Comparison of cytochrome P450 2A6 polymorphism frequencies in Caucasians and African-Americans using a new one-step PCR-RFLP genotyping method

CYP2A6 (cytochrome P450 2A6), which was first identified as the human couma rin 7-hydroxylase, is the most important enzyme in nicotine C-oxidation. Th e enzyme also metabolically activates the tobacco specific nitrosamine 4-{m ethylnitrosamino)-1-(3-pyridyl)-1-butanone {NNK) in vitro. Polymorphisms in the CYP2A6 gene may thus impact on both smoking behavior and lung cancer s usceptibility. Several different genotyping methods have been reported with conflicting results in the frequencies of CYP2A6 polymorphic variants. Thu s we decided to perform a sequence analysis of the entire CYP2A6 gene. Sequ encing confirmed the published CYP2A6 cDNA sequence. However, intron sequen ces differed considerably from the reported sequence of the CYP2Ab*3 (upsil on2) variant. Our analyses revealed that parts of introns shared homologies with the published sequence of CYP2A13. Based on our sequence data we deve loped a one step protocol for specific amplification of exon 3 of CYP2A6. T he resulting PCR product can be used directly for restriction endonuclease digestion with XcmI and DdeI to determine the frequencies of the reported v ariant alleles CYP2A6*2 and CYP2Ab*3. In a population of 305 African-Americ ans and 145 Caucasians, we found allele frequencies of 0.003 (2/610) for CY P2Ab*2 and 0 (0/610) for CYP2A6*3 in African-Americans and allele frequenci es of 0.014 (4/290) and 0 {0/290) in Caucasians. We conclude that both alle les are considerably less frequent in populations than previously reported. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.