A novel VKORC1 promoter mutation found causing warfarin resistance, along with –1639G>A promoter mutation—A pilot study on the genetic variation in patients on warfarin therapy in South India

Abstract

Warfarin is a commonly prescribed oral anticoagulant used for prophylaxis and treatment of deep vein thrombosis, myocardial infarction, heart valve replacement, pulmonary embolisms, and other thromboembolic disorders. Because overdosing of warfarin is fatal to patients and only a few studies are available on the Indian population, the present study was undertaken to develop genotyping assays for the monitoring of patients undergoing warfarin therapy specific to the Indian population. Warfarin dosing is correlated with polymorphisms in VKORC1 (vitamin K epoxide reductase complex 1) and CYP2C9 (cytochrome P450 family 2, subfamily C, polypeptide 9) genes. Hence, this study was undertaken to assess the impact of these genetic variations (SNPs) in VKORC1 and CYP2C9 genes of Indian patients on warfarin therapy. In the present study, genomic DNA samples from 136 individuals (patients on stabilized warfarin therapy) were analyzed through polymerase chain reaction (PCR) and DNA sequencing. Furthermore, the observed SNPs were correlated with the dosage pattern in order to understand the genotype–phenotype correlation significance. Additionally, an amplification refractory mutation system PCR-based genotyping assay was developed for the VKORC1 –1639G>A allele, as a rapid and cost-effective detection tool. The analysis of samples from warfarin-sensitive patients showed that 84.78% of participants had mutant alleles in either the CYP2C9 or the VKORC1 gene. A novel mutation with an insertion of G at 3725 position (Ins-G –1586 with respect to the start codon) in the promoter region of the VKORC1 gene—along with the VKORC1 –1639G>A allele—was observed in four patients, all of whom were on a higher dosage of warfarin (>7 mg/d). Our results clearly indicate that there is a variation in the dosage pattern associated with the VKORC1 –1639G>A genotype in the presence of this novel promoter insertion, further suggesting the need for large-scale studies to be conducted on Indian populations for the validation of warfarin sensitivity tests.