Drug Metab. Pharmacokinet. 22 (2): SNP29 (136)–SNP33 (140) (2007).

SNP Communication Three Novel Single Nucleotide Polymorphisms (SNPs) of CYP2S1 in Japanese Individuals

Yoshiyuki HANZAWA1,2, TakamitsuS ASAKI1,2, Masahiro HIRATSUKA1,2, Masaaki ISHIKAWA2 and Michinao MIZUGAKI1,* 1Department of Clinical Pharmaceutics, Tohoku Pharmaceutical University, Sendai, Japan 2Department of Clinical Pharmacotherapeutics, Tohoku Pharmaceutical University, Sendai, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: We analyzed all nine exons and exon­intron junctions of the CYP2S1 gene in 200 Japanese individuals and identiˆed the following three novel single nucleotide polymorphisms (SNPs): 4612GÀA (Glu147Glu) in exon 3, 5478CÀT (Leu230Leu) and 5479TÀG (Leu230Arg, CYP2S1*5A)inexon5.The allele frequencies were 0.013 for 4612GÀA, 0.058 for 5478CÀT, and 0.003 for 5479TÀG. In addition, a known SNP 1324CÀG (Pro74Pro) was detected at a frequency of 0.300.

Key words: CYP2S1; genetic polymorphism; SNP

were detected in human skin, where CYP2S1 was shown Introduction to be induced by ultraviolet radiation, coal tar, and The cytochrome P450s (CYPs) constitute a large and all­trans retinoic acid.3,4) In the same study, CYP2S1 complex gene superfamily. Currently, 57 active CYP wasobservedtometabolizeall­trans retinoic acid, and 58 pseudogenes are known to present in the indicating that CYP2S1 may be involved in the biotran­ . Most of the CYP genes have the highest sformation of endogenous substrates important for cell expression in the liver, which plays a dominant role in proliferation and diŠerentiation.5) the clearance of foreign compounds. CYP enzymes also The human CYP2S1 gene has recently been shown metabolize many endogenous compounds important to be polymorphic; two amino acid­changing allelic for the maintenance of cellular homeostasis, such as variants CYP2S1*2 (10347CÀT; 13255AÀG) and steroids, retinoids, bile acids, fatty acids, and eicosa­ CYP2S1*3 (13106CÀT; 13255AÀG) have been noids. detected in Caucasians.6) CYP2S1 has been genetically Recently, a novel CYP gene, CYP2S1, has been analyzed in Caucasians but not in Japanese individuals. identiˆed. The CYP2S1 gene is localized in the CYP2 In the present study, nine exons and exon­intron gene cluster on 19q.13.2. Several studies junctionsoftheCYP2S1 gene from 200 Japanese that investigated the tissue distribution of human individuals were screened for genetic polymorphisms by CYP2S1 mRNA demonstrated that it has low expres­ using denaturing HPLC (DHPLC). We identiˆed three sion levels in the liver but is detectable in extrahepatic novel single nucleotide polymorphisms of the CYP2S1 tissues such as those of the respiratory and digestive gene, including a nonsynonymous polymorphism, in systems.1–3) Furthermore, CYP2S1 mRNA and Japanese individuals. Materials and Methods As of December 1, 2006, these SNPs were not found in dbSNP in the National Center for Biotechnology Information (http:WWwww. Venous blood was obtained from 200 unrelated ncbi.nlm.nih.govWSNP W), GeneSNPs at the Utah Genome Center healthy Japanese volunteers and patients admitted to (http:WWwww.genome.utah.eduWgenesnpsW) or the Human CYP Allele TohokuUniversity Hospital. Written informed consent Nomenclature Committee database (http:WWwww.imm.ki.seW CYPallelesW). The CYP2S1 haplotype with 5479TÀG (Leu230Arg) was obtained from all the blood donors, and the study was assigned as CYP2S1*5A by the Human CYP Allele Nomenclature was approved by the Local Ethics Committee of Committee (http:WWwww.imm.ki.seWCYPallelesW). TohokuUniversity Hospital and TohokuPharmaceuti­

Received; December 19, 2006, Accepted; February 13, 2007 *To whom correspondence should be addressed: Michinao MIZUGAKI, Department of Clinical Pharmaceutics, Tohoku Pharmaceutical University, 4­4­1, Komatsushima, Aoba­ku, Sendai 981­8558, Japan. Tel. {81­22­727­0211, Fax. {81­727­0149, E­mail: mizugaki—tohoku­ pharm.ac.jp

SNP29 (136) Novel SNPs in CYP2S1 Gene SNP30 (137)

Table 1. Ampliˆcation and DHPLC conditions for CYP2S1 SNP analysis of genomic DNA

Size Forward primer Reverse primer Annealing PCR DHPLC Exon (bp) (5? to 3?) (5? to 3?) Temp. (9C) cycles Temp. (9C)

1 247 gccgcgcggagcgcctggga ccaggacgtccccagagccc Slowdowna 70.0–55.0 63 65.8, 68.5 2 376 cttggatcgaagaggtcacagc ttgggatttcaggcactagcc 60.0 35 64.3

3 303 caacagagcgagattccgtctc agttttccctttcactcggctg 65.0 35 62.9

4 367 ctctctccctgcgctgtcc gagaagggcagctagttctcatgg 60.0 35 62.6

5 257 tcccatgagaactagctgcc caccatgcccattcagagag 60.0 30 64.7

6 336 taacttgtgtttccgaccccag ctcagcctcccaaagtgctg 65.0 35 59.2

7 436 acaagatgtgtggtctttgggc agaaaaagtcagggagacactgacag 60.0 35 63.0

8 485 tctctcacctcagcctcccac tcagtattcctcacacccaggc 60.0 35 60.5

9 342 tgaggaatactgactcagccctctc acactctggagacattaaccctgtcc 60.0 35 62.4 aSlowdown protocol: The annealing temperature was decreased after cycle 3 by 1.09C every 3 cycles, beginning at 709C and decreased to a ``slowdown'' annealing temperature of 559C, followed by 15 additional cycles with an annealing temperature of 609C. The PCR was used at a ramp rate of 2.59CWs and reached annealing temperature at 1.59CWs.

8–12) cal University. DNA was isolated from K2 EDTA­ USA). Ampliˆed PCR samples (5 mL) were separat­ anticoagulated peripheral blood by using QIAamp ed on a heated C18 reverse­phase column (DNAsep})by DNA Mini Kits (Qiagen, Hilden, Germany) according using 0.1 M triethylammonium acetate (TEAA) in water to the manufacturer's instructions. and 0.1 M TEAA in 25z acetonitrile at a ‰ow rate of Table 1 lists the primer pairs used to amplify nine 0.9 mLWmin. The software provided with the instru­ exons and exon­intron junctions of the CYP2S1 gene. ment selected the temperature for the heteroduplex These primers were designed based on the genomic separation of a heterozygous CYP2S1 fragment. sequence reported in GenBank (NGä000008.7). The Table 1 summarizes the DHPLC running conditions for amplicons for exon 1 were generated using AmpliTaq each amplicon. The linear acetonitrile gradient was Gold PCR Master Mix (Applied Biosystems, Foster adjusted to the retention time of the DNA peak at City, CA, USA). PCR reactions were performed using 4–5 minutes. Homozygous nucleotide exchanges can an iCycler (Bio­Rad, Hercules, CA, USA). Moreover, occasionally be detected due to a slight shift in the the method relied on a combination of the slowdown elution time as compared with that of the reference. The method and the addition of betaine (Sigma­Aldrich, addition of an approximately equal amount of wild­type St.Louis, MO) for this region with high GC content DNA to the samples (1:1) at the denaturation step (À70z).7) The PCR condition comprised denaturation enabled the reliable detection of homozygous altera­ at 959C for 5 minutes, followed by 48 cycles of tionsinexon2.Thiswasperformedforallsamplesin denaturation at 959C for 30 seconds, annealing for 30 order to identify homozygous sequence variations. seconds, extension at 729C for 40 seconds, and ˆnally, Therefore, all samples were analyzed as follows. First, 15 additional cycles at an annealing temperature of equal amounts of four samples were mixed to identify 609C. The amplicons for each exon from 2 to 9 were the heterozygotes, and then, each sample was mixed generated using the AmpliTaq Gold PCR Master Mix. with wild­type DNA to detect the homozygous variants. The PCR conditions comprised denaturation at 959C The resultant chromatograms were compared with those for 10 minutes, followed by 30 or 35 cycles of of the wild­type DNA. denaturation at 959C for 30 seconds, annealing for 30 Both strands of samples in which variants were seconds, extension at 729C for 30 seconds, and a ˆnal detected by DHPLC were analyzed using a CEQ8000} extension at 729C for 7 minutes. The annealing temper­ automated DNA sequencer (Beckman­Coulter Inc., atures and PCR cycles for the screening of CYP2S1 Fullerton, CA, USA). Further, we sequenced all variants are summarized in Table 1. Heteroduplexes samples having chromatographic ˆndings that diŠered were generated by performing thermal cycling as from that of the wild­type to establish links between follows: 959C for 1 minute, followed by a reduction in mutations and speciˆc proˆles. We sequenced the PCR temperature from 959Cby45incrementsof1.59Cper products by ‰uorescent dideoxy termination using a minute. DTCS DNA Sequencing Kit (Beckman­Coulter Inc.) The PCR products were analyzed using the DHPLC according to the manufacturer's instructions. system (WAVE}; Transgenomic Inc., Omaha, NE, SNP31 (138) Yoshiyuki HANZAWA, et al.

Table 2. The location of SNPs and frequencies of the CYP2S1 gene in 200 DNA samples of Japanese individuals

Amino acid SNP ID The number of Observed Frequency (z) Frequency (z)predicted Location Variant change dbSNP (NCBI) each genotype (95z CI) by Hardy­Weinberg law

Exon 2 1324CÀG Pro74Pro rs338599 CWC: 99 49.5 (42.6–56.4) 49.0 CWG: 82 41.0 (34.2–47.8) 42.0 GWG: 19 9.5 (5.4–13.6) 9.0

Exon 3 4612GÀA Glu147Glu — GWG: 195 97.5 (95.3–99.7) 97.5 GWA: 5 2.5 (0.3–4.7) 2.5 AWA: 0 0.0 (0.0) 0.0

Exon 5 5478CÀT Leu230Leu — CWC: 177 88.5 (84.1–92.5) 88.8 CWT: 23 11.5 (7.1–15.9) 10.8 TWT: 0 0.0 (0.0) 0.3

Exon 5 5479TÀG Leu230Arg — TWT: 199 99.5 (98.5–100) 99.5 TWG: 1 0.5 (0–1.5) 0.5 GWG: 0 0.0 (0.0) 0.0

Fig. 1. The nucleotide sequences of the CYP2S1 gene in exon 5. Although sequences are shown for anti­sense strands, both strands were sequenced. Arrows indicate the positions of the variant nucleotide.

chromatographic proˆles that were distinct from that of Results and Discussion the wild­type in exons 2, 3, and 5. We tested the speciˆc­ We found the following three novel SNPs: ity of DHPLC in detecting the variant allele in these exons by comparing the results with those of direct 1) SNP: 061023Hiratsuka10; GENE NAME: sequencing. Four SNPs including three novel and one CYP2S1; known SNP (rs338599) were detected (Table 2). The ACCESSION NUMBER: NGä000008; LENGTH: 25 electrophoretograms of the novel nonsynonymous SNP bases; are shown in Fig. 1. The SNP in exon 5 was 5479TÀG 5?­AGAAGGCGAGGAGWACTGATCCAGGCG­3?. and resulted in an amino acid change of Leu230Arg. 2) SNP: 061023Hiratsuka11; GENE NAME: Among the 200 individuals, one was heterozygous for CYP2S1; the 5479TÀG SNP, suggesting that the allele frequency ACCESSION NUMBER: NGä000008; LENGTH: 25 was 0.003 in the Japanese population. The other novel bases; SNPs 4612GÀA and 5478CÀT were detected at 5?­TTCCTGCGGCCCCWTTGCCAGGCCCCC­3?. frequencies of 0.013 and 0.058, respectively. The se­ 3) SNP: 061023Hiratsuka12; GENE NAME: quences for each sample were obtained from at least two CYP2S1; diŠerent PCR ampliˆcations. ACCESSION NUMBER: NGä000008; LENGTH: 25 The novel SNP 5479TÀG is located in exon 5 of the bases; CYP2S1 gene and results in an amino acid substitution. 5?­TCCTGCGGCCCCTWGGCCAGGCCCCCA­3?. Homology modeling of the human CYP2 family DHPLC analysis of the CYP2S1 gene in the 200 DNA enzymes based on the CYP2C5 crystal structure lead to samples obtained from Japanese individuals revealed speculation that Leu230 is located at the start of the Novel SNPs in CYP2S1 Gene SNP32 (139)

G­helix.13,14) In the CYP2 family, this region is not a P450 CYP2S1: individuality in regulation by therapeutic putative active site.15,16) However, the Pro227 substitu­ agents forpsoriasis and otherskin diseases. Lancet., tion of CYP2C19 (CYP2C19*10) in the same region has 361: 1336–1343 (2003). been clearly identiˆed as being responsible for the 4) Guengerich, F. P., Wu, Z. L. and Bartleson, C. J.: decreased catalytic activity toward S­mephenytoin.17) Function of human cytochrome P450s: characterization Pro220 was also one of several critical amino acids that of the orphans. Biochem. Biophys. Res. Commun., 338: 465–469 (2005). appearto determinethe extent of the speciˆcity of 5) Saarikoski, S. T., Rivera, S. P., Hankinson, O. and CYP2C19 for S­mephenytoin as compared to that of Husgafvel­Pursiainen, K.: CYP2S1: A short review. 18) CYP2C9. Therefore, the start of the G­helix and F­G Toxicol. Appl. Pharmacol., 207: 62–69 (2005). loop of the CYP2 family, including CYP2S1, might be 6) Saarikoski, S. T., Suitiala, T., Holmila, R., Impivaara, associated with enzyme activity. O., Jarvisalo, J., Hirvonen, A. and Husgafvel­Pursiai­ Numerous genetic polymorphisms have been identi­ nen, K.: Identiˆcation of genetic polymorphisms of ˆedinmostCYP genes. In particular, genetic polymor­ CYP2S1 in a Finnish Caucasian population. Mutat. phisms of the CYP2 family are believed to be responsi­ Res., 554: 267–77 (2004). ble for large individual variations. Saarikoski et al.have 7) Bachmann, H. S., SiŠert, W. and Frey, U. H.: Success­ detected two variant alleles CYP2S1*2 and CYP2S1*3 ful ampliˆcation of extremely GC­rich promoter regions using a novel `slowdown PCR' technique. Phar­ in the CYP2S1 gene in the Caucasian population.6) macogenetics, 13: 759–766 (2003). In the present study we identiˆed the novel variant allele 8) Hiratsuka, M., Nozawa, H., Konno, Y., Saito, T., * CYP2S1 5A in the Japanese population. CYP2S1 could Konno, S. and Mizugaki, M.: Human CYP4B1 gene in be involved in the metabolism of all­trans retinoic acid, the Japanese population analyzed by denaturing HPLC. which is used to treat skin diseases. CYP2S1 expression Drug. Metab. Pharmacokinet., 19: 114–119 (2004). was induced in some individuals treated topically with 9) Ebisawa, A., Hiratsuka, M., Sakuyama, K., Konno, Y., all­trans retinoic acid, whereas others showed no Sasaki, T. and Mizugaki, M.: Two novel single nucleo­ response.3) At least in part, such variation may be tide polymorphisms (SNPs) of the CYP2D6 gene in caused by sequence variations aŠecting expression or Japanese individuals. Drug. Metab. Pharmacokinet., 20: activity of CYP2S1. 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