Development of a Real-time PCR-based Method for the Measurement of Relative Allelic Expression, and Identification of CYP2A13 Alleles with Decreased Expression in Human Lung

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, Black, 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. products for TBP and β -actin), a no-template control, and the first-strand cDNA samples. The data were evaluated with the Roche LightCycler Run 5.32 software, with use of the Fit Points method. Standard curves were generated from a minimum of four data points by plotting Ct (threshold cycles) against the log copy number of the templates. The relative levels of CYP2A13 mRNA in various total RNA preparations were normalized as number of copies of CYP2A13 cDNA per 1,000 copies of TBP cDNA, and per 100,000 copies of β -actin cDNA, in each RT product. -1638G>T, and