Introduction

Cytochrome P450 (CYP) enzymes are involved in the metabolism of numerous exogenous and endogenous substances including drugs, environmental chemicals, steroid hormones and bile acids. The CYP2A gene subfamily comprises three genes, CYP2A6, CYP2A7, and CYP2A13, as well as the split pseudogene CYP2A18. CYP2A6 can metabolize nicotine,1 coumarin,2 tegafur,3 losigamone,4 letrozole,5 and nitrosamines such as 4-methylnitrosamino-1-(3-pyridyl)-1-butanone.6 CYP2A7 has been shown to not incorporate heme and to be functionally inactive.2, 7

There are genetic polymorphisms in the CYP2A6 gene. In addition to a variety of single nucleotide polymorphism (SNP)s, several alleles that are produced by crossover with the CYP2A7 gene are known to exist. The CYP2A7 gene is located approximately 25 kb upstream of the CYP2A6 gene with a identity of 96.5% in the coding nucleotide sequence.2 The CYP2A6*1B allele has a gene conversion with CYP2A7 in the 3′-untranslated region.8 In the CYP2A6*4 (CYP2A6*4A, CYP2A6*4B, and CYP2A6*4D) alleles, the entire CYP2A6 gene is deleted and the enzymatic activity is lacking.8, 9, 10, 11 The CYP2A6*1X2 allele has a duplication of the CYP2A6 gene as the reciprocal product of the CYP2A6*4D allele after an unequal crossover event.12 The CYP2A6*12 is a CYP2A7/CYP2A6 hybrid allele created by an unequal crossover in intron 2.13

Recently, we developed a genotyping method to distinguish the CYP2A6*4A, CYP2A6*4D, CYP2A6*1F, and CYP2A6*1G alleles.14 Using this method, we found that 14 out of 187 European-American subjects heterozygously possessed the CYP2A6*4A allele, resulting in an allele frequency of 3.7%. Heterozygotes of the CYP2A6*4 allele have one copy of the CYP2A6 gene. To our surprise, several subjects with the CYP2A6*4A allele were judged as possessing two copies of the CYP2A6 gene, as they were genotyped as heterozygotes for other SNPs with the wild type. From these subjects, we found novel CYP2A7 polymorphic alleles in which the sequences in the 3′-flanking region have undergone conversion with the corresponding CYP2A6 sequences, leading to misgenotyping as the CYP2A6*4A allele.

Results

Sequence analysis of the 3′-flanking region of the CYP2A7 gene

With polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using the primers of 2Aint7F and 2A6R2,14 we found that 14 out of 187 European-American subjects and three out of 176 African-American subjects were heterozygotes of the CYP2A6*4A allele, resulting in allele frequencies of 3.7 and 0.9%, respectively. However, all European-American and one out of three African-American heterozygotes of the CYP2A6*4A allele were genotyped as a heterozygote for SNPs of g.−48T>G (found in CYP2A6*9 allele) or g.51G>A (found in CYP2A6*14, CYP2A6*18B, CYP2A6*20, CYP2A6*21, and CYP2A6*22 alleles) with the wild type, indicating the presence of two copies of the CYP2A6 gene. In contrast, no contradiction in the genotype was observed for 73 Japanese and 42 Korean subjects heterozygously or homozygously possessing the CYP2A6*4A allele.

As we previously reported,14 the primer 2Aint7F can anneal to both the CYP2A6 and CYP2A7 genes, but the primer 2A6R2 specifically anneals to the CYP2A6 gene. If the primer 2A6R2 anneals to the CYP2A7 gene, the PCR-RFLP pattern would cause us to mistakenly genotype it as the CYP2A6*4 allele. To investigate the cause of the contradiction observed in these subjects, sequence analyses of the 3′-flanking region of the CYP2A7 gene were performed. Consequently, three novel CYP2A7 alleles were found (Figure 1a). In an allele termed CYP2A7*1B, the sequences at the region of the primer 2A6R2 are converted with the corresponding sequences of the CYP2A6 gene, that is, a 6-bp deletion at region 2. In another allele termed CYP2A7*1C, the sequences at region 1 as well as region 2 are deleted. In the other allele termed CYP2A7*1D, the sequences at regions 1, 2, and 3 are deleted. This is the first report of the CYP2A7 polymorphic alleles.

Figure 1
figure 1

(a) Sequence alignments of the 3′- flanking region of CYP2A7*1A, CYP2A7*1B, CYP2A7*1C, and CYP2A7*1D alleles, as well as the CYP2A6 gene. The accession number of the genomic DNA sequences of CYP2A7 and CYP2A6 is NG_000008.5. The nucleotide numbering refers to the TGA of the stop codon with the next nucleotide of A as 1 with the reference sequence of the CYP2A7 gene. Deletions are denoted by dashes. The solid boxes represent the regions that are different from those of CYP2A6 gene. The horizontal arrows indicate the location and direction of the primers for the polymerase chain reaction. The recognition sites of Tai I are indicated by vertical arrows with dashed boxes. (b) Schematic and (c) representative polymerase chain reaction-restriction fragment length polymorphism patterns for CYP2A7 alleles. (d) Schematic and (e) representative allele specific-PCR patterns for CYP2A7*1A/CYP2A7*1C or CYP2A7*1A/CYP2A7*1D. In the present study, there was no homozygote of the CYP2A7 polymorphic alleles.

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Frequencies of CYP2A7 polymorphic alleles

With the conventional genotyping method of CYP2A6*4,14 the subject not possessing the CYP2A6*4 allele could not have the CYP2A7 polymorphic allele. Therefore, we performed the genotyping analyses of the CYP2A7 polymorphic alleles (Figure 1b–e) for 14 European-Americans, three African-Americans, 73 Japanese and 42 Koreans who were once genotyped as heterozygotes or homozygotes of the CYP2A6*4 allele. Among the 14 European-Americans, five subjects were genotyped as CYP2A7*1A/CYP2A7*1B; seven subjects were genotyped as CYP2A7*1A/CYP2A7*1C; and two subjects were genotyped as CYP2A7*1A/CYP2A7*1D (Table 1). One African-American subject and one Japanese subject were genotyped as CYP2A7*1A/CYP2A7*1D and CYP2A7*1A/CYP2A7*1B, respectively. In contrast, the CYP2A7 polymorphic alleles were not found in the Korean subjects. The allele frequencies of the CYP2A7 polymorphic allele are summarized in Table 1.

Table 1 Allele frequencies of CYP2A7 alleles and CYP2A6*4A in four populations
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CYP2A6 copy number in the subjects possessing the CYP2A7 polymorphic alleles

To investigate the copy number of the CYP2A6 gene in the subjects with the CYP2A7 polymorphic alleles, we performed quantitative analyses of the PCR-amplified fragments. The genomic DNA samples from the Japanese or Korean subjects who did not have the CYP2A7 polymorphic alleles and were genotyped as CYP2A6*4A/CYP2A6*4A, CYP2A6*1/CYP2A6*4A, CYP2A6*1/CYP2A6*1 or CYP2A6*1/CYP2A6*1X2 were used for the standard curve. The region from exon 3 to intron 3 in both the CYP2A6 and CYP2A7 genes was amplified (Figure 2). The ratios of the PCR products corrected with the fragment lengths of CYP2A6/CYP2A7 for the standard samples were as follows: 0.07 and 0.08 for CYP2A6*4A/CYP2A6*4A (n=2), 0.81–0.93 for CYP2A6*1/CYP2A6*4A (n=6), 1.69–2.06 for CYP2A6*1/CYP2A6*1 (n=6), and 2.81 for CYP2A6*1/CYP2A6*1X2 (n=1). With the standard curve, the ratios of the copy numbers of CYP2A6/CYP2A7 in the 14 European-American, one African-American and one Japanese subjects who possessed the CYP2A7 polymorphic alleles were calculated as 0.88–1.12, indicating the presence of two copies of the CYP2A6 gene.

Figure 2
figure 2

Copy number assay for the CYP2A6 and CYP2A7 genes by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting exon 3. (a) Schematic structures of the CYP2A6 and CYP2A7 genes. Boxes represent exon 3 and lines represent intron 3. Polymerase chain reaction amplification from exon 3 to intron 3 was performed with the primer pair indicated by horizontal arrows. The recognition site of EcoN I is indicated by a vertical arrow. (b) Representative photograph of PCR-RFLP patterns for different CYP2A6 genotypes. The intensities of the 391-bp (CYP2A6) and 273-bp (CYP2A7) fragments were quantified. The ratio of the intensities corrected by the fragment length was calculated as the ratio of the copy numbers of CYP2A6/CYP2A7. Homozygotes of CYP2A6*4A, heterozygotes of CYP2A6*1/CYP2A6*4A, homozygotes of CYP2A6*1, and heterozygotes of CYP2A6*1/CYP2A6*1X2 have 0, 1, 2 and 3 copies of the CYP2A6 gene, respectively. (c) Standard curve of the ratio of the copy numbers of CYP2A6/CYP2A7. (d) The ratios of the copy numbers of CYP2A6/CYP2A7 in the 14 European-American, one African-American and one Japanese subjects who possessed the CYP2A7 polymorphic alleles were calculated using the standard curve shown in (c).

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The region from exon 5 to intron 5 in both the CYP2A6 and CYP2A7 genes was also amplified (Figure 3). The ratios of the PCR products corrected with the fragment lengths of CYP2A6/CYP2A7 for the standard samples were as follows: 0.00 for CYP2A6*4A/CYP2A6*4A (n=2), 0.75–0.82 for CYP2A6*1/CYP2A6*4A (n=6), 1.30–1.53 for CYP2A6*1/CYP2A6*1 (n=6), and 2.10 for CYP2A6*1/CYP2A6*1X2 (n=1). With the standard curve, the ratios of the copy numbers of CYP2A6/CYP2A7 in the 14 European-American, one African-American, and one Japanese subjects who possessed the CYP2A7 polymorphic alleles were calculated as 0.84–1.00, indicating the presence of two copies of the CYP2A6 gene.

Figure 3
figure 3

Copy number assay for CYP2A6 and CYP2A7 genes by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting exon 5. (a) Schematic structures of the CYP2A6 and CYP2A7 genes. Boxes represent exon 5 and lines represent intron 5. Polymerase chain reaction amplification was performed with the primer pair indicated by horizontal arrows. The restriction site of Taq I is indicated by a vertical arrow. (b) Representative photograph of PCR-RFLP patterns for different CYP2A6 genotypes. The intensities of the 330-bp (CYP2A6) and 189-bp (CYP2A7) fragments were quantified. The ratio of the intensities corrected by the fragment length was calculated as the ratio of the copy numbers of CYP2A6/CYP2A7. (c) Standard curve of the ratio of the copy numbers of CYP2A6/CYP2A7. (d) The ratios of the copy numbers of CYP2A6/CYP2A7 in the 14 European-American, one African-American, and one Japanese subjects who possessed the CYP2A7 polymorphic alleles were calculated using the standard curve shown in the (c).

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Allele frequency of CYP2A6*4A

The improved genotyping method for CYP2A6*4A which can exclude the polymorphic CYP2A7 alleles was established (Figure 4) and was applied for the subjects (14 European-Americans, three African-Americans, 73 Japanese, and 42 Koreans) who possessed the CYP2A6*4A allele or the CYP2A7 polymorphic alleles. The subjects possessing the CYP2A7 polymorphic alleles were not genotyped as the CYP2A6*4A allele. Thus, no European-American subject possessed the genuine CYP2A6*4A allele in the present study (n=187). The allele frequencies of CYP2A6*4A in the African-Americans (n=176) and Japanese (n=184) were correctly recalculated as 0.6 and 22.3%, respectively (Table 1).

Figure 4
figure 4

Improved genotyping of CYP2A6*1A, CYP2A6*1B, CYP2A6*4A, and CYP2A6*4D alleles by the combination of allele specific-PCR (AS-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). (a) Schematic structures of the CYP2A6 and CYP2A7 genes. Closed and open boxes represent the exons of CYP2A7 and CYP2A6, respectively and lines represent introns. The primers of 2A6int8F and 2A7int8F specifically anneal to the CYP2A6 and CYP2A7 genes, respectively. The amplified DNA was digested by Acc II. (b) Schematic and (c) representative AS-PCR and PCR-RFLP patterns for the different CYP2A6 genotypes. Three subjects with the CYP2A7 polymorphic allele were confirmed not to have the CYP2A6*4 allele. Subjects 1 and 2 who were previously genotyped as CYP2A6*1A/CYP2A6*4A have been correctly re-genotyped as CYP2A6*1A/CYP2A6*1A and CYP2A6*1A/CYP2A6*1B, respectively. Subject 3 who was previously genotyped as CYP2A6*1B/CYP2A6*4A has been correctly re-genotyped as CYP2A6*1B/CYP2A6*1B.

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In vivo nicotine metabolism in subjects possessing the CYP2A7 polymorphic alleles

In this study, the phenotype of nicotine metabolism in 176 European-Americans and 160 African-Americans was determined. Among them, the data for 163 European-Americans (Figure 5a) and 116 African-Americans (Figure 5b) whose genotypes were relevant to the CYP2A7 polymorphic alleles or the CYP2A6*4A allele are shown. Among the European-Americans, 12 subjects who had been mistakenly genotyped as CYP2A6*1/CYP2A6*4A owing to the polymorphic CYP2A7 alleles were correctly genotyped as CYP2A6*1/CYP2A6*1 (Figure 5a). The cotinine/nicotine ratios in these subjects (6.4±3.5, 1.5–14.9, n=12) were in the range of the ratios in the homozygotes of CYP2A6*1 not possessing the CYP2A7 polymorphic alleles (7.9±5.6, 0.6–36.5, n=115). Two subjects who had been mistakenly genotyped as CYP2A6*4A/CYP2A6*9 or CYP2A6*4A/CYP2A6*14 owing to the polymorphic CYP2A7 alleles were correctly genotyped as CYP2A6*1/CYP2A6*9 or CYP2A6*1/CYP2A6*14, respectively. The cotinine/nicotine ratios in the two subjects (4.3 and 3.7) were in the range of the ratios in the subjects with CYP2A6*1/CYP2A6*9 (5.9±3.2, 0.8–13.7, n=24) and the subjects with CYP2A6*1/CYP2A6*14 (6.7±4.5, 2.5–17.1, n=10), not possessing the CYP2A7 polymorphic alleles, respectively.

Figure 5
figure 5

Cotinine/nicotine ratios in plasma 2 h after chewing one piece of nicotine gum. The cotinine/nicotine ratios in (a) 163 European-Americans and (b) 116 African-Americans who were genotyped for CYP2A6 alleles. The closed circles indicate the subjects who were once genotyped as the heterozygotes of the CYP2A6*4 allele with the conventional genotyping method, but re-genotyped as not CYP2A6*4 allele owing to the CYP2A7 polymorphic alleles. The open circles indicate the subjects not possessing the CYP2A7 polymorphic alleles. The numbers of subjects are shown in parentheses.

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Among the African-Americans, a subject who had been mistakenly genotyped as CYP2A6*4A/CYP2A6*17 was correctly genotyped as CYP2A6*1/CYP2A6*17 (Figure 5b). The cotinine/nicotine ratio in this subject (2.5) was in the range of the ratios in the subjects with CYP2A6*1/CYP2A6*17 not possessing the CYP2A7 polymorphic alleles (5.4±3.0, 1.4–10.7, n=21). The cotinine/nicotine ratios in the subjects with CYP2A6*1/CYP2A6*4A or CYP2A6*4A/CYP2A6*9 were 4.3 and 0.9, respectively. The ratios in the homozygotes of CYP2A6*1 not possessing the CYP2A7 polymorphic alleles were 8.0±5.1 (0.9–30.4, n=92).

If the phenotype data of the subjects possessing the polymorphic CYP2A7 alleles might be mistakenly interpreted as the heterozygotes of the CYP2A6*4 allele, the data analyses of the relationship between the genotype and phenotype would be perverted.

Discussion

In the present study, we found novel CYP2A7 polymorphic alleles (CYP2A7*1B, CYP2A7*1C, and CYP2A7*1D) that have a gene conversion with the CYP2A6 sequence in the 3′-flanking region and confound the genotyping of the CYP2A6*4 allele. These alleles were found in European-Americans with a moderate frequency (0.5–1.9%), whereas they were very rare in the African-Americans, Japanese, and Koreans. By the discrimination of the CYP2A7 alleles and genuine CYP2A6*4 allele, the previous allele frequencies of CYP2A6*4 of 3.7% (European-Americans, n=187), 0.9% (African-Americans, n=176), and 22.6% (Japanese, n=184) were corrected to 0, 0.6, and 22.3%, respectively. Thus, the polymorphisms of CYP2A7 would lead to misgenotyping and overestimation of the allele frequency of CYP2A6*4A, particularly with individuals of European origin.

In the genotyping method by Ariyoshi et al.,15 the primer set of 2A6-B4 and 2A6UTR-AS1 was used (Figure 6a). The 2A6-B4 primer can anneal to exon 8 of both the CYP2A6 and CYP2A7 genes. The 2A6UTR-AS1 primer is the same as the 2A6R2 primer but is 2 bp longer. Therefore, the CYP2A7 polymorphic alleles would be also amplified (Figures 1a and 6a). Loriot et al.16 reported that the allele frequency of CYP2A6*4 was 4.0% in the French population (white subjects) by this genotyping method. The possibility cannot be excluded that the allele frequency of CYP2A6*4 might be overestimated.

Figure 6
figure 6

Summary of previous genotyping methods for the CYP2A6*4 allele. Closed and open boxes represent exons of the CYP2A7 and CYP2A6 genes, respectively and lines represent the introns. The horizontal arrows indicate the location and direction of the primers for the PCR. Dashed lines between the horizontal arrows represent the amplification of PCR. (a) Methods by Ariyoshi et al.15 and Nakajima et al.14 The primers of 2A6-B415 and 2Aint7F14 anneal to both the CYP2A6 and CYP2A7 genes. The primers of 2A6UTR-AS115 and 2A6R214 anneal to the CYP2A7 polymorphic alleles (CYP2A7*1B, CYP2A7*1C and CYP2A7*1D) as well as the CYP2A6 gene, resulting in the misgenotyping of CYP2A6*4. (b) Method by Oscarson et al.8 The primer of 2Aex7F anneals to both the CYP2A6 and CYP2A7 genes. The primer of 2A6R1 anneals to CYP2A7*1D allele as well as the CYP2A6 gene (PCR I). In PCR II, the primer of 2A7ex8F can anneal to the CYP2A7*1D allele as well as to the CYP2A6*4 allele. Therefore, this method also leads to misgenotyping of CYP2A6*4A. (c) Method by Goodz and Tyndale.17 The primer of 2A6R3 specifically anneals to the CYP2A6 gene, but not to the polymorphic CYP2A7 alleles. This method can distinguish between the CYP2A6*4A allele and the CYP2A7 polymorphic alleles.

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In the genotyping method by Oscarson et al.,8 the CYP2A6*4 allele was detected with a two-step PCR analysis. In the first PCR reaction (PCR I), the primer set of 2Aex7F and 2A6R1 was used (Figure 6b). The sense primer was designed to anneal to a common sequence located in exon 7 of both the CYP2A6 and the CYP2A7 genes, whereas the antisense primer was designed to anneal only to the sequence of the 3′-flanking region in the CYP2A6 gene (Figures 1a and 6b). In the second step (PCR II), the allele specific (AS)-PCR was performed with the primer sets of 2A6ex8F or 2A7ex8F and 2A6R2. However, this genotyping method cannot distinguish between CYP2A6*4 and CYP2A7*1D alleles, as the primer of 2A6R1 can anneal to the CYP2A7*1D allele (Figure 6b). Oscarson et al.8 reported that the allele frequencies of CYP2A6*4 in Finns (n=100) and Spaniards (n=100) were 1.0 and 0.5%, respectively. Owing to the presence of the CYP2A7*1D alleles, the allele frequencies of CYP2A6*4 in these populations might be actually lower than those values. The frequency of the CYP2A6*4 allele in Europeans remains to be determined by the improved genotyping method with a large number of subjects.

In the genotyping method by Goodz and Tyndale,17 the CYP2A6*4 allele was detected by the two-step PCR analysis reported by Oscarson et al.8 with a modification of the primer. In the first PCR reaction, the primer 2A6R3 was used instead of 2A6R1 used in the method by Oscarson et al.8 The 2A6R3 primer was designed to anneal only to the sequence of the 3′-flanking region in the CYP2A6 gene (Figures 1a and 6c). As the 2A6R3 primer cannot anneal to any polymorphic CYP2A7 alleles (Figure 6c), this genotyping method could identify the genuine CYP2A6*4A allele. Schoedel et al.18 reported that the frequency of the CYP2A6*4 allele in Canadian white subjects (n=1168) was 1.2% using this genotyping method. However, this genotyping method could not distinguish between the CYP2A6*4A and CYP2A6*4D alleles. In contrast, our improved genotyping method can distinguish the CYP2A6*4A, CYP2A6*4D, and the polymorphic CYP2A7 alleles.

The CYP2A6*4A allele completely lacks the enzymatic activity, as we previously reported with the in vivo phenotyping of nicotine.19, 20 The misgenotyping of the CYP2A6*4 allele results in an inconsistency between the genotype and phenotype. As some heterozygotes of the CYP2A6*4 allele can metabolize nicotine at levels similar to homozygotes of CYP2A6*1A,19, 20 the effects of the misgenotyping as heterozygotes of the CYP2A6*4A owing to the CYP2A7 polymorphic alleles on the phenotype were not dramatic (Figure 5). However, if subjects were misgenotyped as the CYP2A6*4A allele in combination with the other CYP2A6 alleles that are known to be inactive, such as CYP2A6*2, CYP2A6*7, CYP2A6*10, or CYP2A6*20, it would clearly confound the relationship between the genotype and phenotype.

Several studies have demonstrated an association between CYP2A6 genetic polymorphisms and the risk of lung cancer in the Japanese population.21, 22, 23 In contrast, no clear association was observed in Europeans and Chinese populations.16, 24, 25 In those studies, the misgenotyping of the CYP2A6*4 might be one of the possible factors in the contradiction. It is important to investigate the association of the CYP2A6 genetic polymorphisms and the interindividual differences in the enzymatic activity or the risk of cancer with an accurate CYP2A6 genotyping method. The comprehensive and reliable genotyping method for the CYP2A6*4 allele developed in the present study will be essential to improve the results of such population studies.

In conclusion, we found CYP2A7 polymorphic alleles in which the sequences of the 3′-flanking region were converted with the corresponding sequences of the CYP2A6 gene. These CYP2A7 alleles confound the genotyping analysis of CYP2A6*4A. The CYP2A7 polymorphic alleles were found in European-Americans with a moderate frequency but were very rare in African-Americans, Japanese, and Koreans. We must pay attention to the CYP2A7 alleles in the genotyping of the CYP2A6 gene.

Materials and methods

Chemicals and reagents

Taq DNA polymerase and Blend Taq DNA polymerase were obtained from Greiner Japan (Tokyo, Japan) and TOYOBO (Osaka, Japan), respectively. Restriction enzymes were purchased from Fermentas (Hanover, MD, USA), New England BioLabs (Beverly, MA, USA) or Takara (Kyoto, Japan). Primers were commercially synthesized at Hokkaido System Sciences (Sapporo, Japan). All other chemicals and solvents were of 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, USA) and the Ethics Committees of Kanazawa University (Kanazawa, Japan) and Soonchunhyang University Hospital (Chonan, Korea). We recruited 187 European-American, 176 African-American, 184 Japanese and 209 Korean subjects. Written informed consent was obtained from all subjects. Blood samples were collected from a cubital vein. Genomic DNA was extracted from peripheral lymphocytes by use of a Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN, USA).

Genotyping of CYP2A6 alleles

The genotyping of CYP2A6*1B,14 CYP2A6*1F,14 CYP2A6*1G,14 CYP2A6*1X2,26 CYP2A6*2,27 CYP2A6*3,27 CYP2A6*4A,14 CYP2A6*4D,14 CYP2A6*5,19 CYP2A6*6,19 CYP2A6*7,26 CYP2A6*8,26 CYP2A6*9,28 CYP2A6*10,26 CYP2A6*11,26 CYP2A6*12,14 CYP2A6*13,29 CYP2A6*14,29 CYP2A6*15,29 CYP2A6*16,29 CYP2A6*17,30 CYP2A6*18,29 CYP2A6*19,29 and CYP2A6*2031 was performed as described previously. An AS-PCR method for the SNP of g.51G>A was developed in the present study. The primer sets were 2A6-51G or 2A6-51A, and 2A6int1AS (Table 2). The reaction mixture contained the genomic DNA samples (100 ng), 1 × PCR buffer, 1.5 mM MgCl2, 0.4 μ M of each primer, 250 μ M of dNTPs and 1 U of Taq DNA polymerase in a final volume of 25 μl. After initial denaturation at 94°C for 3 min, amplification was performed by denaturation at 94°C for 25 s, annealing at 57°C for 25 s and extension at 72°C for 25 s for 28 cycles, followed by a final extension at 72°C for 5 min. The expected size of the PCR product was 285 bp.

Table 2 Primers used in the present study
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Sequence analyses of 3′-flanking region of the CYP2A7 gene

The PCR product with the primers of 2A6-delS and 2A7 FR-R (Table 2, Figure 1a) was subcloned into pT7Blue T-vector (Novagen, Madison, WI, USA). The PCR reaction mixture was the same as described above except for primers. After initial denaturation at 94°C for 3 min, amplification was performed by denaturation at 94°C for 25 s, annealing at 56°C for 25 s, and extension at 72°C for 2 min for 35 cycles, followed by a final extension at 72°C for 5 min. The plasmid DNA was submitted to DNA sequencing using a Long-Read Tower DNA sequencer (GE Healthcare Bio-Science, NJ, USA).

Detection of CYP2A7 polymorphisms

To detect the CYP2A7 polymorphic alleles (CYP2A7*1B, CYP2A7*1C and CYP2A7*1D), a PCR-RFLP method was developed. The PCR reaction mixture was the same as described above except for the primers (2A6-delS and 2A7-REV). The primers 2A6-delS and 2A7-REV anneal to the CYP2A7 gene (Table 2 and Figure 1). After initial denaturation at 94°C for 3 min, amplification was performed by denaturation at 94°C for 25 s, annealing at 58°C for 25 s and extension at 72°C for 30 s for 35 cycles, followed by a final extension at 72°C for 5 min. The PCR product was digested with Tai I restriction enzyme and electrophoresed on a 4% agarose gel. The wild-type of the CYP2A7 gene (CYP2A7*1A allele) yields 197-, 132- and 101-bp fragments; the CYP2A7*1B allele yields 197-, 132- and 95-bp fragments; the CYP2A7*1C allele yields 197-, 132- and 90-bp fragments; the CYP2A7*1D allele yields 197-, 128- and 90-bp fragments (Figure 1b).

To distinguish between CYP2A7*1A/CYP2A7*1C and CYP2A7*1A/CYP2A7*1D, we established an AS-PCR method. The PCR reaction mixture was the same as described above except for the primer sets (2A7 FR-F and 2A7-REV or 2A6 FR-F and 2A7-REV). The 2A7 FR-F primer anneals to CYP2A7*1A and CYP2A7*1C, whereas the 2A6 FR-F primer anneals to CYP2A7*1D (Table 2 and Figure 1). After initial denaturation at 94°C for 3 min, amplification was performed by denaturation at 94°C for 25 s, annealing at 56°C for 25 s and extension at 72°C for 25 s for 35 cycles, followed by a final extension at 72°C for 5 min. An aliquot (10 μl) of the PCR product was electrophoresed on a 4% agarose gel. The CYP2A7*1A and CYP2A7*1C alleles were amplified with the primer set of 2A7 FR-F and 2A7-REV (122 bp). The CYP2A7*1D allele was amplified with the primer set of 2A6 FR-F and 2A7-REV (118 bp) (Figure 1c).

Determination of the relative gene copy ratio of CYP2A6/CYP2A7

To investigate whether the subjects with the CYP2A7 polymorphic allele have two copies of the CYP2A6 gene, PCR analyses with the quantified genomic DNA (100 ng) were performed. The 2Aex3F and 2A6/7int3AS (Table 2) primers can anneal to both the CYP2A6 and CYP2A7 genes (Figure 2a). After an initial denaturation at 94°C for 3 min, amplification was performed by denaturation at 94°C for 25 s, annealing at 62°C for 25 s, and extension at 72°C for 30 s for 35 cycles, followed by a final extension at 72°C for 5 min. The PCR product derived from the CYP2A7 gene was digested to 273- and 118-bp fragments by EcoN I, whereas that from CYP2A6 gene was not digested (391 bp). These products were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining (Figure 2b). The intensities of the 391-bp (CYP2A6) and 273-bp (CYP2A7) fragments were quantified using ImageQuant TL (GE Healthcare Bio-Science). The ratio of the intensities corrected by the length of the fragment was calculated as the relative gene copy ratio of CYP2A6/CYP2A7. Standard curve was made using genomic DNA samples from the subjects who were genotyped as CYP2A6*4A/CYP2A6*4A (n=2, Japanese), CYP2A6*1/CYP2A6*4A (n=6, Japanese), CYP2A6*1/CYP2A6*1 (n=6, Japanese), and CYP2A6*1/CYP2A6*1X2 (n=1, Korean) (Figure 2c). Using the standard curve, we quantified the relative gene copy ratios of CYP2A6/CYP2A7 for European-American subjects who were genotyped as heterozygotes of the CYP2A6*4A allele.

To confirm the determination of the targeted exon 3, a similar analysis was performed at the different region (from exon 5 to intron 5). The 2A6E5F and 2A6/7int5AS (Table 1) primers were used (Figure 3a). Both primers can anneal to both the CYP2A6 and CYP2A7 genes. After an initial denaturation at 94°C for 3 min, amplification was performed by denaturation at 94°C for 25 s, annealing at 55°C for 25 s and extension at 72°C for 30 s for 35 cycles, followed by a final extension at 72°C for 5 min. The PCR product derived from the CYP2A7 gene was digested to 189- and 141-bp fragments by Taq I, whereas that from CYP2A6 gene was not digested (330 bp). These products were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining (Figure 3b). Standard curve was made with the genomic DNA samples described above (Figure 3c). The relative gene copy ratios of CYP2A6/CYP2A7 for European-American subjects who were genotyped as heterozygotes of the CYP2A6*4A allele were calculated using the standard curve.

Although there is no CYP2A7 gene in the CYP2A6*12 allele, no subject possessed the CYP2A6*12 allele in our study. In addition, as no duplicate or multiplicate alleles have been reported for CYP2A7 gene, the intensity of CYP2A7 fragment was used as control for two copies. As the sequences of CYP2A6 and CYP2A7 genes to which the primers anneal are completely identical, it was assumed that the PCR efficiencies for the two genes would be equal. Even if the PCR efficiencies might be different, it would appear in the slope of the standard curve. Thus, the relative gene copy ratios of CYP2A6/CYP2A7 were calculated using the standard curve.

Improved genotyping method for the CYP2A6*4 alleles

To avoid the misgenotyping of the CYP2A6*4A allele owing to the amplification of the polymorphic CYP2A7 alleles, an improved genotyping method was designed (Figure 4). The antisense primer 2A6reverse (Table 2) specifically anneals to the CYP2A6 gene and does not anneal to CYP2A7 polymorphic alleles. The primers of 2A6int8F and 2A7int8F (Table 2) specifically anneal to the CYP2A6 and CYP2A7 genes, respectively. The reaction mixture contained genomic DNA (100 ng), 1 × PCR buffer, 0.2 mM dNTPs, 0.4 μ M each primer, and 0.5 U of Blend Taq DNA polymerase in a final volume of 25 μl. After initial denaturation at 94°C for 3 min, amplification was performed by denaturation at 94°C for 25 s, annealing at 54°C for 25 s, and extension at 72°C for 2 min for 35 cycles, followed by a final extension at 72°C for 5 min. An aliquot (5 μl) of the PCR product was analyzed by electrophoresis with 0.8% agarose gel. The CYP2A6*1A and CYP2A6*1B alleles were amplified with the primer set of 2A6int8F and 2A6reverse (1937 and 1936 bp, respectively). The CYP2A6*4A and CYP2A6*4D alleles were amplified with the primer set of 2A7int8F and 2A6reverse (1935 and 1936 bp, respectively). These PCR products were digested with Acc II at 37°C for 3 h and electrophoresed on a 2% agarose gel (Figure 4b and c). The PCR products derived from the CYP2A6*1B and CYP2A6*4A alleles were digested by Acc II, but those from CYP2A6*1A and CYP2A6*4D were not.

In vivo phenotyping of nicotine metabolism

Written informed consent was obtained from 187 healthy European-American and 176 healthy African-American non-smokers. The phenotyping of in vivo nicotine metabolism was performed according to the method established in our previous study.19 Briefly, the subjects chewed one piece of nicotine gum 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.32 The cotinine/nicotine ratio of the plasma concentration was calculated as an index of nicotine metabolism. As nicotine and cotinine were detected in the plasma before chewing nicotine gum, 11 European-Americans and 16 African-Americans were judged as smokers. Therefore, the phenotype data from 176 European-Americans and 160 African-Americans were analyzed along with the CYP2A6 genotype data.