantly increased risk of major hemorrhage conferred by variant CYP2C9 genotype but not by variant VKORC1 1173C/T genotype after adjusting for the influence of INR at the time of the event and other clinical covariates. The risk was statistically significant among European Americans but not in African Americans. The latter finding may be due to the lower frequency of the variant genotypes in African Americans. Along with CYP2C9, an elevated INR significantly increased the risk of major hemorrhage. For every unit increase in INR the risk of major hemorrhage increased by 75% (HR 1.74, 95%CI: 1.5, 2.1). In both African American and European American patients these gene-response associations highlight two things; the risk of hemorrhage is higher among patients who possess CYP2C9 variants and the risk of hemorrhage is higher among patients who have elevated INR. These effects are independent of each other.
To our knowledge, our cohort represents the largest population of African Americans genotyped for CYP2C9 and VKORC1. Inclusion of the *5, *6 and *11 variants in the genotyping provides a robust estimate of the CYP2C9 allele frequencies in this previously underrepresented racial group. We examined only four SNPs in the VKORC1 gene (1173C/T, 3730G/A, 2255C/T, 1542G/C). We did not assess the -1639G/A polymorphism (rs9923231) as studies have demonstrated that the 1173C and -1639G allele are in linkage disequilibrium among both African Americans and European Americans. Three of these polymorphisms (1173C/T, 2255C/T, 1542G/C) are part of the haplotype that has been associated with a relatively low hepatic VKORC1 mRNA expression in the liver of European Americans. Furthermore, Rieder et al showed that the 1173C/T polymorphism alone was as informative as VKORC1 haplotypes for predicting warfarin dose in a Caucasian population. The haplotype structure differs significantly between persons of European versus African descent[20, 59] and may differ among African Americans across the US depending on the degree of racial admixture.[60–62] Therefore all four SNPs were included in the initial models for African Americans. Assessment of other VKORC1 polymorphisms will help determine haplotype structure and may identify other influential VKORC1 polymorphisms among African Americans.
We also recognize our sample-size was inadequate to detect significant CYP2C9-VKORC1 interaction in either race group. After adjusting for statistically significant and clinically relevant covariates a post-hoc assessment of power demonstrates the adequacy of the cohort size to detect significant dose differences (between variant and wild-type genotype) for VKORC1 among European Americans and African Americans and for CYP2C9 among European Americans (power >80%). However among African Americans significant dose difference was not detected for CYP2C9 (power ~40%) except when it was modeled on an additive scale for average dose. For most anticoagulation endpoints the risk ratios detected were consistent with the null for both European Americans and African Americans. Only risk ratios for INR >4 demonstrated an increased risk of over-anticoagulation among European Americans with variant VKORC1 genotype (power >80%) but not for variant CYP2C9 genotype (power ~70%). Documentation of vitamin K intake was based on patient report using vitamin K inventory and was not quantified by assay/measurements. However, all measurements were used consistently; therefore, bias if any should be non-differential. We recognize that many factors including changes in vitamin K intake can contribute to INR fluctuation.[45, 63] The inclusion of the Vscore potentially accounts for the changes in unmeasured/unobserved environmental influences. We assessed the influence of only two genes (CYP2C9 and VKORC1) and recognize that other genes may influence warfarin response or modify the effect of these genes. ApoE has recently been shown to influence warfarin dose among African Americans. Other genes such as gamma-glutamyl carboxylase, calumenin, epoxide hydroxylase may influence warfarin dose in this race group. However, the extent to which variability in other genes in the warfarin pathways influences warfarin response is yet to be resolved.
In conclusion the CYP2C9 and VKORC1 variants are associated with lower warfarin dose requirements among both African Americans and European Americans. Although CYP2C9 and VKORC1 genotyping have the potential to facilitate the development of individually tailored warfarin dose in both African and European Americans, the ability to predict risk of over-anticoagulation is limited to European Americans. Perhaps future identification of other genetic/environmental factors that can predict such risk consistently in the racially diverse group of patients encountered in clinical practice will facilitate individually tailored maintenance of warfarin therapy.
Supported in part by a grant from the National Institute of Neurological Disorders and Stroke (Grant Number: K23NS45598-01) and in part by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences
Financial disclosure/ Acknowledgments
The authors thank Joyce Blaisdell for her work with CYP2C9 genotyping. The first author wishes to acknowledge Dr. Edward Faught for his support and mentorship. We are grateful to all the patients that participated in the study. We thank Janice Ware for her untiring efforts with patient recruitment and the staff of the Anticoagulation Clinic at The Kirklin Clinic, the Cooper Green Hospital and Jefferson Clinic P.C for their help with identification of potential participants. We also thank the physicians, especially Drs. Mark Wilson, and Melissa Baird; at the University of Alabama at Birmingham and the Health Service Foundation for their support of this research. Thanks to Steve Duncan and Darlene Green and the Office of Data Resources for their work with the POAT database and quality assurance.
This work was supported in part by a grant from the National Institute of Neurological Disorders and Stroke (Grant number K23NS45598-01) and in part by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences. This study has contributed samples to the NINDS Human Genetics Resource Center DNA and Cell Line Repository (http://ccr.coriell.org/ninds), NINDS Repository sample numbers corresponding to the samples used are ND04466, ND04556, ND04604, ND04605, ND04626, ND04869, ND04907, ND04934, ND04951, ND05036, ND05108, ND05175, ND05176, ND05239, ND05605, ND05606, ND05701, ND05702, ND05735, ND06147, ND06207, ND06385, ND06424, ND06480, ND06706, ND06814, ND06871, ND06983, ND07057, ND07234, ND07304, ND07494, ND07602, ND07711, ND07712, ND08065, ND08596, ND08864, ND08932, ND09079, ND09172, ND09760, ND09761, ND09809.
The Broad Institute Center for Genotyping and Analysis is supported by grant U54 RR020278-01 from the National Center for Research Resources.