A novel polymorphism of human CYP2A6 gene CYP2A6*17 has an amino acid substitution (V365M) that decreases enzymatic activity in vitro and in vivo.

Cytochrome P450 (CYP) 2A6 is a major CYP responsible for the metabolism of nicotine and coumarin in humans. We identified a novel allele, designated CYP2A6*17, which contains A51G (exon 1), C209T (intron 1), G1779A (exon 3), C4489T (intron 6), G5065A (V365M, exon 7), G5163A (intron 7), C5717T (exon 8), and A5825G (intron 8). We developed a genotyping method by polymerase chain reaction–restriction fragment length polymorphism for the CYP2A6*17 allele, targeting the G5065A mutation. The allele frequency in black subjects (n = 96) was 9.4% (95% confidence interval [CI], 5.3%-13.5%). The allele was not found in white subjects (95% CI, 0%-0.9%; n = 163), Japanese subjects (95% CI, 0%-1.6%; n = 92), and Korean subjects (95% CI, 0%-0.7%; n = 209). To examine the effects of the amino acid change in the CYP2A6*17 allele on the enzymatic activity, we expressed a wild-type or variant (V365M) CYP2A6 together with NADPH-CYP reductase in Escherichia coli. For coumarin 7-hydroxylation, the apparent Michaelis-Menten constant value of variant CYP2A6 (1.06 ± 0.11 μmol/L) was significantly (P < .005) higher than that of wild type (0.60 ± 0.05 μmol/L). The maximum velocity values of the wild-type and variant CYP2A6 were 0.61 ± 0.06 and 0.64 ± 0.07 pmol · min−1 · pmol−1 CYP, respectively. For nicotine C-oxidation, the apparent Michaelis-Menten constant values of the wild-type or variant CYP2A6 were 31.6 ± 2.9 μmol/L and 31.3 ± 3.1 μmol/L, respectively. The maximum velocity value of variant CYP2A6 (0.72 ± 0.21 pmol · min−1 · pmol−1 CYP) was significantly (P < .05) lower than that of the wild type (1.80 ± 0.42 pmol · min−1 · pmol−1 CYP). Thus the intrinsic clearance values for coumarin 7-hydroxylation and nicotine C-oxidation by the variant were both significantly (P < .05) decreased to 40% to 60% compared with the wild type. Furthermore, cotinine/nicotine ratios after 1 piece of nicotine gum was chewed, used as an index of in vivo nicotine metabolism, were significantly (P < .05) decreased in heterozygotes of the CYP2A6*17 allele (5.4 ± 2.7, n = 12) compared with homozygotes of the wild type (11.5 ± 10.5, n = 37). A subject with CYP2A6*17/CYP2A6*17 revealed the lowest cotinine/nicotine ratio (1.8). We found a novel allele in black subjects that affects the nicotine metabolism in vitro and in vivo. (Clin Pharmacol Ther 2004;76:xxx-x.) Clinical Pharmacology & Therapeutics (2004) 76, 519–527; doi: 10.1016/j.clpt.2004.08.014