Yasuyuki Agatsuma

Allele and genotype frequencies of CYP2B6 and CYP3A5 in the Japanese population.

HeadingAbstractObjective. The goal of this study was to determine the frequencies of allelic variants of CYP2B6 and CYP3A5 in the Japanese population.Methods. Genotyping of CYP2B6(*2, *3, *4, *5, *6, and *7) and CYP3A5 (*2, *3, *4, *5, and *6) was carried out in 265 unrelated Japanese subjects by polymerase chain reaction (PCR), restriction fragment length polymorphism and allele-specific, real-time PCR assays.Results. Allele frequencies for CYP2B6*2, *3, *4, *5, *6, and *7 in 256 Japanese subjects were 0.047, 0, 0.093, 0.011, 0.164, and 0, respectively. Ethnic variation in allele frequencies relative to that in Caucasian subjects was observed for CYP2B6*4 (0.093 vs 0.040), *5 (0.011 vs 0.109), *6 (0.164 vs 0.256), and *7 (0 vs 0.030). Allele frequencies for CYP3A5*2, *3, *4, *5, and *6 in 265 Japanese subjects were 0, 0.740, 0, 0.004, and 0, respectively. The frequency of the CYP3A5*1 allele is 2.8 times higher in Japanese than in Caucasians.Conclusions. Our results contribute to a better understanding of the molecular basis of ethnic differences in drug response, which may help to improve individualization of drug therapy and offer a preliminary basis for more rational use of drugs that are substrates for CYP2B6 and CYP3A5 in the Japanese population.

Genetic analysis of thiopurine methyltransferase polymorphism in a Japanese population

Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurine drugs such as 6-mercaptopurine, 6-thioguanine, and azathiopurine. Several mutations in the TPMT gene have been identified which correlate with a low activity phenotype. The molecular basis for the genetic polymorphism of TPMT has been established for European Caucasians, African-Americans, Southwest Asians and Chinese, but it remains to be elucidated in Japanese populations. The frequency of the four allelic variants of the TPMT gene, TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A) and TPMT*3C (A719G) were determined in Japanese samples (n=192) using polymerase chain reaction (PCR)-RFLP and allele-specific PCR-based assays. TPMT*3C was found in 0.8% of the samples (three heterozygotes). The TPMT*2, TPMT*3A and TPMT*3B alleles were not detected in any of the samples analyzed. This study provides the first analysis of TPMT mutant allele frequency in a sample of Japanese population and indicates that TPMT*3C is the most common allele in Japanese subjects.

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

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.

A simultaneous LightCycler detection assay for five genetic polymorphisms influencing drug sensitivity.

OBJECTIVES The routine detection of polymorphisms affecting drug sensitivity in patients before treatment is important in the identification of drug responders or nonresponders, and patients at increased risk of drug toxicity. Here, we present an assay for the simultaneous and rapid genotyping of five polymorphisms influencing drug sensitivity. DESIGN AND METHODS We used a hybridization probe assay on the LightCycler to detect five single nucleotide polymorphisms (SNPs): INPP1 (973C>A), ADRB2 (R16G and Q27E), HTR2A (102T>C), and mtDNA (1555A>G). Two fluorescent labeled hybridization probes were designed for the simultaneous detection of the five SNPs and detection of the variant alleles was performed by melting curve analysis. RESULTS All five SNPs were detected with a single thermocycle protocol within 40 min. The genotypes determined in this assay were identical to those obtained with conventional PCR and restriction fragment length polymorphism analysis. CONCLUSIONS To our knowledge, we report here for the first time a method for simultaneous detection of five SNPs, on a single thermocycle protocol by the LightCycler. This method is rapid, highly sensitive, and high-throughput, and is thus suitable for routine clinical use and large-scale epidemiologic studies.