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
we demonstrate the variance in warfarin dose explained is higher when VKORC1
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
(dominant model) was consistent with prior reports.[25
] However, contrary to prior reports and concordant with those of Momary et al,[24
] 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[24
] 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
By defining dose in two ways; dose at first stabilization and average maintenance dose we demonstrated consistent influence of VKORC1
genotype regardless of definition. VKORC1
along with clinical covariates explained up to 55% of the variance in dose in European Americans (up to 40% among African Americans) with VKORC1
accounting for up to 30% (10% among African Americans). Among European Americans these findings are consistent with the prior reports.[10
] 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.[28
] In concordance with their findings, the variability in dose explained by the VKORC11173C/T
was lower in African Americans compared to European Americans.
Consistent with Schellman et al[28
] and Kealy et al,[25
] our findings demonstrate the lack of influence of VKORC1
on time to stabilization in patients of either race. However these findings are discordant with those reported by Schalekamp et al.[7
] 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,[28
] 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[25
]. Although we do not know the reason for the racial differences, we can speculate on the influence and interplay of various factors:
- The contribution of unmeasured genetic/environmental factors to INR fluctuation may differ by race. This was supported by the significance of race-gene interactions in race adjusted analyses for time to stabilization (VKORC1 1173C/T x race, p=0.03) and risk of over anticoagulation (VKORC1 1173C/T x race, p=0.002; CYP2C9 x race, p=0.09). Recognizing race specific differences we chose to conduct stratified analyses.
- We had to combine the ‘CT and TT’ genotypes for multivariable analyses because of the small number of patients with the ‘TT’ genotype thereby diluting the effect of VKORC1 polymorphism on risk of over-anticoagulation.
- The association between the VKORC1 polymorphisms studied and the causative polymorphism(s) that determines warfarin response is weaker in African Americans compared with European Americans because of different haplotype structures.
- Genetic and environmental factors other then those studied influence the risk of over-anticoagulation in African Americans. This idea is supported by the higher intra-individual variation in INR among African Americans compared to European Americans.
Among European Americans, the risk ratios in our study were lower than those reported by Kealy et al and Schellamn et al.[25
] 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
] 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.[31
] 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
. 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[28
] and European Americans.[49
] 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.[49
] 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.[49
] The haplotype structure differs significantly between persons of European versus African descent[20
] and may differ among African Americans across the US depending on the degree of racial admixture.[60
] 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.[34
] 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
] 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 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.