Duplications and Defects in the CYP2A6 Gene: Identification, Genotyping, and In Vivo Effects on Smoking YUSHU RAO, EWA HOFFMANN, MOHAMMAD ZIA, LAURENT BODIN, MARILYN ZEMAN, EDWARD M. SELLERS, and RACHEL F. TYNDALE Centre for Addictions and Mental Health (Y.R., E.H., M.Zi., L.B., E.M.S., R.F.T.), Centre for Research in Women’s Health (M.Ze., E.M.S., R.F.T.), and Departments of Pharmacology (Y.R., E.H., M.Zi., L.B., E.M.S., R.F.T.), Psychiatry (E.M.S.), and Medicine (E.M.S.), University of Toronto, Toronto, Canada Received November 8, 1999; accepted June 30, 2000 This paper is available online at http://www.molpharm.org ABSTRACT In humans, 80% of nicotine is metabolized to the inactive metabolite cotinine by the enzyme CYP2A6, which can also activate tobacco smoke procarcinogens (e.g., 4-(methylnitro- samino)-1-(3-pyridyl)-1-butanone). Previously, we demon- strated that individuals who are nicotine-dependent and have defective CYP2A6 alleles (*2, *3) smoked fewer cigarettes; how- ever, we recognize that the genotyping method used for the CYP2A6*3 allele gave a high false-positive rate. In the current study we used improved genotyping methods to examine the effects of the defective CYP2A6*2 and CYP2A6*4 alleles on smoking behavior. We found that those with the defective alleles (N = 14) smoked fewer cigarettes per day than those homozygous (N = 277) for wild-type alleles (19 versus 28 cigarettes per day, P  .001). In addition, we identified a duplicated form of the CYP2A6 gene, corresponding to the gene deletion CYP2A6*4 allele, developed a genotyping assay, assessed the gene copy number, and examined its prevalence in Caucasian smokers (N = 296). We observed an ascending rank order for plasma cotinine and breath carbon monoxide levels (an index of smoke inhalation) in individuals with null (CYP2A6*2 and CYP2A6*4) alleles (N = 14), those homozygous for wild-type (CYP2A6*1/*1) alleles (N = 277), and those with our newly identified CYP2A6 gene duplication (N = 5). The phenotype, as determined by plasma nicotine/cotinine ratios, had a descending rank order for these three genotype groups that did not reach significance. Although further characteriza- tion is required for the duplication gene variant, these results extend our previous findings and suggest a substantial influ- ence of CYP2A6 genotype and phenotype on smoking behav- ior. Genetic variation of CYP2A6 alters coumarin and nicotine (NIC) metabolism (Yamano et al., 1990; Iscan et al., 1994; Messina et al., 1997). Initially, a wild-type (CYP2A6*1) and two defective alleles (CYP2A6*2 and CYP2A6*3) were iden- tified. CYP2A6*2 is a null allele with no activity toward probe substrates, although the methodology for detection, function, and allele frequency of the CYP2A6*3 allele are controversial (Yamano et al., 1990; Fernandez-Salguero et al.,1995; Oscarson et al., 1998; Benowitz et al., 2000). Re- cently, a CYP2A6 gene deletion (CYP2A6*4) was character- ized (Yokoi and Kamataki, 1998; Nunoya et al., 1999; Oscar- son et al., 1999b); the mechanism proposed for the creation of the deleted allele is similar to that found for the deleted (CYP2D6*5) and duplicated (CYP2D6*2X2) alleles of CYP2D6, involving unequal crossover between CYP2D6 and adjacent CYP2D genes (Gaedigk et al., 1991). The existence of a CYP2A6 gene deletion variant infers the existence of a CYP2A6 gene duplication (Fig. 1A). In humans, 80% of NIC is inactivated by metabolism to cotinine (COT; Benowitz et al., 1994). Determining the vari- ation in NIC inactivation is important because of NIC’s role in producing tobacco dependence and regulating smoking behavior. We, and others, have demonstrated that CYP2A6 is responsible for the majority of the metabolic inactivation of NIC to COT (Nakajima et al., 1996b, 2000; Messina et al., 1997; Benowitz et al., 2000) and for the metabolism of COT to trans-3-hydroxyCOT, 5'-hydroxyCOT and possibly norCOT (Nakajima et al., 1996a; Murphy et al., 1999). Dependent smokers adjust their smoking behavior to maintain constant blood and brain NIC levels (McMorrow and Foxx, 1983; Russel, 1987). Consistent with this, we pre- viously found that heterozygotes for defective (CYP2A6*2 or CYP2A6*3) alleles smoked fewer cigarettes (CIGs) per week than smokers homozygous for wild-type CYP2A6*1 alleles (129 versus 159 CIGs per week) and were less likely to become NIC dependent (Pianezza et al., 1998). Repeating the Supported in part by Grant DA06889 from the National Institute of Drug Abuse, Nicogen Research Inc., and the Centre for Addictions and Mental Health (Toronto, Canada). ABBREVIATIONS: NIC, nicotine; CIG, cigarette; COT, cotinine; CO, carbon monoxide; PCR, polymerase chain reaction; dNTP, deoxyribonucle- otide triphosphate; bp, base pair(s); ppm, parts per million. 0026-895X/00/040747-09$3.00/0 MOLECULAR PHARMACOLOGY Vol. 58, No. 4 Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics 13079/854112 Mol Pharmacol 58:747–755, 2000 Printed in U.S.A. 747 at ASPET Journals on October 13, 2017 molpharm.aspetjournals.org Downloaded from