Detection of CYP2C9 polymorphism based on the polymerase chain reaction in Chinese

Abstract

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 Cys144/Arg, Tyr358/Cys, Leu359/Ile, and Gly417/Asp. PCR-endonuclease digestion methods have been developed to detect these four possible polymorphisms. The T416 —>C mutation in exon 3 of CYP2C9 (Cys144 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(Arg144). A Tyr358Cys 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 Tyr358 Ile359. A mismatched PCR primer pair was then designed to detect codon C1061A (Leu359 —> Ile) mutation. In 115 subjects tested, 111 samples showed a complete Nsi I digestion (Ile359) and four samples showed heterozygous results. Another mismatched PCR primer pair was used to confirm the C1061 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 G1236 —>A mutation in exon of CYP2C9 (Gly417 —> Asp) creates a Hph I site. In all 46 subjects,homozygous Gi236 (Gly417) was found. Most Chinese subjects actually haveArg144 Tyr358 Ile359 Gly417 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 Cys144/Arg and Leu359/Ile are the two possible sites showing inter-racial polymorphism.