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Homologous Unequal Cross-Over Within the Human CYP2A Gene Cluster As a Mechanism for the Deletion of the Entire CYP2A6 Gene Asso

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Homologous Unequal Cross-Over Within the Human CYP2A Gene Cluster As a Mechanism for the Deletion of the Entire CYP2A6 Gene Asso J. Biochem. 126, 402-407 (1999) Homologous Unequal Cross-Over within the Human CYP2A Gene Cluster as a Mechanism for the Deletion of the Entire CYP2A6 Gene Associated with the Poor Metabolizer Phenotype1 Ken-ichi Nunoya,* Tsuyoshi Yokoi,* Yuki Takahashi,* Kanzo Kimura,•õ Moritoshi Kinoshita,•ö and Tetsuya Kamataki*,2 *Laboratory of Drug Metabolism , Graduate School of Pharmaceutical Sciences, Hokkaido University, N12W6, Sapporo 060-0812; •õClinical Research and Development Division, Sumitomo Pharmaceuticals Co., Ltd., Osaka 541-0045; and tDiagnostics Division, Otsuka Pharmaceutical Co., Ltd., Tokushima 771-0130 Received May 12, 1999; accepted June 5, 1999 To clarify the molecular mechanisms involved in the generation of the CYP2A6 gene deletion (E-type variant), we analyzed the CYP2A7 gene, which is located in the 5?-flanking region of the CYP2A6 gene, from individuals with the E-type variant and compared it with the sequences of wild type CYP2A7 and CYP2A6 genes. The 3?-downstream sequence (up to 324 by from the SacI site in exon 9) of the CYP2A7 gene of the E-type variant is identical to that of the wild CYP2A7 gene. However, the 3?-downstream sequence (starting from 325 by from the SacI site in exon 9) of the CYP2A7 gene of the E-type variant is identical to that of the wild CYP2A6 gene, indicating that the 3?-downstream region of CYP2A7 and the 3?-downstream region of CYP2A6 linked directly eliminating the whole CYP2A6 gene. PCR analysis using primers specific to the CYP2A7 gene and the CYP2A6 and CYP2A7 genes confirmed that all DNA samples obtained from 7 individuals carrying the E-type variant possessed the same break points. These results indicate that the breakpoint of the CYP2A6 gene deletion lies in the 3?-downstream region of the CYP2A7 and CYP2A6 genes. Key words: coumarin, genetic polymorphism, gene analysis, pharmacogenetics, RFLP. Cytochrome P450 (CYP) is a heme-containing enzyme found new variant alleles that account for the large interin responsible for the metabolism of exogenous compounds dividual variations of CYP2A6 activity in a Japanese such as drugs, environmental pollutants, and dietary population using SM-12502 as a new probe drug. The chemicals, and endogenous compounds such as steroids, disposition of SM-12502 was investigated in the plasma or fatty acids, and prostaglandins (1). Among the forms of urine from 28 healthy Japanese volunteers after a single CYP, CYP2A6 has been recognized as a form with unique intravenous administration of SM-12502. Three of the 28 catalytic properties. CYP2A6 was first shown to catalyze Japanese were phenotyped as PM and 25 were EM (15). coumarin 7-hydroxylation (2-5) . Studies by us have shown The CYP2A gene cluster contains five tandemly arranged that it also catalyzes SM-12502 S-oxidation (6) and coti genes including three complete genes, CYP2A6, CYP2A7, nine 3?-hydroxylation (7). This molecular form of CYP is and CYP2A13, and two pseudogenes, CYP2A7PC and also capable of metabolically activating genotoxins includ CYP2A7PT, on chromosome 19q13.2 (16, 17). The ing aflatoxin B1 (8), NDEA (9), and the tobacco-specific CYP2A7 and CYP2A13 genes have DNA sequences similar NNK (10). It has been noted that there is great interin to that of the CYP2A6 gene (2, 16), while no substrates for dividual variation in the activity of the coumarin 7-by- CYP2A7 or CYP2A13 have been reported. droxylase in vivo (11-13). Recently, Nunoya et al. (14,15) We proposed in a previous paper that there are at least five variants, namely A, B, C, D, and E, as judged by 1 This study was supported in part by a Grant-in-Aid from the SacI-RFLP. Among them, the E-type variant shows ex Ministry of Education, Science, Sports and Culture of Japan, and also tremely low S-oxidase activity to SM-12502. The E-type from the Ministry of Health and Welfare for the second-term Com variant was also shown to be caused by the whole deletion prehensive2To whom correspondence10-Year Strategy should for Cancer be addressed Control. of the CYP2A6 gene (15). Moreover, the SacI-D-type . Tel: +81-11-706- seemed to be a significant factor in the poor metabolic 3233, Fax: +81-11-706-4978, E-mail: [email protected]. ac.jp capacity of SM-12502 in human liver microsomes (14) . The Abbreviations: CYP, cytochrome P450; dATP, deoxyadenosine 5?- frequency of CYP2A6 gene deletions (D-type and E-type) triphosphate; dCTP, deoxycytidine 5?-triphosphate; dGTP, deoxygu in the Japanese population is 4.5% (15). The frequency of anosine 5?-triphosphate; dNTP, deoxynucleotide 5'-triphosphates; the SacI-E-type variant is 3.2%. We also observed that dTTP, deoxythymine 5?-triphosphate; EM, extensive metabolizer; 6.4-, 4.5-, and 2.6-kb fragments, which are present in the NDEA, N-nitrosodiethylamine; NNK, 4-(methylnitrosamino)-1-(3- wild type gene, are absent in the E-type variant of SacI- pyridyl)-1-butanone; PM, poor metabolizer; SM-12502, (+)-cis-3,5- dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride. RFLP. The lack of a fragment about 13-kb in size as compared with the wild type allele was also noted in c 1999 by The Japanese BiochemicalSociety. SphI-RFLP. 402 J. Biochem . Mechanism for the Deletion of CYP2A6 Gene 403 In the present study, we analyzed the sequence of the DNA from subjects PM3 and EM3 was extracted from CYP2A7 gene, which locates in the 5?-flanking region of the peripheral lymphocytes with phenol-chloroform followed CYP2A6 gene, from individuals with the E-type variant by ethanol precipitation (18). The examination of CYP2A6 and compared it with the sequences of the CYP2A7 and genotypes and phenotypes as judged by the in vivo metabo CYP2A6 genes from wild type individuals. Break points in lism of SM-12502 has already been reported (15). Subject the gene were found to be present at about 300 by 3?-down EM3 was judged to show the wild type phenotype and to be stream from the stop codon of the CYP2A7 and CYP2A6 a homozygote of the wild genotype or a heterozygote of gene, generating the deletion of the entire CYP2A6. wild/mutant genotypes. On the other hand, subject PM3 showed the PM phenotype and was judged to be a homo- MATERIALS AND METHODS zygote of the CYP2A6 gene deletion. Genomic DNAs from subjects PM3 and EM3, partially digested with Sau3AI, Materials-Restriction endonuclease was obtained from were fractionated by a sucrose density gradient (10-38%) Toyobo (Osaka), Takara Shuzo (Kyoto), or New England centrifugation. Fractions containing fragments with mean Biolabs (Beverly, MA), nylon membranes (Nytran NY13) lengths of 15-kb were partially filled-in with dGTP and were from Schleicher & Schuell (Dassel, Germany), dATP, and ligated to a lambda FIXII vector, which had been lambda FIXII, Pfu DNA polymerase, and Gigapack III Gold digested with XhoI and partially filled-in with dTTP and Packaging Extracts were from Stratagene (La Jolla, CA), dCTP (Stratagene, CA, USA). The ligated product was T4 DNA polymerase was from Takara Shuzo (Kyoto), packaged in vitro using Gigapack III Gold Packaging Sequenase Ver. 2 was from United States Biochemical Extracts. This library was screened by the plaque hybridi zation method using the 1.6-kb fragment of human CYP- (Cleveland, OH), and Ampli Taq DNA Polymerase was from Perkin Elmer Cetus (Norwalk, CT). All other chemi 2A6 cDNA as a probe. Phage DNAs were purified from cals and solvents were of the highest grade commercially positive plaques. The inserted DNA fragments were digest- available. ed with various restriction enzymes, and subcloned into Synthesis of Oligonucleotide-Oligonucleotides were pUC18, M13mp18, or M13mp19. The nucleotide se synthesized with a DNA synthesizer (Model 381A; Applied quences of the positive clones were analyzed by the dideoxy Biosystems, Foster City, CA). The sequences and locations method with Sequenase Ver. 2 or an Applied Biosystems of the synthesized CYP2A oligonucleotides are shown in Model 373A DNA sequencer following the protocol pro Table I. vided by the manufacturer. Preparation of Genomic DNA and Southern Blot Anal PCR Analysis of the 5?-Flanking Region of the CYP2A6 Gene-To confirm that the E-type variant contains the yses-Genomic DNA was prepared from peripheral lym 5?-flanking region of the CYP2A6 gene, PCR amplification phocytes by phenol-chloroform extraction followed by was carried out. Primers 2A6-kaS1, 2A6-kaAS1, 2A6- ethanol precipitation (18). The DNA preparations (10ƒÊg) were digested with restriction endonucleases. The digested kuS1, and 2A6-kuAS1 were designed and used to amplify DNA was subjected to electrophoresis in 0.6% agarose gels. the 5?-flanking region of the CYP2A6 gene (Table I). PCR The gels were treated with 0.5M NaOH containing 1.5M was performed in a reaction mixture containing 10mM Tris-HC1 buffer (pH 8.3), 50mM KCl, 1.5mM MgCl2, NaCl, neutralized with 0.5M Tris-HCl buffer (pH 8.0) 0.001% (w/v) gelatin, each dNTP (200ƒÊM), primer (0.5ƒÊ containing 1.5M NaCl, and equilibrated with 0.3M sodium citrate containing 3M NaCl prior to the transfer of the M), genomic DNA (500ng), and 1 U of Ampli Taq DNA DNA to nylon membranes. The membranes were baked at polymerase in a final volume of 50ƒÊl. Thirty cycles of amplification were performed using a DNA Thermal Cycler 80°C for 2h, prehybridized at 65•Ž for 2 h, and then under the following conditions: denaturation at 94•Ž for 2 hybridized at 65•Ž for 8h. Hybridization was performed in min, annealing at 48•Ž for 2 min, and extension at 72•Ž for reaction mixtures containing the 1.6kb fragment of a 32P -labeled human CYP2A6 cDNA, 50mM Tris-HCl buffer 2 min.
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