title: Development of a real-time polymerase chain reaction-based method for the measurement of relative allelic expression and identification of CYP2A13 alleles with decreased expression in human lung.
abstract: CYP2A13 is a human cytochrome P450 monooxygenase that is efficient in the metabolic activation of tobacco-specific nitrosamines. Sequence variations that affect CYP2A13 expression may contribute to interindividual differences in susceptibility to tobacco-related tumorigenesis. The aim of this study was to identify any impact of CYP2A13 single-nucleotide polymorphisms (SNPs) on CYP2A13 expression in human lung. Expression levels of CYP2A13 mRNA in normal lung displayed significant interindividual variation (>50-fold). Preliminary sequence analysis of CYP2A13 RNA-polymerase chain reaction (PCR) products suggested that a 7520C > G variation, located in the 3'-untranslated region, could be associated with low transcript abundance. Subsequently, we developed a method for the measurement of relative allelic expression, by taking advantage of the capability for melting-curve analysis in real-time PCR. Quantitative analyses using this method indicated that transcripts from the 7520G-containing alleles were >10-fold less abundant than those from the 7520C-containing alleles in 14 of 16 samples examined. The frequencies of the 7520C > G variation in anonymous White, African American, Hispanic, and Asian newborns from New York State were found to be 5.2, 26.8, 17.7, and 4.3%, respectively. The 7520C > G SNP was previously known to be present in both CYP2A13(*)1H and (*)3 alleles. However, analyses of SNP distribution indicated that, in 15 of the 16 heterozygous DNA samples, the 7520C > G SNP belonged to new CYP2A13(*)1 haplotypes. These findings provide a basis for further studies that associate CYP2A13 haplotypes with incidences of smoking-related lung tumors and for studies on the mechanisms of the low-expression phenotype of the 7520G-containing allele.
authors.affiliation: Wadsworth Center, New York State Department of Health, Empire State Plaza, Box 509, Albany, NY 12201-0509, USA.
authors.forename: Xiuling
authors.initials: X
authors.lastname: Zhang
authors.forename: Michele
authors.initials: M
authors.lastname: Caggana
authors.forename: Tracey L
authors.initials: TL
authors.lastname: Cutler
authors.forename: Xinxin
authors.initials: X
authors.lastname: Ding
mesh_terms: D000328:Adult; D000483:Alleles; D001189:Aryl Hydrocarbon Hydroxylases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D005006:Ethnicity / Q000235:genetics; D015870:Gene Expression; D005787:Gene Frequency; D006579:Heterozygote; D006801:Humans; D008168:Lung / Q000201:enzymology*; D010641:Phenotype; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D012333:RNA, Messenger; D001189:Aryl Hydrocarbon Hydroxylases; C414206:CYP2A13 protein, human