Short Communication A Novel Duplication Type of CYP2A6 Gene in African-American Population

Human CYP2A6 is responsible for the metabolism of nicotine and its genetic polymorphisms affect smoking behavior and risk of lung cancer. In the present study, we identified a novel type of CYP2A6 gene duplication that is created through an unequal crossover event with the CYP2A7 gene at 5.2 to 5.6 kilobases downstream from the stop codon. The novel duplication type of CYP2A6 was found in African Americans (n 176) at an allele frequency of 1.7%, but was not found in European-American (n 187), Korean (n 209), or Japanese (n 184) populations. The plasma cotinine/ nicotine ratio in subjects possessing the novel CYP2A6 gene duplication with the CYP2A6*1 allele (10.8 7.0, n 4) was 1.4-fold higher than that in homozygotes of the wild type (8.0 5.0, n 87), although the difference was not statistically significant. The findings in the present study suggested that the novel duplicated CYP2A6 allele, which is specific for African Americans, would increase nicotine metabolism and may affect smoking behavior. Cytochrome P450 is a superfamily of hemoproteins, many of which can metabolize xenobiotics such as clinically used drugs, procarcinogens, and environmental pollutants. The CYP2A gene subfamily comprises three genes, CYP2A6, CYP2A7, and CYP2A13, as well as a split pseudogene, CYP2A18. Among them, the genes coding functional enzymes are CYP2A6 and CYP2A13 (Yamano et al., 1990; Su et al., 2000). CYP2A6 is mainly expressed in liver and is responsible for nicotine metabolism (Nakajima et al., 1996). In addition, it can metabolize some pharmaceutical agents such as losigamone, letrozole, halothane, valproic acid, and disulfiram, and activates some procarcinogens such as 4-methylnitrosoamino-1-(3-pyridyl)-1-butanone and N-nitrosodiethylamine (Nakajima et al., 2002). Genetic polymorphisms in the CYP2A6 gene are associated with large interindividual variability in nicotine metabolism, smoking behavior, and the risk of lung cancer (Fujieda et al., 2004). Several CYP2A6 alleles possessing a single nucleotide polymorphism (SNP), which leads to an amino acid change, have decreased or no enzymatic activity. Other CYP2A6 alleles such as CYP2A6*1B or CYP2A6*12 are created by gene conversion or an unequal crossover event with the CYP2A7 gene (Ariyoshi et al., 2000; Oscarson et al., 2002). The CYP2A7 gene is located approximately 25 kb upstream of the CYP2A6 gene and shows an identity of 96.5% in the coding sequences (Yamano et al., 1990). Rao et al. (2000) reported a duplicated CYP2A6 allele, CYP2A6*1 2. This allele is created through an unequal crossover with the CYP2A7 gene at intron 8-exon 9 and the CYP2A6*4D allele is produced as the reciprocal product (Oscarson et al., 1999a). CYP2A6*4A and CYP2A6*4B alleles have also been reported as entire gene-deleted alleles (Oscarson et al., 1999b; Ariyoshi et al., 2004). In a previous study (Nakajima et al., 2006), we found an AfricanAmerican subject possessing the CYP2A6*1A, CYP2A6*1D (g.1013A G), and CYP2A6*1H (g.-745A G) alleles, envisaging the existence of three copies of the CYP2A6 gene. However, genotyping analysis did not assign the CYP2A6*1 2 allele reported by Rao et al. (2000). In the present study, we investigated the CYP2A locus and identified the novel duplication type of CYP2A6 gene. Materials and Methods Chemicals and Reagents. Long and accurate (LA) TaqDNA polymerase and Blend TaqDNA polymerase were obtained from Takara (Shiga, Japan) and Toyobo (Osaka, Japan), respectively. Primers were commercially synthesized at Hokkaido System Sciences (Sapporo, Japan). The restriction enzymes were purchased from Takara, Toyobo, New England Biolabs (Ipswich, MA), and MBI Fermentas (Hanover, MD). Nicorette (nicotine gum containing 2 mg of nicotine) was obtained from Pfizer Japan (Tokyo, Japan). All other chemicals and solvents were of analytical or the highest grade commercially available. Genomic DNA. This study was approved by the Human Studies Committee of Washington University School of Medicine (St. Louis, MO) and the Ethics Committees of Kanazawa University (Kanazawa, Japan) and Soonchunhyang University Hospital (Chonan, Korea). Written informed consent was obtained from 187 European-American, 176 African-American, 209 Korean, and 184 Japanese subjects. Blood samples were collected from a cubital vein. Genomic DNA was extracted from peripheral lymphocytes using a Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN). Genotyping of CYP2A6 Alleles. The genotyping of CYP2A6*1A (Nakajima et al., 2006), CYP2A6*1B1 (Nakajima et al., 2006), CYP2A6*1B2 (Nakajima et al., 2006), CYP2A6*1B13 (Nakajima et al., 2006), CYP2A6*1D (Nakajima et al., 2006), CYP2A6*1F (Nakajima et al., 2004), CYP2A6*1G (Nakajima et al., 2004), CYP2A6*1H (Nakajima et al., 2006), CYP2A6*1J (Nakajima et al., 2006), conventional CYP2A6*1 2 (Yoshida et al., 2002), CYP2A6*2 (Nakajima et al., 2000), CYP2A6*3 (Nakajima et al., 2000), CYP2A6*4A (Fukami et al., 2006), CYP2A6*4D (Fukami et al., 2006), This study was supported in part by a grant from Japan Health Sciences Foundation with Research on Health Science Focusing on Drug Innovation, by a grant from the Smoking Research Foundation in Japan. T.F. was supported as a Research Fellow of the Japan Society for the Promotion of Science. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.106.013557. ABBREVIATIONS: SNP, single nucleotide polymorphism; kb, kilobase(s); LA, long and accurate; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; bp, base pair(s). 0090-9556/07/3504-515–520$20.00 DRUG METABOLISM AND DISPOSITION Vol. 35, No. 4 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 13557/3194853 DMD 35:515–520, 2007 Printed in U.S.A. 515 at A PE T Jornals on M ay 8, 2017 dm d.aspurnals.org D ow nladed from CYP2A6*5 (Nakajima et al., 2001), CYP2A6*6 (Yoshida et al., 2002), CYP2A6*7 (Yoshida et al., 2002), CYP2A6*8 (Yoshida et al., 2002), CYP2A6*9 (Nakajima et al., 2006), CYP2A6*10 (Yoshida et al., 2002), CYP2A6*11 (Yoshida et al., 2002), CYP2A6*12 (Nakajima et al., 2004), CYP2A6*13 (Fukami et al., 2005b), CYP2A6*14 (Fukami et al., 2005b), CYP2A6*15 (Fukami et al., 2005b), CYP2A6*16 (Fukami et al., 2005b), CYP2A6*17 (Fukami et al., 2004), CYP2A6*18 (Fukami et al., 2005b), CYP2A6*19 (Fukami et al., 2005b), CYP2A6*20 (Fukami et al., 2005a), CYP2A6*21 (Nakajima et al., 2006), and CYP2A6*22 (Nakajima et al., 2006) was performed as described previously. Determination of the Relative Gene Copy Ratio of CYP2A6/CYP2A7. To determine the copy number of the CYP2A6 gene, polymerase chain reaction (PCR) analyses were performed as described previously (Fukami et al., 2006). DNA samples from the subjects who were genotyped as CYP2A6*4A/ CYP2A6*4A (n 2, Japanese), CYP2A6*1/CYP2A6*4A (n 4, Japanese), CYP2A6*1/CYP2A6*1 (n 4, Japanese), and CYP2A6*1/conventional CYP2A6*1 2 (n 1, Korean) were used for the standard curve. PCR-RFLP to Identify the Crossover Region. To determine the crossover region of the novel duplicated CYP2A6 allele with the CYP2A7 gene, PCRrestriction fragment length polymorphism (RFLP) was performed at the 3 flanking region. The primer sets used were 2A6FR-S and 2A6FR13.8kR, 2A7FR-S and 2A7FR16.9kR, or 2A6FR-S and 2A7FR16.9kR (Table 1). The reaction mixture contained genomic DNA (200 ng), 1 LA-PCR buffer, 2.5 mM MgCl2, 0.4 mM deoxynucleoside-5 -triphosphates, 0.4 M each primer, and 1 U of LA TaqDNA polymerase in a final volume of 25 l. After an initial denaturation at 94°C for 1 min, the amplification was performed by denaturation at 98°C for 20 s, annealing and extension at 68°C for 12 min (with prolongation for 15 s per 1 cycle during 13–26 cycles) for 26 cycles, followed by a final extension at 72°C for 10 min. The LA-PCR product was digested with AleI, EcoRI, EcoRV, or FspI at 37°C for 3 h. The digestion patterns were determined by electrophoresis in a 0.8% agarose gel. Sequence Analysis of the 3 -Flanking Region of the CYP2A6 Gene. Sequence analysis was performed to examine the nucleotide sequences of the crossover region of the novel duplicated CYP2A6 allele. The PCR mixture contained genomic DNA (100 ng), 1 PCR buffer, 0.2 mM deoxynucleoside5 -triphosphates, 0.4 M 2A6FR4.2kS and 2A7FR7.8kR primers (Table 1), and 0.5 U of Blend TaqDNA polymerase in a final volume of 25 l. After an initial denaturation at 94°C for 3 min, the amplification was performed by denaturation at 94°C for 25 s, annealing at 57°C for 25 s, and extension at 72°C for 3 min for 28 cycles, followed by a final extension at 72°C for 5 min. The PCR product was submitted to DNA sequencing using a Thermo Sequenase Cy5.5 Dye Terminator Cycle Sequencing kit (GE Healthcare Bio-Science, Chalfont St. Giles, UK) with a Long-Read Tower DNA sequencer (GE Healthcare Bio-Science). Genotyping Method for the Novel Duplicated CYP2A6 Allele. Allelespecific-PCR was applied for the genotyping with the primer sets 2A6FR4.2kS and 2A6FR6.8kR or 2A7FR7.8kR (Table 1). The reaction mixture and PCR condition were the same as described above except for the primers. The CYP2A6*1 allele was amplified with the primer set 2A6FR4.2kS and 2A6FR6.8kR (2681 bp), and the novel duplicated CYP2A6 allele was amplified with the primer set 2A6FR4.2kS and 2A7FR7.8kR (2387 bp). In Vivo Phenotyping of Nicotine Metabolism. Since 16 of 176 African Americans were judged as smokers based on the baseline presence of nicotine and cotinine, phenotyping of in vivo nicotine metabolism was not performed in these subjects. Phenotyping analysis was performed in 160 African Americans in our previous study (Nakajima et al., 2006). In brief, the subjects chewed one piece of nicotine gum (Nicorette) for 30 min, chewing for 10 s per 30 s. Blood samples were collected from a cubital vein just before and 2 h after the start of chewing. The concentrations of nicotine and cotinine in the plasma samples were determined by high-performance liquid chromatography as described previously (Nakajima et al., 2000). The cotinine/nicotine ratio of the plasma concentration was calculated as an index of nicotine metabolism. Statistical analysis of the cotinine/nicotine ratios between the different genotypes was performed by Mann-Whitney U test. A value of P 0.05 was considered statistically significant.