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 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 C416 (Arg144). A Tyr358-->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 Tyr358Ile359. A mismatched PCR primer pair was then designed to detect codon C1061-->A (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 8 of CYP2C9 (Gly417-->Asp) creates a Hph I site. In all 46 subjects, homozygous G1236 (Gly417) was found. Most Chinese subjects actually have Arg144 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 NIa III site.(ABSTRACT TRUNCATED AT 250 WORDS)
Wang
S L
SL
Department of Pharmacology, National Cheng Kung University, Medical College, Taiwan, Republic of China.
Huang
J
J
Lai
M D
MD
Tsai
J J
JJ
D000483:Alleles; D001189:Aryl Hydrocarbon Hydroxylases; D044466:Asian People / Q000235:genetics; D001483:Base Sequence; D002681:China; D065729:Cytochrome P-450 CYP2C9; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D005091:Exons; D014644:Genetic Variation; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D039181:Steroid 16-alpha-Hydroxylase; D013624:Taiwan
D017931:DNA Primers; D003577:Cytochrome P-450 Enzyme System; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D039181:Steroid 16-alpha-Hydroxylase