DETECTION OF CYP2C9 POLYMORPHISM BASED ON THE POLYMERASE CHAIN-REACTION IN CHINESE

Cytochrome P450 (CYP) 2C9 catalyses the metabolism of a wide range of drugs. Previous studies have shown the differences in the amino acid composition among CYP2C9 variants at Cys(144)/Arg, Tyr(358)/Cys, Leu(359)/Ile, and Gly(417)/Asp. PCR-endonulclease digestion methods have been developed to detect these four possible polymorphisms. The T-416 --> C mutation in exon 3 of CYP2C9 (Cys(144) --> Arg) creates an Ava II site, In the 135 subjects we tested, all leukocyte DNA samples showed a complete Ava II digestion indicating homozygous C-416 (Arg(144)). A Tyr(358) --> Cys mutation will create a Nsi I site at codon 1057-1063 in exon 7. In 40 subjects tested, all samples-showed negative results. DNA sequencing on a few samples showed Tyr(358)lle(359). A mismatched PCR primer pair was then designed to detect codon C-1061 --> A (Leu(359) --> Ile) mutation. In 115 subjects tested, 111 samples showed a complete Nsi I digestion (Ile(359)) and four samples showed heterozygous results. Another mismatched PCR primer pair was used to confirm the C-1061 codon in heterozygous subjects. The four heterozygous subjects showed partial digestion with endonuclease Kpn I, which confirmed the heterozygous Ile/Leu at amino acid 359. The G(1236) --> A mutation in exon 8 of CYP2C9 (Gly(417) --> Asp) creates a Hph I site. In all 46 subjects, homozygous G(1236) (Gly(417)) was found, Most Chinese subjects actually have Arg(144) Tyr(358) Ile(359) Gly(417) in CYP2C9 as previously reported human-2. Furthermore, we found an A --> T (+12 position in intron 2) mutation in our CYP2C9 sequencing process. The mutation creates a Nla III site. In the 44 subjects we tested, all showed complete digestion, In conclusion, we found small inter-individual Variation in CYP2C9 sequence among Chinese subjects, and that Cys(144)/Arg and Leu(359)/Ile are the two possible sites showing inter-racial polymorphism.