Identification of a novel CYP2C9 gene mutation (1400T>C) and assessment of its enzymatic activity in vitro

Abstract

To perform a pharmacogenetic study of a patient with warfarin hypersensitivity and determine the biological function of a novel CYP2C9 mutation. Genomic DNAs were extracted from the warfarin hypersensitive patient and used for genetic screening of CYP2C9, VKORC1 and CYP4F2 by direct sequencing. Acquired sequences were aligned and blasted with public nucleotide database to clarify whether site mutations were present in these 3 genes. Full-length cDNA fragments of CYP2C9*2, *3, *13 and 1400T>C were obtained by polymerase chain reaction (PCR) site-directed mutagenesis and used for the insect expression vector construction. According to the manufacturer's instruction, insect cell microsomes expressing 5 CYP2C9 variants were obtained with Bac-to-Bac Baculovirus Expression System. Then CYP2C9 protein expression level for each variant was quantified by Western blot and tolbutamide used as a probing substrate for assessing the in vitro metabolic characteristics of each CYP2C9 variant. Vmax and Km values were calculated and used for evaluating the biological impact of novel 1400T>C mutant. One novel nucleotide mutation 1400T>C was detected in DNAs of tested patient. And this site mutation resulted in one amino acid change at position 467 of CYP2C9 protein (L467P). Other two candidate genes VKORC1 and CYP4F2 were found to be the wild-type alleles. When expressed in insect cell microsomes, the relative clearance values of CYP2C9 *2, *3, *13 and L467P variants to tolbutamide were 91.58%, 13.55%, 0.11% and 1.59% to that of wild-type protein respectively. 1400T>C mutation of CYP2C9 gene greatly reduces the in vitro enzymatic activity of CYP2C9. While taking the drugs metabolized by CYP2C9, the patients carrying this mutated allele should take more cautions because their metabolic rates are slower than those of wild-type carriers. Ensuing drug accumulation in vivo may cause potentially serious adverse reactions.