Abstract
Warfarin is a widely prescribed anticoagulant but the doses required to attain the optimum therapeutic effect exhibit dramatic inter-individual variability. Pharmacogenomics-guided warfarin dosing has been recommended to improve safety and effectiveness. We analyzed the cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes among 120 patients taking warfarin. A new coding variant was identified by sequencing CYP2C9. The novel A > G mutation at nucleotide position 14,277 led to an amino acid substitution of isoleucine with valine at position 213 (I213V). The functional consequence of the variant was subsequently evaluated in vitro. cDNA of the novel variant was constructed by site-directed mutagenesis and the recombinant protein was expressed in vitro using a baculovirus–insect cell expression system. The recombinant protein expression was quantified at apoprotein and holoprotein levels. Its enzymatic activities toward tolbutamide, warfarin and losartan were then assessed. It exhibited changed apparent Km values and increases of 148%, 84% and 67% in the intrinsic clearance of tolbutamide, warfarin and losartan, respectively, compared to wild-type CYP2C9*1, indicating dramatically enhanced in vitro enzymatic activity. Our study suggests that the amino acid at position 213 in wild-type CYP2C9*1 may be important for the enzymatic activity of CYP2C9 toward tolbutamide, warfarin and losartan. In summary, a patient taking high-dose warfarin (6.0 mg/day) in order to achieve the target international normalized ratio was found to have a mutation in the CYP2C9 gene.
Highlights
Warfarin is the most frequently prescribed oral coumarin-derived anticoagulant drug for the prevention and treatment of arterial and venous thromboembolism
The data for the study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB40955
We found a novel mutation in exon four of the cytochrome P450 2C9 (CYP2C9) gene in a patient taking warfarin
Summary
Warfarin is the most frequently prescribed oral coumarin-derived anticoagulant drug for the prevention and treatment of arterial and venous thromboembolism It has a narrow therapeutic index and high inter-patient variability regarding the dose required to achieve the target international normalized ratio (INR) of 2–3 for most indications (Ageno et al, 2012). In 2007, the United States Food and Drug Administration (FDA) updated the warfarin labeling to include information on its pharmacogenomics for the first time (Gage and Lesko, 2008), allowing the therapeutic warfarin dose to be estimated by using pharmacogenomics data on cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) single-nucleotide polymorphisms Statements from both the US FDA and the Clinical Pharmacogenetics Implementation Consortium (CPIC) further confirmed the importance of genetic factors when determining optimum warfarin dosing (Klein et al, 2009; Johnson et al, 2017; Drozda et al, 2018). VKORC1 and CYP2C9 genotyping has been routinely included in pharmacogenomic algorithms for warfarin dosing, along with the two revisions of the warfarin labeling regarding pharmacogenomics
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