Rapid Detection of CYP2C18 Genotypes by Real-time Fluorescence Polymerase Chain Reaction

Abstract

In man, CYP2C19, a liver enzyme, plays an important role in the metabolism of several drugs. Mutation of the CYP2C19 gene results in a poor metaboliser phenotype. S-Mephenytoin hydroxylation genetic polymorphism is due to two mutations of the CYP2C19 gene, namely CYP2C19*2, located in exon 5, and CYP2C19*3, located in exon 4. CYP2C18 is also polymorphically expressed. The mutant alleles of this enzyme are CYP2C18m1, located in exon 2 and CYP2C18m2, located in the 5'-flanking region. We have developed an allele-specific TaqMan polymerase chain reaction (PCR) assay with which to detect CYP2C18 mutant alleles. This assay combines hybridization of the TaqMan probe and allele-specific amplification primers to the target DNA. The TaqMan probe is labelled with 6-carboxyfluorescein at the 5' end and 6-carboxytetramethylrhodamine together with a phosphate at the 3' end. Genotypes are separated according to the different threshold cycles of the wild type and mutant primers. We applied this procedure to DNA extracted from the blood or saliva of 144 healthy Japanese volunteers. The wt/wt, wt/m1, wt/m2, m1/m1, m1/m2 and m2/m2 genotypes of the CYP2C18 alleles detected by the assay were consistent with the results obtained from restriction enzyme cleavage. In accordance with a previous report, the genotypes of CYP2C18m1 and CYP2C18m2 coincided with those of CYP2C19*3 and CYP2C19*2, respectively. Therefore, detection of CYP2C18 mutant alleles also allows that of CYP2C19 mutant alleles. Among 19 poor metabolisers, eight showed the homozygous CYP2C19*2/CYP2C19*2, two the homozygous CYP2C19*3/CYP2C19*3 and nine the compound heterozygous CYP2C19*2/CYP2C19*3 genotype. We found the allele-specific TaqMan PCR assay rapid, simple and cost-effective, as well as suitable for high-throughput applications in a routine laboratory. This assay allows the fast and reliable detection of inherited disorders that might influence diagnosis and treatment.

The effects of lansoprazole on erosive reflux oesophagitis are influenced by CYP2C19 polymorphism

“…Leucocyte nuclei were prepared from the patient's venous blood. The CYP2C19 genotype was determined by a rapid detection method using real‐time fluorescence polymerase chain reaction 27 . The patients were grouped according to their CYP2C19 genotype.…”

“…Poor metabolizers (PMs), however, have two mutation alleles in both exons 4 and 5. In our study, an allele‐specific TaqMan polymerase chain reaction assay for the detection of point mutations of drug metabolizing enzymes was used to genotype the specimens obtained from all patients 27 …”

Cytochrome P450 2C19 polymorphism influences the preventive effect of lansoprazole on the recurrence of erosive reflux esophagitis

“…Leukocyte nuclei were prepared from venous blood samples and the CYP2C19 genotype was determined by a rapid detection method using real‐time fluorescence polymerase chain reaction (PCR) 21 . This method used an allele‐specific TaqMan PCR (Applied Biosystems, CA, USA) to detect point mutations of drug metabolizing enzymes.…”

“…Leukocyte nuclei were prepared from venous blood samples and the CYP2C19 genotype was determined by a rapid detection method using real-time fluorescence polymerase chain reaction (PCR). 21 This method used an allele-specific TaqMan PCR (Applied Biosystems, CA, USA) to detect point mutations of drug metabolizing enzymes. The patients were grouped according to CYP2C19 genotype as follows: homo-EM, wild-type alleles in both exons, 4 and 5 of the CYP2C19 gene; hetero-EM, mutation of one allele in either exon 4 or exon 5; or PM, mutation of two alleles in both exons 4 and 5.…”

Recommendations from the EGAPP Working Group: testing for cytochrome P450 polymorphisms in adults with nonpsychotic depression treated with selective serotonin reuptake inhibitors

“…For CYP2D6 and CYP2C19 polymorphisms, nine published articles and two FDA summaries reported on performance of genotyping methods 3,4,[23][24][25][26][27][28][29][30][31] ; only three provided a comparison to the gold standard of DNA sequencing. 3,4,25 • Analytic sensitivity (how effectively the test identifies specific polymorphisms that are present in a sample) was high for all common polymorphisms, ranging from 94% to 100%.…”