Pharmacogenetics of warfarin

Warfarin is one of the most commonly used oral anticoagulants worldwide and is highly efficacious for the treatment and prevention of thromboembolic disorders. However, due to its narrow therapeutic index and large interindividual variability, it remains a challenging drug to prescribe. Genetic factors (CYP2C9 and VKORCI), together with clinical factors (age and body weight), account for up to 60% of warfarin dose variance but the remaining ~40% variability remains unexplained. A polymorphism rs2108622 in CYP4F2, a vitamin K oxidase, has previously been associated with increased warfarin stable dose requirements, accounting for 1-7% dose variability. In our cohort of prospectively recruited patients (n = 311), we were unable to confirm these results. Interestingly, after fine mapping of the CYP4F2 gene region, we found a SNP rs2189784, which is in LD with rs2108622, to be associated with time to therapeutic INR (Pc = 0.03). Further fine mapping of the CYP4F gene cluster together with the utilisation a bank of well characterized Caucasian surgical liver samples (n = 149) and data from a genome-wide association study (n = 714), showed that CYP4F2 rs2108622 and rs2189784 SNPs were found to be associated with increasing CYP4F2 and decreasing CYP4FII or CYP4Fl2 mRNA expression, respectively. Interestingly, a CYP4Fll variant rsl060467 (in LD with rs2108622) was associated with reduced CYP4F2 rnRNA expression. Furthermore, rsl060467 contributes to 2.5% of warfarin dose variability and was associated with reduced warfarin dose requirement (~1 mg/day, Pc = 0.003), an effect in the opposite direction previously reported with CYP4F2 rs2108622 by Caldwell et al. (2008) and other studies. Warfarin-resistant patients have been reported to harbour VKORCI missense mutations. Extended regions of VKORCI were sequenced in our patients (n = 65) with resistance to warfarin, defined by clinical and pharmacodynamic criteria. Seven novel heterozygous mutations were identified and in silica analyses predicted the promoter c.-160G>C mutation creates a putative Spl transcription factor binding site and that the missense mutation c.79C>G to be deleterious. To confirm these predictions, in vitro functional studies were carried out using EMSA, transient transfection assays, and DNA methylation. c.-160G>C was found to create a weak binding site for Spl transcription factor, and caused an increase in promoter activity by ~20% (P = 0.003). The c.79C>G mutation reduced levels of PIVKA-II by ~10%. Associations of VKORCI genotypes with DNA methylation did not remain significant after correction for multiple testing. The effect of warfarin on the rate of decline of vitamin K-dependent clotting factors, and the role of SNPs in the clotting factor genes, is not known. Using a large prospective cohort of patients (n = 619), SNPs in F7 and F 10 genes showed association with variability in factor VII levels. The rate at which the plasma levels of factors II, X and protein C decline affect how patients respond to warfarin, in particular the achievement of warfarin stable dose and time to therapeutic INR. Furthermore, the change in clotting factor X level accounted for 1.4% of warfarin dose variability. In conclusion, the results presented in this thesis demonstrate that multiple genetic, clinical and biochemical factors account for the variability in warfarin response. Further understanding of such complex interactions, along with the advent of genomics technologies and development of new computational and conceptual tools, will yield insights to the accurate prediction of drug efficacy and toxicity, which will hopefully translate into improved outcomes for patients.EThOS - Electronic Theses Online ServiceGBUnited Kingdo