Drug Metabolism and Disposition Fast Forward
First published on November 12, 2007; DOI: 10.1124/dmd.107.019141
0090-9556/08/3602-349-352$20.00
DMD 36:349-352, 2008
Genetic Polymorphisms and Haplotype Structures of the Human CYP2W1 Gene in a Japanese Population
Yoshiyuki Hanzawa,
Takamitsu Sasaki,
Michinao Mizugaki,
Masaaki Ishikawa, and
Masahiro Hiratsuka
Department of Clinical Pharmaceutics, Tohoku Pharmaceutical University, Sendai, Japan (Y.H., T.S., M.I., M.H.); and Department of Clinical Pharmacotherapeutics, Tohoku Pharmaceutical University, Sendai, Japan (Y.H., T.S., M.M., M.H.)
(Received October 8, 2007;
Accepted November 8, 2007)
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Abstract
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A novel human cytochrome P450, designated CYP2W1, has recently been identified and is found to be present mainly in tumor cells, particularly in colon cancer cells. In the present study, we report the first systematic investigation of polymorphisms in the human CYP2W1 gene. Based on denaturing high performance liquid chromatography analyses of polymerase chain reaction products, we analyzed nine exons and exon-intron junctions of the gene in DNA samples from 200 Japanese subjects and identified six single nucleotide polymorphisms (SNP). Three of the novel nonsynonymous SNPs were as follows: 173A>C (Glu58Ala) in exon 1 and 5432G>A (Val432Ile) and 5584G>C (Gln482His) in exon 9. Two previously known nonsynonymous SNPs, that is, 2008G>A (Ala181Thr) in exon 4 and 5601C>T (Pro488Leu) in exon 9, were also found. On haplotype analyses, in addition to the wild-type CYP2W1*1A (frequency, 0.295) allele, other alleles, namely, CYP2W1*1B (0.318), CYP2W1*2 (0.005), CYP2W1*3 (0.005), CYP2W1*4 (0.008), CYP2W1*5 (0.003), and CYP2W1*6 (0.368), were also characterized. The most common allele, CYP2W1*6, exhibited the amino acid substitution Pro488Leu. These results were in good agreement with the expected genotype distributions that were calculated using the Hardy-Weinberg equation. The data on variant alleles and comprehensive haplotype structures would be useful for predicting the metabolic phenotypes of CYP2W1 substrates in the Japanese population.
Cytochromes P450 (P450s) are mono-oxygenases that play an important role in the oxidative metabolism of many therapeutic drugs and endogenous compounds such as fatty acids, vitamins, and steroids. The P450 enzymes are expressed in high levels in the liver, but these enzymes are also found in extrahepatic tissues. Extrahepatic tissues with high P450 expression levels are the respiratory and gastrointestinal tracts that are exposed to foreign compounds entering the body. The extrahepatic P450 enzymes can be important for tissue-specific metabolic activation or the inactivation of xenobiotic compounds.
CYP2W1, an extrahepatic P450, was recently identified. In adult human tissues, CYP2W1 mRNA was either not expressed or expressed at very low levels (Choudhary et al., 2005
; Aung et al., 2006; Karlgren et al., 2006). In human tumor tissues, CYP2W1 mRNA is expressed in the colon and adrenal gland (Karlgren et al., 2006). In particular, high CYP2W1 mRNA and protein expressions were found in HepG2 cells (Karlgren et al., 2006). Several studies on CYP2W1 have recently investigated the activity of CYP2W1 heterologously expressed in Escherichia coli and mammalian cells. Karlgren et al. (2006) reported that the CYP2W1 expressed in HEK293 cells catalyzes the oxidative metabolic conversion of arachidonic acid into 8,9-dihydroxyeicosatrienoic acid (DHET), 11,12-DHET, and 14,15-DHET at a small but significant rate. Wu et al. (2006) reported that recombinant CYP2W1 expressed in E. coli metabolizes benzphetamine and catalyzes the activation of several procarcinogens, particularly polycyclic hydrocarbon diols. Yoshioka et al. (2006) also showed that CYP2W1 expressed in E. coli catalyzes the oxidation of indoles.
Wide interindividual differences in metabolic capacity have been detected in many CYP enzymes. For CYP2W1, several single nucleotide polymorphisms (SNPs) have been reported thus far, but three of them are located only within exons (http://www.ensembl.org and http://www.hapmap.org). The SNP 166C>T that is located in exon 1 is silent, but both the other 2 SNPs, namely, 2008G>A in exon 4 and 5601C>T in exon 9, give rise to the amino acid exchanges Ala181Thr and Pro488Leu, respectively.
In the present study, we systematically investigated the variants of CYP2W1 in a population sample that comprised 200 Japanese subjects. To analyze the protein-coding region of all of the 9 exons and find novel genetic variations, we used a denaturing high performance liquid chromatography (DHPLC) system. In addition, cloning methods were used to determine these haplotypes of CYP2W1.
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Materials and Methods
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Subjects and DNA Samples. Venous blood was obtained from 200 unrelated healthy Japanese volunteers, and the patients were admitted to Tohoku University Hospital. Written informed consent was obtained from all blood donors, and the study was approved by the Local Ethics Committee of Tohoku University Hospital and Tohoku Pharmaceutical University. DNA was isolated from K2EDTA-anticoagulated peripheral blood by using QIAamp DNA Mini Kits (QIAGEN, Hilden, Germany) in accordance with the manufacturer's instructions.
PCR Amplification. Table 1 lists the primer pairs that were used to amplify the entire coding sequence and the exon-intron junctions of the CYP2W1 gene. These primers were designed based on the genomic sequence reported in GenBank (accession number NT_007819
[GenBank]
). Amplicons of exon 1 were generated using the AmpliTaq Gold PCR Master Mix (Applied Biosystems, Foster City, CA). PCR reactions were performed using the iCycler (Bio-Rad, Hercules, CA). Moreover, the method relied on the combination of the slowdown method (Bachmann et al., 2003) and the addition of betaine (Sigma-Aldrich, St. Louis, MO) for the region with high GC content (>70%). The PCR protocol was as follows: denaturation at 95°C for 5 min followed by 48 cycles of denaturation at 95°C for 30 s, annealing for 30 s, extension at 72°C for 40 s, and, finally, 15 additional cycles with an annealing temperature of 60°C. The amplicons for exons 2 to 9 were generated using the AmpliTaq Gold PCR Master Mix and betaine addition. The PCR protocol was as follows: denaturation at 95°C for 10 min followed by 30 or 35 cycles of denaturation at 95°C for 30 s, annealing for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 7 min. The annealing temperatures and PCR cycles for the screening of CYP2W1 variants are summarized in Table 1.
DHPLC Analysis. The PCR products were analyzed using the DHPLC system (WAVE; Transgenomic, Omaha, NE) (Hiratsuka et al., 2004a,b; Ebisawa et al., 2005; Hiratsuka et al., 2005; Hiratsuka et al., 2006; Sasaki et al., 2006; Hanzawa et al., 2007). Unpurified PCR samples (5 µl) were separated on a heated C18 reverse-phase column (DNASep) by using 0.1 M triethylammonium acetate in water and 0.1 M triethylammonium acetate in 25% acetonitrile at a flow rate of 0.9 ml/min. The software provided with the instrument selected the temperature for the heteroduplex separation in the heterozygous CYP2W1 fragment. Table 1 summarizes the DHPLC running conditions for each amplicon. The linear acetonitrile gradient was adjusted to the retention time of the DNA peak at 4 to 5 min.
Homozygous nucleotide exchanges can occasionally be distinguished because of a slight shift in the elution time compared with the reference. The addition of an approximately equal amount of wild-type DNA to the samples (1:1) before the denaturation step enabled the reliable detection of homozygous alterations. This was performed routinely for all samples to identify homozygous sequence variations. Therefore, all the samples were analyzed as follows: first, the sample alone to detect heterozygotes; then, after mixing each sample with wild-type DNA to detect homozygous variants. The resultant chromatograms were compared with those of the wild-type DNA.
Direct Sequencing. Both strands of samples with variants as determined by DHPLC were analyzed using a CEQ 8000 automated DNA sequencer (Beckman Coulter, Fullerton, CA). We also sequenced all samples with chromatographic findings that differed from the wild type to establish links between mutations and specific profiles. We sequenced the PCR products by fluorescent dideoxy termination by using the DTCS DNA Sequencing Kit (Beckman Coulter) in accordance with the manufacturer's instructions.
Haplotype Analysis. To determine the linkage among the polymorphisms identified in this study, PCR reactions were used to amplify long fragments that were obtained from individuals who were heterozygous at sites of interest. Long amplicons (5701 base pairs) were generated with LA Taq DNA polymerase (Takara, Kyoto, Japan). The PCR protocol was as follows: denaturation at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 1 min, extension at 68°C for 5 min, and a final extension at 72°C for 7 min. The forward and reverse primers were 5'-ggacggggcccaggaggggagtgga-3' and 5'-gctgggaggggagtggtcaggagga-3', respectively. The fragment was run on a low-melting gel, gel-purified, and ligated to the pCR-XL-TOPO vector (Invitrogen, Carlsbad, CA). After ligation, the plasmid was transfected into E. coli strain TOP10 (Invitrogen), and single colonies (each containing a plasmid with only one of the two alleles) were grown and subjected to plasmid isolation and sequencing using the CEQ 8000 automated DNA sequencer. In cases of samples having both heterozygous 5601C>T and 166C>T, eight samples were cloned and sequenced to ascertain whether they are found together on the same chromosome.
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Results
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DHPLC and Sequence Analysis. DHPLC analysis of the CYP2W1 gene for the 200 DNA samples obtained from Japanese individuals revealed chromatographic profiles that were distinct from those of the wild type for exons 1, 4, and 9. Direct sequencing analysis of the deviant DNAs detected by DHPLC revealed a total of 6 different polymorphisms, including 3 novel nonsynonymous polymorphisms (Glu58Ala, Val432Ile, and Gln482His), 2 known nonsynonymous polymorphisms (Ala181Thr and Pro488Leu), and 1 silent SNP (Leu56Leu). The locations and frequencies of these polymorphisms are described in Table 2. We did not observe any variants affecting the recognition sequences of the exon-intron splice site or any variants that would create new putative splice sites near the exon-intron boundaries.
Haplotype Analysis. Based on the concomitant occurrence of the SNPs among the individuals studied and the linkage analysis by the cloning method, the different SNPs can be deduced to comprise 7 haplotypes, as described in Fig. 1. A genomic reference sequence of accession number NT_007819
[GenBank]
was defined as the wild-type allele CYP2W1*1A. The other alleles were named according to the recommendations of the CYP allele nomenclature committee (http://www.cypalleles.ki.se). The allelic variants (CYP2W1*1B–CYP2W1*6) discovered in this study have been submitted to the CYP alleles web page.
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FIG. 1. Allelic variants of the human CYP2W1 gene. Schematic representation of "wild-type" allele for CYP2W1 (CYP2W1*1A) and six variant alleles. The numbering is based on the first nucleotide of the initiation codon that is represented as 1. CYP2W1* alleles were named in accordance with the established nomenclature for the P450 alleles. The frequency of each CYP2W1* allele has been estimated on the population of 200 Japanese volunteers.
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Allele and Genotype Frequencies. The frequency of the alleles and genotypes discovered are listed in Fig. 1 and Table 3, respectively. CYP2W1*6 was the most common among all the alleles. It occurred at a frequency of 36.8% in our study population (Fig. 1). The frequencies of the various genotypes observed in our Japanese population followed those predicted by the Hardy-Weinberg law (Table 3). These results were in good agreement with the expected genotype distributions that were calculated using the Hardy-Weinberg equation.
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Discussion
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In the present study, we performed a comprehensive investigation of the genetic variations in CYP2W1 gene. To screen for SNPs in the coding region of this gene, we developed a PCR-DHPLC assay that allows molecular analysis of each exon of the gene. The sequence designated in the National Center for Biotechnology Information as the genome reference sequence with the accession number NT_007819
[GenBank]
was defined as the wild-type allele CYP2W1*1A. Five of the six SNPs detected in the CYP2W1 gene in the DNA samples of 200 Japanese subjects resulted in amino acid substitutions. The alleles carrying these alterations were named CYP2W1*2–CYP2W1*6 by the CYP allele nomenclature committee. The variant allele carrying the silent SNP (166C>T, Leu56Leu) was defined as CYP2W1*1B. The most frequent variant allele was CYP2W1*6, followed by CYP2W1*1B and CYP2W1*1A, and their frequencies were observed to be 0.368, 0.318, and 0.295, respectively. Three novel nonsynonymous SNPs, 173A>C (Glu58Ala), 5432G>A (Val432Ile), and 5584G>C (Gln482His), identified in this study were rare, with allele frequencies <1%.
Homology modeling of the human CYP2 family enzymes based on the CYP102 crystal structure (Lewis, 1998) lead to speculation that Val432Ile is located at the heme-binding region. The important region called "P450 signature motif region" comprises the 10-residue P450 signature motif that contains the invariant cysteine that forms the proximal heme ligand. This region and the following L-helix represent one of the most highly conserved sections. This homologous segment is associated with the binding of both heme and redox partners with the common motif Phe-X-X-Gly-X-Arg-X-Cys-X-Gly. In wild-type CYP2W1*1A, this motif is Phe-Ser-Ala-Gly-Arg-Arg-Val-Cys-Val-Gly; in CYP2W1*4 and CYP2W1*5, including Val432Ile, the first valine is changed to isoleucine (Phe-Ser-Ala-Gly-Arg-Arg-Ile-Cys-Val-Gly). For example, the Pro428Thr substitution of CYP2B6 (CYP2B6*21) in the same region has been clearly identified as being responsible for the decreased protein stability (Klein et al., 2005). Therefore, the homologous segment of the CYP2 family, including CYP2W1, might be closely associated with enzyme activity. Further studies, such as the use of the recombinant CYP2W1 protein, are required to confirm the function of these variant enzymes.
Karlgren et al. (2006) have reported that CYP2W1 mRNA was detected by real-time PCR of human tumor cells; the highest expression was in colon tumors, but moderate expression was also observed in several adrenal gland tumor cells. They also suggest that CYP2W1 is potentially an important drug target or a useful molecular marker for cancer therapy and diagnosis (Karlgren and Ingelman-Sundberg, 2007). If this is the case, further understanding of the nature of CYP2W1 would be important for cancer therapy.
In summary, this comprehensive investigation of the polymorphisms in the coding region of the CYP2W1 gene identified 6 variant CYP2W1 alleles (CYP2W1*1A–CYP2W1*6). In vitro analysis of recombinant mutated cDNAs as well as phenotyping studies will help in determining the functions of the identified variants.
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Footnotes
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This work was supported by the High-Tech Research Center Program of the Ministry of Education, Culture, Sports, Science and Technology of Japan and a Grant-in-Aid from the Ministry of Health, Labor and Welfare of Japan.
On September 18, 2007, the SNPs detected in this study were not present in dbSNP in the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/SNP), GeneSNPs at the Utah Genome Center (http://www.genome.utah.edu/genesnps), or the Human CYP Allele Nomenclature Committee database (http://www.cypalleles.ki.se).
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.107.019141.
ABBREVIATIONS: P450, cytochrome P450; SNP, single nucleotide polymorphism; DHET, 8,9-dihydroxyeicosatrienoic acid; DHPLC, denaturing high performance liquid chromatography; PCR, polymerase chain reaction; CI, confidence interval.
Address correspondence to: Dr. Masahiro Hiratsuka, Department of Clinical Pharmacotherapeutics, Tohoku Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai 981-8558, Japan. E-mail: mhira{at}tohoku-pharm.ac.jp
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Abstract
Materials and Methods
