ABSTRACT
The CYP4A fatty acid monooxygenases oxidize endogenous arachidonic acid to 20-hydroxyeicosatetraenoic acid that acts as a regulator of blood pressure. Among the isoforms of the CYP4A subfamily, the human CYP4A22 was recently identified. In this study, we report the comprehensive investigation of polymorphisms in the CYP4A22 gene. To investigate genetic variation in CYP4A22 in 191 Japanese subjects, we used denaturing HPLC (DHPLC) and direct sequencing. Our investigation has enabled the identification of 13 sequence variations in the CYP4A22 coding region, thereby demonstrating for the first time that this gene is subject to polymorphism. Two of these sequence variations correspond to silent mutations located in exons 8 (His323His) and 9 (Gly390Gly). Nine of these sequence variations correspond to missense mutations located in exons 1 (Arg11Cys), 3 (Arg126Trp), 4 (Gly130Ser and Asn152Tyr), 5 (Val185Phe), 6 (Cys231Arg), 7 (Lys276Thr), 10 (Leu428Pro), and 12 (Leu509Phe). One of these sequence variations corresponds to nonsense mutations located in exon 9 (Gln368stop). The 13th mutation corresponds to a nucleotide deletion (G7067del) that causes a frameshift and consequently results in a stop codon 80 nucleotides downstream. In addition to the wild-type CYP4A22*1 allele, 20 variants, namely CYP4A22*2-15, were characterized by haplotype analysis. Based on these data, we concluded that allelic variants of the human CYP4A22 gene exist and speculated that some of these variants may be functionally relevant.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Polymorphism, Genetic , Alleles , Base Sequence , Cytochrome P-450 CYP4A , DNA Primers/genetics , Exons , Gene Frequency , Genetic Variation , Genotype , Haplotypes , Humans , Japan , Polymorphism, Single NucleotideABSTRACT
Human CYP4B1 is a CYP4 enzyme with activity towards xenobiotics. Five alleles of human CYP4B1 have been identified in French Caucasians, but allelic variants of enzyme have not been determined in the Japanese population. To establish a rapid and sensitive means of detecting variant CYP4B1 alleles, we analyzed those of 192 Japanese individuals using denaturing HPLC (DHPLC). We then determined the optimal conditions required to detect SNPs for each PCR fragment. Analysis by DHPLC revealed the novel alleles, CYP4B1(*)6 (517C>T and 1033G>A) and CYP4B1(*)7 (AT881-882-del, 993G>A, and 1018C>T), as well as 3 known alleles. The frequencies of the CYP4B1(*)1, (*)2, (*)3, (*)4, (*)5, (*)6, and (*)7 alleles in 192 Japanese individuals were 0.490, 0.328, 0.154, 0, 0.016, 0.008, and 0.005, respectively. The allele frequencies among Japanese relative to those in French Caucasians for CYP4B1(*)1 (0.490 vs. 0.724) and CYP4B1(*)2 (0.328 vs. 0.147) significantly differed. Our results suggest that high throughput DHPLC can rapidly detect pharmacologically important variants in CYP genes.
Subject(s)
Aryl Hydrocarbon Hydroxylases/genetics , Alleles , Aryl Hydrocarbon Hydroxylases/chemistry , Chromatography, High Pressure Liquid , DNA/genetics , DNA Primers , France/epidemiology , Gene Frequency , Genotype , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Japan/epidemiology , Protein Denaturation , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
We sequenced all exons and exon-intron junctions of the CYP2B6 gene from 200 Japanese individuals. We found three novel single nucleotide polymorphisms (SNPs) (1375A>G, 1427G>A and 1454A>T) causing amino acid substitutions (Met(459)Val, Gly(476)Asp and Gln(485)Leu in exon 9), respectively. The detected SNP was as follows: 1) SNP, 031226Hiratsuka01; GENE NAME, CYP2B6; ACCESSION NUMBER, AC023172; LENGTH, 25 base; 5'-CAGAACTTCTCCA/GTGGCCAGCCCCG-3'. 2) SNP, 031226Hiratsuka02; GENE NAME, CYP2B6; ACCESSION NUMBER, AC023172; LENGTH, 25 base; 5'-CCCAGGAGTGTGG/ATGTGGGCAAAAT-3'. 3) SNP, 031226Hiratsuka03; GENE NAME, CYP2B6; ACCESSION NUMBER, AC023172; LENGTH, 25 base; 5'-CCCCAACATACCA/TGATCCGCTTCCT-3'.
