Rapid detection of CYP2C18 genotypes by real-time fluorescence polymerase chain reaction. 2000

M Mizugaki, and M Hiratsuka, and Y Agatsuma, and Y Matsubara, and K Fujii, and S Kure, and K Narisawa
Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan. mizugaki@mail.cc.tohoku.ac.jp

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.

UI MeSH Term Description Entries
D003577 Cytochrome P-450 Enzyme System A superfamily of hundreds of closely related HEMEPROTEINS found throughout the phylogenetic spectrum, from animals, plants, fungi, to bacteria. They include numerous complex monooxygenases (MIXED FUNCTION OXYGENASES). In animals, these P-450 enzymes serve two major functions: (1) biosynthesis of steroids, fatty acids, and bile acids; (2) metabolism of endogenous and a wide variety of exogenous substrates, such as toxins and drugs (BIOTRANSFORMATION). They are classified, according to their sequence similarities rather than functions, into CYP gene families (>40% homology) and subfamilies (>59% homology). For example, enzymes from the CYP1, CYP2, and CYP3 gene families are responsible for most drug metabolism. Cytochrome P-450,Cytochrome P-450 Enzyme,Cytochrome P-450-Dependent Monooxygenase,P-450 Enzyme,P450 Enzyme,CYP450 Family,CYP450 Superfamily,Cytochrome P-450 Enzymes,Cytochrome P-450 Families,Cytochrome P-450 Monooxygenase,Cytochrome P-450 Oxygenase,Cytochrome P-450 Superfamily,Cytochrome P450,Cytochrome P450 Superfamily,Cytochrome p450 Families,P-450 Enzymes,P450 Enzymes,Cytochrome P 450,Cytochrome P 450 Dependent Monooxygenase,Cytochrome P 450 Enzyme,Cytochrome P 450 Enzyme System,Cytochrome P 450 Enzymes,Cytochrome P 450 Families,Cytochrome P 450 Monooxygenase,Cytochrome P 450 Oxygenase,Cytochrome P 450 Superfamily,Enzyme, Cytochrome P-450,Enzyme, P-450,Enzyme, P450,Enzymes, Cytochrome P-450,Enzymes, P-450,Enzymes, P450,Monooxygenase, Cytochrome P-450,Monooxygenase, Cytochrome P-450-Dependent,P 450 Enzyme,P 450 Enzymes,P-450 Enzyme, Cytochrome,P-450 Enzymes, Cytochrome,Superfamily, CYP450,Superfamily, Cytochrome P-450,Superfamily, Cytochrome P450
D004252 DNA Mutational Analysis Biochemical identification of mutational changes in a nucleotide sequence. Mutational Analysis, DNA,Analysis, DNA Mutational,Analyses, DNA Mutational,DNA Mutational Analyses,Mutational Analyses, DNA
D005838 Genotype The genetic constitution of the individual, comprising the ALLELES present at each GENETIC LOCUS. Genogroup,Genogroups,Genotypes
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001189 Aryl Hydrocarbon Hydroxylases A large group of cytochrome P-450 (heme-thiolate) monooxygenases that complex with NAD(P)H-FLAVIN OXIDOREDUCTASE in numerous mixed-function oxidations of aromatic compounds. They catalyze hydroxylation of a broad spectrum of substrates and are important in the metabolism of steroids, drugs, and toxins such as PHENOBARBITAL, carcinogens, and insecticides. Microsomal Monooxygenases,Xenobiotic Monooxygenases,Hydroxylases, Aryl Hydrocarbon,Monooxygenases, Microsomal,Monooxygenases, Xenobiotic
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D016133 Polymerase Chain Reaction In vitro method for producing large amounts of specific DNA or RNA fragments of defined length and sequence from small amounts of short oligonucleotide flanking sequences (primers). The essential steps include thermal denaturation of the double-stranded target molecules, annealing of the primers to their complementary sequences, and extension of the annealed primers by enzymatic synthesis with DNA polymerase. The reaction is efficient, specific, and extremely sensitive. Uses for the reaction include disease diagnosis, detection of difficult-to-isolate pathogens, mutation analysis, genetic testing, DNA sequencing, and analyzing evolutionary relationships. Anchored PCR,Inverse PCR,Nested PCR,PCR,Anchored Polymerase Chain Reaction,Inverse Polymerase Chain Reaction,Nested Polymerase Chain Reaction,PCR, Anchored,PCR, Inverse,PCR, Nested,Polymerase Chain Reactions,Reaction, Polymerase Chain,Reactions, Polymerase Chain

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