Characterization of the VKORC1 -1639G&gt;A (rs9923231) polymorphism-associated genotypes v1
Authors: Causevic, Honours BSc, PhD Mirsada 
Published: October 2023

The procoagulant factors (FII, FVII, FIX, FX) and anticoagulant factors (proteins C and S), which play an important role in blood coagulation, are vitamin K-dependent factors. That means that their activation depends on the optimum level of the reduced form of vitamin K in the human body. The formation of the reduced form of vitamin K is controlled by the enzyme called vitamin K epoxide reductase (VKOR), which transforms the oxidized form of vitamin K, the vitamin K epoxide, into the reduced form, the vitamin K hydroquinone. Then, the vitamin K hydroquinone functions as a cofactor in the activation of blood factors which are essential for blood clotting. The VKOR is the enzyme which represents the molecular target of warfarin and other coumarin-type anticoagulants, also called vitamin K antagonists (VKAs). They inhibit the VKOR enzyme thereby indirectly reducing the levels of the functional clotting factors and, thus, achieving appropriate anticoagulation. The VKOR enzyme is encoded by the vitamin K epoxide reductase complex subunit 1 (VKORC1) gene, which is a polymorphic gene. A non-coding, single nucleotide polymorphism (SNP), in the promoter region of the VKORC1 gene, regulates the transcription of the VKORC1 and, hence, the translation and the expression level of the VKOR protein/enzyme. This, in turn, affects the level of the functional clotting factors, in an individual's body, which has consequences for an individual's endogenous, physiological thrombogenicity or antithrombosis, as well as his/her sensitivity to anticoagulant drugs. Many studies have demonstrated an association between a gene sequence in the promoter region of the VKORC1 gene (genotype) and the resulting levels of the VKOR enzyme and functional clotting factors (phenotype) - GG or VKORC1 -1639GG genotype: the presence of the wild-type sequence or GG genotype, in an individual, results in high or standard level of the VKOR enzyme, high or standard level of the functional clotting factors, high or standard endogenous thrombogenicity, which requires high or standard dosing of an anticoagulant drug, e.g. warfarin; GA or VKORC1 -1639GA genotype: the presence of the heterozygous, variant sequence or GA genotype results in an intermediate level of the VKOR enzyme, intermediate level of the functional clotting factors, intermediate endogenous thrombogenicity, which may require reduced dose of an anticoagulant drug; and AA or VKORC1 -1639AA genotype: the presence of the homozygous, variant sequence or AA genotype results in low level of the VKOR enzyme, low level of the functional clotting factors, low level of endogenous thrombogenicity, which requires low dose of an anticoagulant drug. This protocol provides a detailed, step-by-step guide to characterization of the VKORC1 -1639G>A (rs9923231) polymorphism-associated genotypes by using a previously published protocol by Elizabeth A. Sconce and colleagues (DOI: 10.1182/blood-2005-03-1108), with modifications. It utilizes polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, followed by direct, Sanger sequencing.