Of the 621 eligible patients asked to participate in the study, 43 (6.9%) declined participation. The enrolled cohort was comprised mainly of African–Americans (47.2%) and European–Americans (52.2%). The cohort characteristics and prevalence of variant CYP2C9
genotypes, detailed in previous reports [26
] are summarized in . There were no significant differences in the proportion of women, length of follow-up or BMI across race groups. European–Americans were older, more physically active, and more likely to be light drinkers (1–7 drinks/week); to have medical insurance, higher education and higher income. Stroke and venous thromboembolism were more common indications for therapy among African–Americans, while atrial fibrillation and valvular heart disease were more common in European–Americans. More European–Americans had undergone coronary artery bypass grafting or percutaneous coronary angioplasty, had hyperlipidemia and malignancies while the prevalence of end stage renal disease and renal insufficiency was higher among African–Americans. The use of nonsteroidal analgesics, CYP2C9
inhibitors or substrates did not differ across racial groups [26
Sociodemographic and lifestyle characteristics of the POAT study participants at baseline*.
Although we planned to assess all polymorphic VKORC1 SNPs with reported MAF greater than or equal to 2%, four SNPs (rs13337470, rs17881535, rs17878521, rs17880887) could not be assessed due to assay limitations/failure. The influence of these SNPs was captured through assessment of other SNPs in strong linkage disequilibrium (LD) (rs17878521 = rs17878338 – r2 = 0.8; rs17881535 and rs17880887 = rs17708472 – r2 = 0.7; rs13337470 = rs13336384 – r2 = 0.6). Two additional African–American specific SNPs (rs17883590, rs17884766) were not assessed. An additional four SNPs (rs17882023, rs17878259, rs17884388, rs17884982) with reported MAF greater than or equal to 2% were found to be monomorphic in both race groups in our population. One SNP (rs17883590) with a reported MAF of 9% was found to have a lower prevalence (MAF 1.8%) in our cohort. HWE assumptions were met for all SNPs assessed in both race groups (all p-values >0.5). The distribution of SNPs (MAF ≥2%) varied significantly by race group (). The influence of eight common VKORC1 SNPs (MAF ≥5%) among European–Americans and eleven SNPs among African–Americans on warfarin dose was statistically evaluated.
Minor allele frequencies of VKORC1 variants by race among European–Americans and African–American participants of the POAT cohort.
Influence of individual VKORC1 SNPs on warfarin maintenance dose
Univariate associations for each common VKORC1 SNP (MAF ≥5%) revealed that among European–Americans all eight SNPs (with MAF ≥5%) were significantly associated with dose (p < 0.0002). Among African–Americans only four (rs17882368, rs2359612, rs9934438, rs9923231) of the eleven SNPs were significantly associated with dose (p < 0.001). The striking variation of MAF and differences in predictive power of individual VKORC1 SNPs across racial groups supports the use of race-stratified multivariable analyses. For both race groups the variability in warfarin dose explained was higher when VKORC1 or CYP2C9 genotype was modeled on an additive scale compared with dominant scale (). The variability in dose explained by VKORC1 (all SNPs) and CYP2C9 was significantly higher among European–Americans compared with African–Americans after adjustment for clinical covariates. The VKORC1 1173C/T (rs9934438) explained the highest variability in warfarin dose among both African– and European–Americans in the multivariable model with CYP2C9 and clinical covariates.
Multivariable evaluation of the association of individual VKORC1 SNPs with log warfarin maintenance dose among POAT participants by race.
Among European–Americans regression analyses identified five highly correlated (r2 >0.9) VKORC1 SNPs (rs2359612, rs8050894, rs9934438, rs9923231, rs7196161) that explained 17–19% of the variability (10.6–13.6% for dominant model) in warfarin dose. The addition of CYP2C9 improved the explanatory power to 28–31% (18.7–21.7% for dominant model, .
Among African–Americans two sets of SNPs (rs9934438 and rs9923231; rs17883591 and rs17886199) analyzed in this study were in strong LD (r2 > 0.9) while the LD between the other SNPs was weaker (r2 ranging from 0.2 to 0.6). In multivariable regression analyses VKORC1 variants were significantly associated with warfarin dose, explaining 1–5% of the variability in dose. Addition of CYP2C9 genotype improved the explanatory power up to 8% (6.2% for dominant model, ).
Influence of VKORC1 haplotypes & haplotype groups on maintenance dose
Among European–Americans, six haplotypes inferred from eight common SNPs accounted for 97.5% of all haplotypes. Four haplotypes with frequency of 5% or greater, assessed in multivariable analyses explained 16.3% of the variability in warfarin dose (p < 0.0001). Adjusted mean doses (and 95% CI) for each haplotype are presented in . Two haplotypes (WHT3 and WHT4) were found to be independently associated with low warfarin dose after adjustment for CYP2C9 and clinical covariates. Pairwise comparisons demonstrate the emergence of two distinct haplotype groups with WHT3 and WHT4 haplotypes associated with significantly lower warfarin dose compared with WHT1 and WHT2. Tukey–Kramer adjustments for multiple comparisons did not alter the significance of this association (p < 0.0001).
VKORC1 haplotype frequency and influence on warfarin dose among European–American and African–American participants.
Among European–Americans the genealogic tree showing the relationship among the four common haplotypes indicates the emergence of two divergent haplotype groups () differentiated by a set of five SNPs (rs2359612, rs8050894, rs9934438, rs9923231 and rs7196161). Haplotypes WHT3 and WHT4 (Group A: low-dose haplotype group) were associated with lower warfarin dose compared with WHT1 and WHT2 (Group B: high-dose haplotype group) after adjustment for CYP2C9 and clinical covariates (). VKORC1 haplotype groups accounted for 16.2% variability in warfarin dose (p < 0.0001) in multivariable regression analyses.
Effect of VKORC1 haplotype groups on warfarin dose among EuropeanAmerican and AfricanAmerican patients
Among African–Americans, twelve haplotypes inferred from 11 common SNPs accounted for 98.4% of all haplotypes. Eight haplotypes with frequency of 5% or greater, assessed in multivariable analyses explained 4.7% of the variability in warfarin dose (p < 0.05). Adjusted mean doses (and 95% CI) for each haplotype are presented in . Two haplotypes (BHT4, BHT6) were found to be independently associated with lower warfarin dose after adjustment for CYP2C9 genotype and clinical covariates (). However, after Tukey–Kramer adjustments only BHT6 was significantly associated with warfarin dose (p < 0.05).
Among African–Americans the genealogic tree showing the relationship among the eight common haplotypes is presented in . Haplotypes BHT4 and BHT6 were associated with lower warfarin dose, with haplotype BHT6 demonstrating the strongest influence. We designated the BHT6 group as low-dose haplotype group (Group A) and the others as high-dose haplotype group (Group B). As shown in , a set comprised of two SNPs (rs9934438 and rs9923231) distinguished the two haplotype groups. Multivariable analyses adjusting for clinical covariates and CYP2C9 indicated significantly lower dose requirements by the presence of the low-dose Group A haplotype () accounting for 7.0% of the variability in warfarin dose (p < 0.0001).
Comparison of predictive power of single VKORC1 polymorphisms haplotypes & haplotype groups on warfarin dose
Percent variability in warfarin dose explained by VKORC1 haplotypes and haplotype groups was similar to that explained by a multivariable model with a single informative VKORC1 SNP () for both African–Americans and European–Americans. Based on these results the most parsimonious model includes a single informative VKORC1 SNP model (rs9934438 or rs9923231) along with CYP2C9 and a limited set of clinical variables.
Multivariable evaluation of the association of VKORC1 and warfarin maintenance dose.
We utilize this model to demonstrate the dose reductions associated with genetic and clinical covariates in . The presence of CYP2C9 and VKORC1 1173C/T variants is associated with warfarin dose reduction among both African–Americans and European–Americans.
Influence of genetic and nongenetic covariates on warfarin dose among European–Americans and African–Americans.