Simultaneous genotyping of CYP2C9*2, *3, and VKORC1 polymorphisms in a Italian population sample using a new minisequencing multiplex single-base extension analysis

Sixty six years since its discovery and fifty-four years since its commercial introduction in 1954, warfarin remains the mainstay of oral anticoagulant therapy. Despite proven efficacy, refinements and standardization in the monitoring of warfarin, its underutilization is widespread. What is clear is that the capricious nature of the response to any given dose of warfarin demands sophisticated and resource intensive methods to determine the warfarin dose and maintain therapeutic anticoagulation. The intricacies of warfarin metabolism, the complexities of the coagulation system and the multitude of factors affecting level of anticoagulation do not make this a straight forward task. Individual responses to warfarin therapy can greatly differentiate among individuals, and these differing responses have a genetic basis that in part has been recognized. Pharmacogenetics is the study of genetic differences and their effects on drug metabolism. The application of pharmacogenetics in identifying individuals with the polymorphisms that produce various responses in patients and the concomitant adjustment of their warfarin dose is expected to confer substantial benefits to patients and to healthcare system overall. The evolution of our understanding of warfarin pharmacodynamics and pharmacokinetics and the recognition of genetic regulation of warfarin response has stimulated efforts aimed at quantifying this influence. Although investigations have identified the influence of several genes on warfarin response, the bulk of the evidence supports the influence polymorphisms in two genes; Cytochrome P450 2C9 (CYP2C9) and Vitamin K epoxide reductase complex 1 (VKORC1) contribute approximately 60% to genetic differences between individuals. Genetics variations in cytochrome P-450, family 2, sub family C, polipeptide 9 (CYP2C9) and vitamin K epoxide reductase complex, subunit 1 (VKORC1) genes have been shown to contribute to impaired metabolism of warfarin. The genetically polymorphic enzyme cytochrome P450 2C9 (CYP2C9), which belongs to the superfamily of cytochrome P-450, influences the metabolism of a wide variety of clinically important drugs, including phenytoin, tolbutamide, warfarin, losartan, and many non-steroidal anti-inflammatory drugs. Point mutations causing single nucleotide polymorphisms (SNPs) in the CYP2C9 gene result in the expression of three main variants: CYP2C9*1 (wild type) CYP2C9*2 (C430T), and CYP2C9*3 (A1075C). Both CYP2C9*2, in which cysteine substitutes for arginine at amino acid residue 144 in the protein, and CYP2C9*3, in which leucine substitutes for isoleucine at amino acid residue 359, exhibit altered catalytic properties compared with the wild-type enzyme: CYP2C9*2 has about 12% of the activity of the wild-type enzyme, whereas the CYP2C9*3 variant has less than 5% of its activity. The allele frequency of CYP2C9*2 in Caucasian populations is about 11% and that of CYP2C9 *3 is about 7%. Although the influence of several VKORC1 polymorphisms (3730G/A, 2255C/T, 1542G/C, 1173C/T and -1639G/A on warfarin response has been investigated, 1173C/T have been the most widely studied [7]. We present here a sperimental study that was carried out to determine the prevalence of relevant CYP2C9 and VKORC polymorphisms in a sample of Italian population, rapresented by 100 individuals living in Ferrara and under oral anticoagulant therapy at the Sant’Anna Hospital. We used a new minisequencing single-base extension (SBE) fluorescent method through the application of multiplex SBE analysis. This SBE technique consists of a modified version of a commercially available fluorescent primer extension analysis called SNaPshot, which accurately genotypes multiple SNPs simultaneously in the same reaction with minimum optimization.