Identification and Enzymatic Activity Evaluation of a Novel CYP2C9 Allelic Variant Discovered in a Patient

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.

Role of Cytochrome P450 2C9 in COVID-19 Treatment: Current Status and Future Directions

“…CYP2C9 is a highly polymorphic enzyme, with 62 allelic variants identified and named thus far by the PharmVar Consortium, and the recently identified novel CYP2C9 variant (I213V) [ 63 ]. The most well-studied CYP2C9 alleles are CYP2C9*2 (rs1799853) and CYP2C9*3 (rs1057910), associated with the reduced metabolism of CYP2C9 substrates in vivo, such as warfarin and phenytoin [ 64 ].…”

Pharmacogenetic Perspective for Optimal Gout Management

“…The CYP2C9 is a phase-I cytochrome P450 drugmetabolizing enzyme isoform involved in oxidizing endogenous and exogenous compounds. The CYP2C9 is a major metabolizing enzyme accounting for more than 25% of NSAIDs metabolic clearance and commonly prescribed drugs [81]. In vivo, losartan [82], diclofenac, flurbiprofen, and tolbutamide are commonly utilized as probe drugs for CYP2C9 phenotyping assays [83][84][85].…”