The Activities Of Human CYP2C19 And CYP2D6 Two Novel Nonsynonymous Variant In Vitro And The Genotyping Of CYP2C19 Two Important Alleles

Cytochrome P450 (Cytochrome P450, CYP450) is the major enzyme responsible for the metabolism of the exnobitics in human body, and catalyze many kinds of biotransformations, mostly oxidations, such as metabolism the most of the drugs, active the carcinogen, clear the toxicant. CYP2C19 and CYP2D6 belong to CYP450 superfamily, which also perform an important function in the metabolism of xenobiotics and drugs. CYP2C19*2 and CYP2C19*3 account for 99% of the defective alleles in Oriental poor metabolizers (PMs). Study the polymorphisms of CYP450s have many significances, such as provide valuable data for studying the effects of polymorphic genes and predict in vivo drug-drug interaction and accelerate the development of clinical individualized medicine.In this study, we first identified the enzyme catalytic activity of two novel nonsynonymous variant including CYP2C19_T302R (cDNA:905C＞G) and CYP2D6_R441H (gDNA:4045T>C) which were systematically screened the polymorphisms of the whole CYP2C19 and CYP2D6 gene in the populations of four different geographical locations in China, namely, Shanghai, Shantou, Shenyang, and Xi'an, using a sample of 100 subjects from each population in the year 2008. CYP2C19, CYP2D6 wild types and their mutations were obtained using recombinant method in vitro, and electrotransformed into yeast Saccharomyces cerevisiaee, expressed protein through galactose induction and then identified the catalytic activity of these recombinant enzymes, and analyze the influence of SNP to the enzyme catalytic activity. Secondly, we use the traditional method PCR-RFLP to detect the variant frequency of CYP2C19*2 and CYP2C19*3 which were the highest variant frequency in Chinese. We analysized the variant frequency of seventy-four samples. Our study can help to give medication instruction to the patients whose genotype was CYP2C19*2 or CYP2C19*3 when they are ready to take the medicine which was mainly metabolized by CYP2C19.In order to analyze the influence of these two SNPs to the enzyme activity, we introduced the mutant into the wild type CYP2C19 and CYP2D6 gene by PCR mutagenesis method. And the cDNAs of CYP2C19 wildtype and CYP2D6 wildtype were ready-made. We use the cDNA of WT as template to introduce polymorphisms CYP2C19_T302R and CYP2D6_R441H by site-directed mutagenesis and were cloned into the same vector pYES2/CT. Transformed yeasts produced plentiful of recombinant enzymes which were validated by Western-blot.We used fluorogenic substrate AMMC (for CYP2D6) and CEC (for CYP2C19) to detect the kinetic constants of enzymes in real-time assays. The other reaction were measured by HPLC for precise detection, using specificity drug probe substrate S-mephenytion for CYP2C19 and bufuralol for CYP2D6.The results showed that two SNPs enzyme catalytic activity were notable discrepancy compared with the wild type when their amino acids were alterd. Not only fluorogenic detection but also HPLC experiment both showed T302R almost lose its all activity and R441H was catalytically inactive. We used the traditional method PCR-RFLP to detect the variant frequency of CYP2C19*2 and CYP2C19*3 in seventy-four Beijing samples. The results showed that the frequency of CYP2C19*2 and CYP2C19*3 were approximately the same as the pre-report literature, CYP2C19*2 account 30.4% and CYP2C19*3 account 6.8%.