TY - JOUR
T1 - 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
AU - Zhang, Xiuling
AU - Caggana, Michele
AU - Cutler, Tracey L.
AU - Ding, Xinxin
PY - 2004/10
Y1 - 2004/10
N2 - 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.
AB - 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.
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U2 - 10.1124/jpet.104.069872
DO - 10.1124/jpet.104.069872
M3 - Article
C2 - 15175424
AN - SCOPUS:4644227631
SN - 0022-3565
VL - 311
SP - 373
EP - 381
JO - Journal of Pharmacology and Experimental Therapeutics
JF - Journal of Pharmacology and Experimental Therapeutics
IS - 1
ER -