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SHORT COMMUNICATION

HAPLOTYPE STRUCTURE AND ALLELE FREQUENCIES OF CYP2B6 IN A KOREAN POPULATION

Clinical Pharmacology Unit, Department of Pharmacology, College of Medicine, Seoul National University, Seoul, South Korea

(Received June 20, 2004; accepted September 15, 2004)

	Abstract

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Cytochrome P450 2B6 (CYP2B6) metabolizes a number of therapeutic drugs and its metabolic activity varies markedly in human liver. Although genetic polymorphisms of CYP2B6 have been reported in noncoding and coding regions, little information is available regarding single nucleotide polymorphisms (SNPs) and their haplotypes in noncoding regions in Asians. Fourteen previously reported SNPs were determined by polymerase chain reaction-restriction fragment length polymorphism or SNaPshot analysis in a Korean population and their haplotypes were inferred from genotype data using an expectation-maximization algorithm. The most common haplotypes were haplotype I, the reference sequence (frequency 0.35), haplotype II (0.19), haplotype III (0.19), and haplotype V (0.12), which together accounted for 85% of all haplotypes. The frequency of haplotype III, which contains -2320C, -1778G, -1186G, -750C, and 15582T, was found to be 2.4-fold higher than that of the ^*1J allele in Caucasians, and the frequency of haplotype V, which contains -8207C, -1456C, -750C, 516T, and 785G, was 55% of that of the ^*6B allele in Caucasians. Moreover, haplotype V, the ^*6B allele, appeared to be completely linked to -8207 within a putative nuclear receptor binding motif, suggesting that lower expressions of the ^*6B allele may be associated with the presence of noncoding SNPs such as -8207G>C linked to nonsynonymous SNPs. In conclusion, we found 11 previously described polymorphisms and identified four major haplotypes of CYP2B6 in Koreans. The frequencies of the ^*1J or ^*6B alleles, which may reduce CYP2B6 enzyme expression, were found to be significantly different between Koreans and Caucasians.

Lang et al. (2001) first identified five nonsynonymous polymorphisms of the CYP2B6 gene in Caucasians. Lamba et al. (2003) and Hesse et al. (2004) have reported additional polymorphisms in the coding regions, introns, and the 5'-flanking region of this gene, which could have a substantial influence on CYP2B6 expression. In addition, these two authors constructed haplotypes of the CYP2B6 gene in Caucasian, African-American, and Hispanic populations. Although Hiratsuka et al. (2002) determined the frequencies of SNPs only in the coding region of CYP2B6 gene in the Japanese, SNPs of CYP2B6 and their haplotypes reported recently in noncoding regions have not been studied in Asians. Therefore, to compare the frequencies of the SNPs and haplotypes of CYP2B6 gene in Asians and other populations, we genotyped known CYP2B6 polymorphisms in a large Korean population and reconstructed haplotypes from this genotype data.

	Materials and Methods

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Materials. QIAamp DNA Blood Mini Kits were purchased from QIAGEN GmbH (Hilden, Germany), and restriction enzymes were obtained from New England Biolabs (Beverly, MA). Exonuclease I and shrimp alkaline phosphatase (exo/SAP) were from USB (Cleveland, Ohio, USA), and AmpliTaq DNA polymerase and the ABI Prism SNaPshot multiplex kits were purchased from Applied Biosystems (Foster City, CA, USA). All other reagents were of the highest grade commercially available.

DNA Samples. Blood samples were obtained from 358 unrelated healthy Korean volunteers (242 men, 116 women) for genotyping analysis. Among them, 158 subjects were randomly chosen for haplotype analysis. All subjects provided written informed consent, and the study was approved by the ethical committee of Seoul National University Hospital. Genomic DNA was extracted from peripheral whole blood using a QIAamp DNA Blood Mini Kit.

Genotyping of CYP2B6 Alleles. The GenBank accession numbers used as a CYP2B6 reference sequence in this study were NG_000008 [GenBank] and NM_000767 [GenBank] , respectively, for the CYP2B6 gene and its mRNA. For SNPs in the CYP2B6 coding region, genotyping of 64C>T, 516G>T, 777C>A, 785A>G, and 1459C>T was carried out by PCR-restriction fragment length polymorphism (RFLP) assay, as described previously (Lang et al., 2001). Additional reported SNPs, namely, -8427T>C, -8207G>C, -2320T>C, -1778A>G, -1456T>C, -1186C>G, -750T>C, 415A>G, and 15582C>T, were analyzed by SNaPshot analysis or by PCR-RFLP. All PCR reactions were performed in a 20-µl volume consisting of 1.5 mM MgCl₂, 250 µM deoxynucleoside-5'-triphosphates, 0.5 pmol of each primer, and 0.25 unit of AmpliTaq DNA polymerase. After an initial denaturation at 94°C for 10 min, DNA was amplified over 30 cycles (denaturation 95°C for 30 s, annealing for 1 min, and extension at 72°C for 1 min) and then extended at 72°C for 7 min. Details of the primer sets used for the PCR amplification, annealing temperatures, PCR product size, and of the PCR-RFLP restriction enzymes are given in Table 1. For the -1456T>C and -1186C>G polymorphisms, digestion with AccI and BseRI for 16 h at 37°C, yielded the following fragments: 81 and 400 bp for -1456TT, 481 bp for -1456CC, 125 and 356 bp for -1186CC, and 481 bp for -1186GG.

For SNaPshot analysis, exo/SAP-purified PCR products were mixed with AmpliTaq DNA polymerase, four fluorescently labeled dideoxynucleoside-5'-triphosphates, and the reaction buffer contained in an ABI Prism SNaPshot multiplex kit. The analysis was performed by following the manufacturer's protocol. The details of the internal reverse primers used for single base extension are also shown in Table 1. The primers were extended over 25 cycles of 96°C for 10 s, 50°C for 5 s, and 60°C for 30 s. Amplicons were then purified with exo/SAP and analyzed on an ABI Prism 3700 Automated Sequencer (Applied Biosystems, Foster City, CA). The DNA sequences near polymorphic sites were confirmed by direct sequencing.

Statistical Analysis. The frequencies of SNPs were calculated from the observed numbers of minor alleles at each polymorphic locus. Hardy-Weinberg equilibrium tests were done using a modified Markov-chain random walk algorithm in the population genetics data analysis program Arlequin, version 2.000 (University of Geneva, Switzerland). Haplotype structures and their frequencies were estimated from the observed number of genotypes using an expectation-maximization algorithm in Arlequin (Excoffier and Slatkin, 1995).

The CYP2B6^*1A allele was assumed when any of the above-tested SNPs did not exist. Differences in allele frequencies between Korean and other populations were determined using chi-square tests. A p value of <0.05 was considered statistically significant.

	Results and Discussion

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Fourteen SNPs of CYP2B6, as identified previously in Caucasians, were investigated in 358 Korean subjects by using PCR-RFLP or the SNaPshot method, as shown in Table 1. Seven SNPs in the 5'-flanking region of CYP2B6 were genotyped. Among them, four SNPs at -8427, -8207, -2320, and -750 were reported previously to lie within the four putative transcription factor-binding sites (Lamba et al., 2003). Also, an SNP in intron 3 branch site (15582) and six nonsynonymous SNPs were genotyped. As shown in Table 2, the frequencies of -8207G>C, -2320T>C, -1778A>G, -1456T>C, -1186C>G, -750T>C, 64C>T (R22C), 15582C>T (intron 3), 516G>T (Q172H), 785A>G (K262R), and 1459C>T (R487C) SNPs were 0.14, 0.47, 0.21, 0.15, 0.21, 0.62, 0.03, 0.47, 0.14, 0.19, and 0.01, respectively. All of the above polymorphisms were in Hardy-Weinberg equilibrium. The frequencies of SNPs -2320T>C, -1778A>G, -1186C>G, -750T>C, 64C>T, 15582C>T, 516G>T, 785A>G, and 1459C>T differed significantly from those of Caucasians as reported by Lamba et al. (2003). The frequencies of -8207G>C and -1456T>C in Koreans were lower than those in Caucasians but not statistically significant because the number of alleles evaluated in Caucasians was relatively smaller than that of Koreans in this study. The frequencies of coding SNPs in Koreans were found to be similar to those of the Japanese, as reported by Hiratsuka et al. (2002), except that of the 785A>G SNP. In the present study, the -8427T>C, 415A>G (K139E), and 777C>A (S259R) SNPs were not found in any subject.

For haplotype analysis of the CYP2B6 gene, genotype data of 158 Korean subjects were used since some SNPs with the high frequencies were genotyped in 158 subjects only. We found no difference between the frequencies of 7 commonly genotyped SNPs in our pool of 158 versus 358 subjects (data not shown). The 10 most common SNPs in more than 1% of subjects were included in the analysis to enhance model stability. Five major haplotypes with frequencies >5% could be inferred using the expectation-maximization algorithm as shown in Table 3. The most common haplotypes were haplotype I, the reference sequence (frequency 0.35), haplotype II (0.19), haplotype III (0.19), and haplotype V (0.12), which together accounted for 85% of all Korean haplotypes. Haplotype II containing -2320C, -750C, and 15582T corresponded to CYP2B6^*1H; haplotype III with -2320C, -1778G, -1186G, -750C, and 15582T to ^*1J, and haplotype V with -8207C, -1456C, -750C, 516T, and 785G to ^*6B. The frequencies of haplotypes III and V in Koreans were 2.4-fold higher and at 55% of their respective levels in Caucasians as reported by Hesse et al. (2004) (Table 3).

Previous studies have suggested that the promoter SNPs -2320T>C and -750T>C, included in both CYP2B6^*1H and ^*1J alleles, may affect the transcriptional regulation of CYP2B6 by disrupting the GATA and hepatic nuclear factor 1 binding sites, respectively (Lamba et al., 2003; Hesse et al., 2004). Lamba et al. (2003) also suggested that the 15582C>T SNP located at the intron 3 branch point site may affect mRNA splicing and thereby negatively impact the CYP2B6 phenotype. Haplotypes II, III, and IV in this study commonly included three noncoding SNPs, -2320T>C, -750T>C, and 15582C>T, which appeared to be in linkage disequilibrium to a high degree. The frequencies of haplotypes II, III, and IV totaled 0.43 in Koreans, whereas those of the CYP2B6^*1H and ^*1J alleles including the above three SNPs totaled 0.31 in Caucasians. In contrast, the frequencies of haplotype V (^*6B) in Koreans and Caucasians were 0.12 and 0.22, respectively.

Although the in vitro expressed CYP2B6 variants, Q127H, K262R, and CYP2B6.6 (Q127H and K262R), were found to have enhanced catalytic activity of this enzyme (Ariyoshi et al., 2001; Jinno et al., 2003; Xie et al., 2003), most recent studies have reported the association of the ^*6 allele with a low level of CYP2B6 expression. Xie et al. (2003) have shown that the ^*6 allele was associated with a trend toward lower CYP2B6 protein expression and cyclophosphamide hydroxylation activity in the human liver. Moreover, several clinical pharmacokinetic studies have recently reported that the ^*6 allele was correlated with reduced bupropion clearance in healthy subjects and with high plasma concentrations of efavirenz metabolized by CYP2B6 in human immunodeficiency virus type 1 patients (Kirchheiner et al., 2003; Tsuchiya et al., 2004). In another study, the ^*6B allele, including -1456C and -750C in linkage disequilibrium with the two common nonsynonymous polymorphic sites, 516T and 785G, was found to be correlated with reduced CYP2B6 activity (Hesse et al., 2004). Similar to the results of Lamba et al. (2003), we also found that the ^*6B allele is completely linked to another upstream site (-8207) within a putative nuclear hormone receptor binding motif (DR2) in addition to -1456 and -750 loci (Podvinec et al., 2002). We speculated that the lower expression of the ^*6B allele could be related to the presence of noncoding SNPs such as -8207G>C and -1456T>C linked to nonsynonymous SNPs.

A recent report described the four very low or null activity variants M46V, G99E, K139E, and I391N, and their frequencies were below 1% in Caucasians (Lang et al., 2004). Although the 415A>G (K139E) was not found in Koreans in the present study, additional studies were needed to determine the frequencies of M46V, G99E, and I391N in Koreans.

In this study, we determined the frequencies of 14 genetic polymorphisms of CYP2B6 and estimated the frequencies of four major haplotypes in Koreans. The frequencies of both ^*1J and ^*6B alleles, which may be associated with variable levels of this enzyme expression, were found to be significantly different between Koreans and Caucasians. Although the estimates of haplotype structure and frequencies will require confirmation by molecular methods, such ethnic differences in the frequencies of defective alleles may contribute to different therapeutic responses of CYP2B6-metabolized drugs among ethnic groups and further reflect the difficulties associated with the global development, evaluation, and use of therapeutic agents.

	Acknowledgments

 
We thank Hyo-Bum Seo and Hwa-Sook Kim for excellent technical assistance.

	Footnotes

 
This study was supported by a grant of the Korea Health 21 R&D Project, Ministry of Health & Welfare, R.O.K (03-PJ10-PG13-GD01-0002).

doi:10.1124/dmd.104.001107.

ABBREVIATIONS: SNP, single nucleotide polymorphism; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; exo/SAP, exonuclease I and shrimp alkaline phosphatase.

Address correspondence to: In-Jin Jang, Department of Pharmacology, College of Medicine, Seoul National University, 28 Yongon-Dong, Chongno-Gu, Seoul, 110-799, South Korea. E-mail: ijjang{at}snu.ac.kr

	References

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This Article Abstract Full Text (PDF) All Versions of this Article: dmd.104.001107v1 32/12/1341    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cho, J.-Y. Articles by Jang, I.-J. Search for Related Content PubMed PubMed Citation Articles by Cho, J.-Y. Articles by Jang, I.-J.