A rapid and simple detection of genetic defects responsible for the phenotypic polymorphism of cytochrome P450 2C19

Abstract

A rapid and simple cytochrome P450 (CYP) 2C19 genotyping system was established by making several modifications in previously reported procedures. PCR conditions were modified to be capable of simultaneous amplification of CYP2C19m1 and CYP2C19m2 regions. Intensive bands of 169 bp for the CYP2C19m1 region and 329 bp for the CYP2C19m2 region with low background were obtained by using PCR condition involving an initial denaturation of 5 min at 94°C, 35 cycles of 1 min at 94°C; 1 min at 53°C; 1 min at 72°C, and final extension of 5 min at 72°C. Next, the optimal restriction enzyme digestion conditions were determined by using PCR products from a subject of wt/wt genotype. Both products were completely digested with 5 U of the corresponding enzyme (Sma I for CYP2C19m1 and Bam HI for CYP2C19m2) for 1 h incubation at an optimal temperature. The incidence (16%; 5/32) of subjects homozygous for mutant alleles determined by an established assay system agreed well with the incidence of the poor metabolizer (PM) phenotype in the Japanese population. The established genotyping system would, therefore, be applicable to the clinical laboratory testing of patients with a PM phenotype of CYP2C19 to select appropriate and effective medication.