Single nucleotide polymorphisms and haplotypes of CYP1A2 in a Japanese population.

In order to identify genetic polymorphisms and haplotype frequencies of CYP1A2 in a Japanese population, the enhancer and promoter regions, all the exons with their surrounding introns, and intron 1 were sequenced from genomic DNA from 250 Japanese subjects. Thirty-three polymorphisms were found, including 13 novel ones: 2 in the enhancer region, 5 in the exons, and 6 in the introns. The most common single nucleotide polymorphism (SNP) was -163C>A (CYP1A2*1F allele) with a 0.628 frequency. In addition to six previously reported non-synonymous SNPs, three novel ones, 125C>G (P42R, CYP1A2*15 allele, MPJ6_1A2032), 1130G>A (R377Q, *16 allele, MPJ6_1A2033), and 1367G>A (R456H, *8 allele, MPJ6_1A2019), were found with frequencies of 0.002, 0.002, and 0.004, respectively. No polymorphism was found in the known nuclear transcriptional factor-binding sites in the enhancer region. Based on linkage disequilibrium analysis, the CYP1A2 gene was analyzed as one haplotype block. Using the 33 detected polymorphisms, 14 haplotypes were unambiguously identified, and 17 haplotypes were inferred by aid of an expectation-maximization-based program. Among them, the second major haplotype CYP1A2*1L is composed of -3860G>A (*1C allele), -2467delT (*1D allele), and -163C>A (*1F allele). Network analysis suggested that relatively rare haplotypes were derived from three major haplotypes, *1A, *1M, and *1N in most cases. Our findings provide fundamental and useful information for genotyping CYP1A2 in the Japanese, and probably Asian populations.

Haplotype structures of EPHX1 and their effects on the metabolism of carbamazepine-10,11-epoxide in Japanese epileptic patients.

OBJECTIVE: Microsomal epoxide hydrolase (mEH) is an enzyme that detoxifies reactive epoxides and catalyzes the biotransformation of carbamazepine-10,11-epoxide (CBZ-epoxide) to carbamazepine-10,11-diol (CBZ-diol). Utilizing single nucleotide polymorphisms (SNPs) of the EPHX1 gene encoding mEH, we identified the haplotypes of EPHX1 blocks and investigated the association between the block haplotypes and CBZ-epoxide metabolism. METHODS: SNPs of EPHX1 were analyzed by means of polymerase chain reaction amplification and DNA sequencing using DNA extracted from the blood leukocytes of 96 Japanese epileptic patients, including 58 carbamazepine-administered patients. The plasma concentrations of CBZ and its four metabolites were determined using high-performance liquid chromatography. RESULTS: From sequencing all 9 exons and their surrounding introns, 29 SNPs were found in EPHX1. The SNPs were separated into three blocks on the basis of linkage disequilibrium, and the block haplotype combinations (diplotypes) were assigned. Using plasma CBZ-diol/CBZ-epoxide ratios (diol/epoxide ratios) indicative of the mEH activity, the effects of the diplotypes in each EPHX1 block were analyzed on CBZ-epoxide metabolism. In block 2, the diol/epoxide ratios increased significantly depending on the number of haplotype *2 bearing Y113H (P=0.0241). In block 3, the ratios decreased depending on the number of haplotype *2 bearing H139R (P=0.0351). Also, an increasing effect of a *1 subtype, *1c, was observed on the ratio. CONCLUSION: These results show that some EPHX1 haplotypes are associated with altered CBZ-epoxide metabolism. This is the first report on the haplotype structures of EPHX1 and their potential in vivo effects.

Six novel nonsynonymous CYP1A2 gene polymorphisms: catalytic activities of the naturally occurring variant enzymes.

Six novel nonsynonymous nucleotide alterations were found in the cytochrome P450 1A2 gene in a Japanese population, which resulted in the following amino acid substitutions: T83M, E168Q, F186L, S212C, G299A, and T438I. These individuals were heterozygous for the amino acid substitutions. The potential functional alterations caused by the amino acid substitutions were characterized by a cDNA-mediated expression system using Chinese hamster V79 cells. Among the six CYP1A2 variants, F186L showed the most profound and statistically significant reduction in O-deethylation of phenacetin and 7-ethoxyresorufin. Kinetic analyses performed for the ethoxyresorufin O-deethylation revealed that the Vmax of the F186L variant was approximately 5% of that of the CYP1A2 wild type, despite a 5-fold lower Km value of the variant, the consequence of which was reduced enzymatic activity toward the substrate. Thus, for the first time, phenylalanine at residue 186 is suggested to be a critical amino acid for catalytic activity.

Five novel single nucleotide polymorphisms in the EPHX1 gene encoding microsomal epoxide hydrolase.

Five novel single nucleotide polymorphisms (SNPs) were found in the EPHX1 gene from 96 Japanese epileptic patients. The detected SNPs were as follows: 1) SNP, MPJ6_EX1009; GENE NAME, EPHX1 ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-CCTCACTTCAGTG/ACTGGGCTTTGCC-3'. 2) SNP, MPJ6_EX1013; GENE NAME, EPHX1; ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-TCCGCAGCCAGGG/CAGGACGACAGCA-3'. 3) SNP, MPJ6_EX1026; GENE NAME, EPHX1; ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-GTTCTCCCTGGAC/TGACCTGCTGACC-3'. 4) SNP, MPJ6_EX1028; GENE NAME, EPHX1; ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-AGGCAGGGGGACG/AGCCAGTCTTGGG-3'. 5) SNP, MPJ6_EX1030; GENE NAME, EPHX1; ACCESSION NUMBER, NT_004525.12; LENGTH, 25 bases; 5'-TGAAAAGTGGGTG/AAGGTTCAAGTAC-3'. The frequencies were 0.016 for MPJ6_EX1028 (IVS8+54G>A) and 0.005 for the other SNPs. The SNP MPJ6_EX1013 (130G>C) results in an amino acid alteration (E44Q). The other three SNPs in the coding region, MPJ6_EX1009 (30G>A), MPJ6_EX1026 (1056C>T), and MPJ6_EX1030 (1239G>A) result in synonymous changes (V10V, D352D, and V413V, respectively).

Eleven novel single nucleotide polymorphisms in the NR1I2 (PXR) gene, four of which induce non-synonymous amino acid alterations.

Eleven novel single nucleotide polymorphisms (SNPs) were found in the NR1I2 (PXR/SXR) gene from 205 Japanese subjects. The detected SNPs were as follows: 1) SNP, MPJ6_1I2001; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-TTTCTACCTCTAC/TTATTGAAAGGGC-3'. 2) SNP, MPJ6_1I2004; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-AGGCCCAAATGTG/AAGTGATGCATAG-3'. 3) SNP, MPJ6_1I2007; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-TGCCAGGCCTGCC/TGCCTGCGCAAGT-3'. 4) SNP, MPJ6_1I2008; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-GAGTGAGCAGTGG/CGCGCGCGGGCGG-3'. 5) SNP, MPJ6_1I2010; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-CAGAGGAGCAGCG/AGATGATGATCAG-3'. 6) SNP, MPJ6_1I2011; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-CTGGAAGTGGCCA/GGGAGGTTCAAAG-3'. 7) SNP, MPJ6_1I2013; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-TCTTCCTCTCGCC/TCCCAACTTCTGG-3'. 8) SNP, MPJ6_1I2017; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-ATTGAATGCAATC/TGGCCCCAGCCTG-3'. 9) SNP, MPJ6_1I2018; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-GGTGAGCACAGCA/GGGGGGTGAGGAC-3'. 10) SNP, MPJ6_1I2019; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-GAGCTCCGCAGCA/GTCAATGCTCAGC-3'. 11) SNP, MPJ6_1I2021; GENE NAME, NR1I2; ACCESSION NUMBER, AF364606; LENGTH, 25 bases; 5'-GGTGACACCTCCG/AAGAGGCAGCCAG-3'. The frequencies were 0.0293 for MPJ6_1I2021, 0.0073 for MPJ6_1I2011, and 0.0024 for the other 9 SNPs. All SNPs were found as heterozygous. Among these SNPs, MPJ6_1I2007, MPJ6_1I2010, MPJ6_1I2017 and MPJ6_1I2019 induce non-synonymous amino acid alterations (R98C, R148Q, R381W and I403V, respectively, in PAR1).