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

Xiuling Zhang, Michele Caggana, Tracey L. Cutler, Xinxin Ding

Research output: Contribution to journalArticle

23 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)373-381
Number of pages9
JournalJournal of Pharmacology and Experimental Therapeutics
Volume311
Issue number1
DOIs
StatePublished - Oct 1 2004
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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