This study provides strong evidence of the increased risk of major hemorrhage among patients with a variant CYP2C9 genotype throughout the duration of warfarin therapy after accounting for VKORC1 1173C/T genotype, demographics, and clinical covariates. To our knowledge, this is the largest prospective study aiming to define the association of variant CYP2C9 genotype with major hemorrhage among both European Americans and African Americans.
Our study is the first to examine the effect of the CYP2C9*5, CYP2C9*6
, and CYP2C9*11
allele in a large African-American population in addition to CYP2C9*2
were the most common CYP2C9
variant alleles in both racial groups, with observed frequencies consistent with previous reports.17,18
On the basis of variant allele frequencies, the combined poor-metabolizer genotypes are significantly more frequent among European Americans than African Americans (29.8 vs 9.73%, P
<0.0001). Inclusion of the *5, *6
, and *11
alleles in determining CYP2C9
genotype resulted in a higher poor-metabolizer genotype frequency (9.7%) among African Americans than that reported previously.19
The frequency of VKORC1 1173C/T
variant was similar for European Americans (60%) but slightly higher for African Americans (19.6 vs 15.4%) than that reported by Schelleman et al
Among European Americans, the risk conferred by the variant CYP2C9
genotype is consistent with previous reports.9,10
To our knowledge, this is the first report of CYP2C9
genotype—major hemorrhage association in African Americans. Population stratification is often cited as a potential confounder in genetic association studies.15
For it to introduce confounding bias, three conditions must be met.20
First, the allele frequencies must differ across race; second, the occurrence of disease (hemorrhage) must vary by race; third, the genetic marker should not be in the casual pathway. In our study, although CYP2C9
allele frequencies show substantial differences across race (P
<0.0001), the occurrence of major or minor hemorrhage does not. Therefore, population stratification does not bias the association of CYP2C9
genotype and risk of hemorrhage in our study. This is further emphasized by the lack of significant differences in the race-specific HRs (P
-value for gene×race interaction=0.27). The lower frequency of the variant genotype in African Americans suggests that the population-attributable risk of this allele may be lower among African Americans than European Americans.
The incidence rate for major hemorrhagic complications in this study was similar to that reported previously.10,21
However, the incidence rate for minor hemorrhagic complications in our population was higher than that reported previously (14.5 per 100 patient-years; 95% CI: 13.7–16.22),21
perhaps because of inclusion of patients with minor hemorrhage who required additional testing or clinic visits.
Several studies have reported an increased risk of hemorrhage among patients with a variant CYP2C9
genotype. However, most have defined the event as “over-anticoagulation” rather than a hemorrhagic event.13,22-28
Four studies measured the occurrence of hemorrhagic events.9-11,29
Of these, only three reported bleeding risk by genotype.10,11,29
Aithal et al
reported bleeding risk for patients in a predefined low-dose category (<1.5 mg/day) rather than by genotype. Ogg et al
reported the influence of a sole variant, CYP2C9*3
variant, on risk of bleeding. A recent meta-analysis30
summarized the bleeding risk by genotype. Patients with at least one copy of a variant allele had an increased risk of bleeding compared with non-carriers for CYP2C9*2
(relative risk (RR) 1.9, 95% CI: 1.2–3.2), CYP2C9*3
(RR 1.8, 95% CI: 1.1–2.9), and CYP2C9*2
(RR 2.3, 95% CI: 1.4–3.7). Our results of increased risk (HR 3.0, 95% CI: 1.1, 8.0) are consistent with these reports.
Previous studies have not been able to compare the influence of individual variant alleles on the risk of major hemorrhage because of the rarity of the event and limited sample size. In our study, patients with the CYP2C9*3 variant had a higher, although not statistically significant, risk of major hemorrhage than those with CYP2C9*2 variant (RR 1.7, 95% CI: 0.47–5.8, P=0.42). The small number of events prevented the determination of the risk of major hemorrhage conferred by the other variant alleles.
As reported by Higashi et al
patients with the variant genotype had a higher risk of major hemorrhage before stabilization of anticoagulation. However, this association could not be assessed in a multivariable model because of the small number (n
=5) of events. The availability of longitudinal data enabled us to define periods of “stable and unstable” anticoagulation for each patient. Patients with the variant genotype continue to have a higher risk throughout the duration of therapy, including periods of “unstable” anticoagulation across the INR range after accounting for the effects of covariates. These findings highlight that the risk conferred by the variant genotype does not dissipate after stabilization of therapy.
This study has several strengths including a 2-year follow-up in a clinically well-characterized cohort, adjustment for time-varying covariates, drug interactions and comorbidity, objective documentation of incident complications, separation of the genotype—phenotype data until time of analyses, and event adjudication by an expert without knowledge of patients’ genotype. Besides the large African-American subgroup, the ability to model time-varying covariates is a major strength. The inclusion of patients from initiation of therapy in this prospective study and the high participation rate (93.1%) minimize selection bias. The generalizability of our findings is further improved by the representativeness of the study cohort as indicated by the similarity in age, gender, and racial distribution as compared to patients treated at University of Alabama at Birmingham (UAB) from the Birmingham metropolitan area.
We also recognize limitations of this study. Documentation of vitamin K intake was based on patients’ report using vitamin K inventory,31
not quantified by assay/measurements. However, all measurements were used consistently among all participants; therefore, bias if any should be non-differential. We also recognize that drug response is influenced by multiple genes. At least one other gene, VKORC1
, has been shown to influence significantly warfarin dose in European Americans.25,32-36
Although it has been hypothesized that VKORC1
may influence the risk of hemorrhage, all studies have aimed at quantifying its influence on risk of severe over-anticoagulation (defined as INR of 6 or higher).25-27,37
Schalekamp et al
found that among acenocoumarol users the possession of CYP2C9*3
allele was associated with an increased risk of over-anticoagulation compared to the CYP2C9*2
alleles. However, among phenprocoumon users both CYP2C9*2
were associated with an increased risk of over-anticoagulation.27
Accounting for the effects of VKORC1
, the risk of over-anticoagulation was increased by a combination of VKORC1
genotypes. Carriers of a combination of CYP2C9
polymorphism had an increased risk of severe over-anticoagulation compared with subjects with no polymorphism or only one polymorphism among both acenocoumarol (HR 3.8, 95% CI: 1.6–9.0)26
and phenprocoumon (HR 7.2, 95% CI: 2.1–24.7) users.25
One study by Reitsma et al
suggests an increased risk of bleeding in the phenprocoumon users (crude odds ratio 2.6, 95% CI: 1.2–5.7), but not in acenocoumarol users (crude odds ratio 1.2, 95% CI: 0.6–2.3). Of note, this association is not consistent across the coumarin anticoagulants and did not account for other factors known to increase the risk of hemorrhage, such as CYP2C9
genotype, INR, drug interactions, and so on. Our results indicate a higher (but not statistically significant) hemorrhagic risk of VKORC1 1173C/T
variants after adjusting for CYP2C9
genotype, clinical and demographic variables. Results of ongoing investigations will help better define the influence of 1173C/T
and other VKORC1
variants (which may differ by race) on the risk of hemorrhagic events among patients on chronic warfarin therapy.
Results of this prospective study provide evidence of an association of variant CYP2C9 genotype and risk of major hemorrhage in both African-American and European-American patients after accounting for the effects of VKORC1 1173C/T, age, gender, warfarin dose, INR, drug interactions, vitamin K intake, and comorbidity. Compared to patients with the wild-type genotype, patients with the variant CYP2C9 genotype exhibit an increased risk of major hemorrhage, which persists even after stabilization of anticoagulation therapy.