| Objective: To identify any genetic polymorphisms of the human CYP2A13 gene, which may alter the metabolic capacities of the enzyme. To determine the frequencies of the variant alleles and to analyze the significance of the single nucleotide polymorphisms.Method: Human genomic DNA was isolated from 32 subjects. All of the 32 subjects examined in this study were Chinese donors with various head and neck diseases: 16 individuals with laryngeal or laryngopharyngeal cancer, two with nasal tumors, five with nasal polyps, and nine with other nontumor head-and-neck diseases.Amplification of CYP2A13 Gene by Long-Distance PCR. Primers were designed according to the CYP2A13 gene sequence from the completed human genome data base (GenBank accession no. AC008962 [GenBank] ). Primers 5'-Fl and 3'-R2 were used to amplify the full-length CYP2A13 gene. A genomic fragment containing the human CYP2A13 gene was amplified from each of 32 genomic DNA samples. The amplified fragment spans 9456 bp,starting at 1117 bp before the ATG start codon and ending at 869 bp after the TGA stop codon. All PCR products were then sequenced using primers. The sequenced regions covered about 1 kilobase before the ATG start codon, all exons and exon-intron junctions, and about 100 bp after the TGA stop codon. All long-PCR products were gel-purified using a QIAquick Gel Extraction kit (QIAGEN) and then subjected to direct sequencing using an automated DNA sequencer.Results: A total of 14 SNPs were detected, all variant alleles were detected as heterozygotes. Of the 14 SNPs, four are located in the coding region, whereas the other 10 are located in noncoding regions: two in the 5'-flanking region, two in the 3'-untranslated region, and six in the introns.Among the four SNPs detected in the coding region, one (Arg257Cys, caused by a 3375C > T missense mutation, with an apparent allele frequency of 10.9% was the same as had been previously identified, whereas the other three (one each in exons 1, 2, and 3) had not been previously reported. The SNP in exon 1 was a 74G > A missense mutation, leading to a predicted amino acid change from Arg to Gin at position 25. This SNP was detected in seven subjects, with an apparent allele frequency of 10.9%. The SNP detected in exon 2 was a 578C > T missense mutation, resulting in an amino acid change from an Arg to a stop codon at position 101. This SNP was detected in two subjects, with an apparent allele frequency of 3.2%. The SNP in exon 3, which was detected in only one subject (1.8%), was a missense mutation (1706C > G), which is expected to cause a conserved Aspl58Glu substitution.None of the 10 SNPs found in the noncoding regions had been detected in the previous study. The two SNPs in the 5'-flanking region, -729C > T (at 729 bp before ATG) and -411G > A, had apparent allele frequencies of 4.7% and 15.6%, respectively, whereas the two SNPs in the 3'-untranslated region, 7520C > G and 7571G > C, had apparent allele frequencies of 4.7 % and 10.9%, respectively. The other six SNPs were detected in introns 1, 2, 3, 5, 6,and 8, with apparent allele frequencies between 1.8% to 15.6%.Conclusion: There are single nucleotide polymorphisms of the human CYP2AI3 gene.A total of 14 SNPs were detected. We have identified 13 additional SNPs in the CYP2A13 gene in the present study, which increases the total number of known variants in this gene to 20.Four are located in the coding region, leading to a predicted amino acid change, may alter the metabolic capacities of the enzyme.
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