A nicotine C-oxidase gene (CYP2A6) polymorphism important for promoter activity

In humans, several polymorphic variants have been described for the gene encoding the major nicotine C-oxidase, cytochrome P450 2A6 (CYP2A6), which is to a great extent responsible for the large interindividual differences seen at the enzymatic and activity levels. Hitherto, mainly polymorphic variants in the open reading frame have been identified. In the present study, we identified a novel single nucleotide polymorphism (SNP) located in the 5' flanking region of the CYP2A6 gene. Sequencing of 1.4 kb of the 5'-upstream region of the CYP2A6 gene from eight individuals revealed a c.-1013A>G polymorphism defining two new alleles, CYP2A6*1D and CYP2A6*1E, lacking or having also the CYP2A7 3'-UTR. Analysis of genomic DNA from 32 Swedish and 109 Turkish subjects by dynamic allele-specific hybridization (DASH) showed that, in both groups, the variants carrying the c.-1013A>G SNP represent approximately 70% of the total number of alleles. Transfection of HepG2 cells with luciferase reporter constructs containing 1019 bp of the CYP2A6 5'-regulatory sequence showed that the region between c.-1005 and c.-1019 elicited a strong enhancer effect and that the CYP2A6*1D promoter had significantly reduced expression as compared to CYP2A6*1A carrying c.-1013A. Electrophoretic mobility shift assays (EMSA) showed that nuclear proteins from HepG2 and B16A2 cells exhibited a higher binding affinity to the probe harboring c.-1013A as compared to the c.-1013G probe, although the transcription factor(s) responsible for this binding could not be identified. In conclusion, our results indicate the presence of a strong enhancer or promoter responsive element between c.-1005 and c.-1019 in the CYP2A6 gene and that a c.-1013A>G polymorphism in this region affects CYP2A6 transcription.