Evaluation of VKORCL haplotypes in a cohort of patients treated with warfarin / Ebtesam Ali Taeb Beshna

Introduction: Coumarin derivatives such as warfarin are the therapy of choice for the long-term treatment and prevention of thromboembolic events. Warfarin is administered orally as a racemic mixture of two enantiomers. It has very narrow therapeutic windows and large individual variability. The amount of dose needed to achieve therapeutic efficacy varies across the population due to differences in the genetic makeup of individual patients. Polymorphism of the VKORC1 gene is one of the genetic factors that caused altered sensitivity to warfarin or warfarin resistance. Ten common SNPs have been identified, at positions 381, 861, 2653, 3673, 5808, 6009, 6484, 6853, 7566, and 9041 of the VKORC1 reference sequence (Gen Bank accession number AY587020). Five major haplotypes were derived and haplotypes HI and H2 were assigned to group A which requires low warfarin dose while haplotypes H7, H8 and H9 were assigned to group B and require high-warfarin dose. Objective: To develop genotyping method for detection of ten variants of VKORC1 gene and establish the distribution of VKORC1 haplotypes among patients prescribed with warfarin and study the relationship of haplotypes and warfarin dose, INR and bleeding. Method: A total of 156 warfarin patients were recruited after they were screened for inclusion and exclusion criteria. Five ml of blood was drawn from each patient and DNA was extracted for genotyping of VKORC1 variants. Two steps PCR method was developed to identify all the 10 SNPs. Two pairs of primers were designed specifically to detect each of the SNPs and the properties of the primers were checked using Oligo Analyzer software. The optimized method was validated by direct sequencing. Result: Twelve haplotypes were inferred from 10 common SNPs. Four haplotypes HI, H6, H7 and H10 were found to be more frequent in among the patients. The haplotype with the highest frequency was HI (72.9%) followed by H6 (4.2%), H7 (8.9%) and HI0 (4.5%). The most common haplotype pairs was HI-HI (55.1%) followed by H1-H7 (16%), H1-H6 (7.1%) and HIH10 (4.5%). Two haplotype pairs, HI-HI and H1-H10 were associated with a low warfarin dose requirement per day (2.93 mg, 95% CI: 2.70 - 3.16, MLR; P< 0.05 and 3.58 mg, 95% CI: 1.98 - 5.19, MLR; P> 0.01) while H1-H6 and H1-H7 were associated with high dose requirement (4.59 mg, 95% CI: 3.97 -5.22, MLR; P< 0.01 and 5.05 mg, 95% CI: 3.89 - 6.21, MLR; P< 0.01). The average INR was approximately 2.77 and did not differ significantly among the patients classified according to VKORC1 haplotype groups (MLR; P> 0.05). The multiple regression analysis (Enter method) indicated that VKORC1 haplotypes accounted for 28.1% of the variation in warfarin dose at the end of 6 months follow ups. Conclusion: VKORC1 haplotype provided a good correlation for different warfarin dose requirements. Genotyping of patients before warfarin is thus useful to predict more accurate dose required.