Mechanisms of differential expression of the CYP2A13 7520C and 7520G alleles in human lung: Allelic expression analysis for CYP2A13 heterogeneous nuclear RNA, and evidence for the involvement of multiple Cis-regulatory single nucleotide polymorphisms

Objective: To identify the mechanisms underlying the decreased allelic expression of a common CYP2A13 allele (7520C>G) in the human lung; CYP2A13 is expressed selectively in the respiratory tract and is highly efficient in the metabolic activation of several chemical carcinogens. Methods: The 7520C/G alleles were compared for mRNA stability in cells and relative heterogeneous nuclear RNA (hnRNA) levels in human lungs. Promoter region single nucleotide polymorphisms (SNPs) were identified and analyzed through in-vitro reporter gene assays and gel-shift assays, to uncover the causative SNPs responsible for the decreased allelic expression. Results (i) The 7520C>G SNP does not influence CYP2A13 mRNA stability in CYP2A13-transfected human lung or nasal epithelial cells; (ii) levels of the 7520G hnRNA were consistently lower (<10%) than the levels of the 7520C hnRNA in lung samples from nine heterozygous individuals; (iii) three SNPs (-1479T>C, -3101T>G and -7756G>A) in linkage disequilibrium with the 7520C>G variation were found to cause altered interaction with DNA-binding proteins and decreases in promoter activity; (iv) the suppressive effects of the -1479T>C, -3101T>G, and -7756G>A SNPs on the CYP2A13 promoter were additive, whereas the negative effects of the -1479T>C SNP were enhanced by methylation of -1479C. Conclusion: The decrease in the expression of 7520G allele was because of the cumulative suppressive effects of multiple SNPs, with each by itself having a relatively small effect on CYP2A13 transcription.