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 micromol/L) was significantly (P < .005) higher than that of wild type (0.60 +/- 0.05 micromol/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 micromol/L and 31.3 +/- 3.1 micromol/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.
Fukami
Tatsuki
T
Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kanazawa 920-1192, Japan.
Nakajima
Miki
M
Yoshida
Ryoko
R
Tsuchiya
Yuki
Y
Fujiki
Yuto
Y
Katoh
Miki
M
McLeod
Howard L
HL
Yokoi
Tsuyoshi
T
D000483:Alleles; D019943:Amino Acid Substitution; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism*; D044383:Black People; D002462:Cell Membrane / Q000201:enzymology / Q000235:genetics; D002681:China / Q000453:epidemiology; D065637:Cytochrome P-450 CYP2A6; D004247:DNA / Q000235:genetics; D005091:Exons / Q000235:genetics; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D007438:Introns / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007564:Japan / Q000453:epidemiology; D007723:Korea / Q000453:epidemiology; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D009538:Nicotine / Q000378:metabolism / Q000493:pharmacokinetics; D018722:Nicotinic Agonists / Q000378:metabolism / Q000493:pharmacokinetics; D010957:Plasmids / Q000235:genetics; D011110:Polymorphism, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014481:United States / Q000453:epidemiology; D001741:Black or African American
D007527:Isoenzymes; D018722:Nicotinic Agonists; D009538:Nicotine; D004247:DNA; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6