Association study between the genetic polymorphisms of glutathione-related enzymes and schizophrenia in a Japanese population.код для вставкиСкачать
RESEARCH ARTICLE Neuropsychiatric Genetics Association Study Between the Genetic Polymorphisms of Glutathione-Related Enzymes and Schizophrenia in a Japanese Population Daisuke Matsuzawa,1,2 Kenji Hashimoto,3* Tasuku Hashimoto,1 Eiji Shimizu,2 Hiroyuki Watanabe,1 Yuko Fujita,3 and Masaomi Iyo1 1 Department of Psychiatry, Chiba University Graduate School of Medicine, Chiba, Japan Integrative Neurophysiology, Chiba University Graduate School of Medicine, Chiba, Japan 2 3 Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, Chiba, Japan Received 19 November 2007; Accepted 27 March 2008 Several lines of evidence suggest that oxidative stress plays a role in the pathogenesis of schizophrenia, and that glutathione (GSH) plays a crucial role in antioxidant defense mechanisms. In this study, we performed association studies between GSH-related genes (GSTM1, GSTP1, GSTO1, GSTT1, GSTT2, GPX1, and GCLM) and schizophrenia in a Japanese population. The overall distributions of the genotypes and alleles of each gene were not different between schizophrenic patients and controls. Subjects with residual-type schizophrenia showed different distributions in the analysis of GSTM1 genotype and in the combination analysis of GSTs, GPX1, and GCLM genotypes although the small sample size should be considered as a limitation of this study. In addition, our findings revealed that there were large ethnic differences in the genotype distributions of those GSHrelated genes. The present study suggests that GSH-related genes may not play a major role in the pathogenesis of schizophrenia in a Japanese population. However, a dysregulation of GSH metabolism may be one of the vulnerability factors contributing to the development of a certain type of schizophrenia, and it is likely that the ethnic background should be considered in further study for those GSH-related genes. 2008 Wiley-Liss, Inc. Key words: schizophrenia; oxidative stress; glutathione; susceptibility; polymorphism INTRODUCTION Schizophrenia is a devastating psychiatric disease with a complex genetic etiology. Several lines of evidence suggest that oxidative stress plays a role in the pathogenesis of schizophrenia [Mahadik and Mukherjee, 1996; Schulz et al., 2000; Yao et al., 2001; Carter, 2006]. Dopamine (DA) is a major source of reactive oxygen species (ROS) in the central nervous system, as excess DA can easily autooxidize to produce DA-quinone [Baez et al., 1997; Smythies, 1997]. Glutathione (GSH), one of the major non-protein antioxidants and redox regulators, detoxifies ROS and thus plays a major role in protecting neural tissues [Dringen, 2000b; Schulz et al., 2000]. Interestingly, in a study by Do et al. , GSH levels in the 2008 Wiley-Liss, Inc. How to Cite this Article: Matsuzawa D, Hashimoto K, Hashimoto T, Shimizu E, Watanabe H, Fujita Y, Iyo M. 2009. Association Study Between the Genetic Polymorphisms of Glutathione-Related Enzymes and Schizophrenia in a Japanese Population. Am J Med Genet Part B 150B:86–94. cerebrospinal fluid of drug-free patients with schizophrenia were significantly decreased as compared with those in control groups, and non-invasive proton magnetic resonance spectroscopy revealed a significant reduction of GSH in the medial prefrontal cortex of schizophrenic patients, suggesting that a deficit in GSH and GSH-related enzymes might play a role in the pathophysiology and might constitute a major risk factor of schizophrenia. Given the role of GSH in the anti-oxidative process, the genes encoding the proteins known as polymorphic glutathione S-transferases (GSTs: Enzyme Commission (EC) number 188.8.131.52), glutathione peroxidase-1 (GPX1: EC 184.108.40.206), and glutamate cysteine ligase (GCL: EC 220.127.116.11) are clearly worthy of investigation [Hayes and Strange, 2000; Forsberg et al., 2001; Yoshimura et al., 2003; Tosic et al., 2006]. The GSTs are a family of multifunctional enzymes that catalyze the conjugation of reduced GSH with electrophilic groups of a wide Grant sponsor: Minister of Education, Culture, Sports, Science, and Technology of Japan. *Correspondence to: Dr. Kenji Hashimoto, Ph.D., Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, 1-8-1 Inohana, Chiba 2608670, Japan. E-mail: firstname.lastname@example.org Published online 30 April 2008 in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/ajmg.b.30776 86 MATSUZAWA ET AL. variety of compounds, including carcinogens, environmental contamination, and products of oxidative stress [Mannervik, 1985; Hayes and Strange, 2000]. Genes of human cytosol GST consist of at least eight distinct gene families (alpha, mu, theta, pi, zeta, sigma, kappa, omega) and, among them, several classes of GST genes have been shown to be polymorphic: GSTM (mu) 1 and M3 on chromosome 1p13.3, GSTT (theta) 1 and T2 on 22q11.2, GSTO (omega) 1 on 10q24.3, and GSTP (pi) 1 on 11q13 [Strange et al., 2001; Whitbread et al., 2005]. Homozygous deletion of GSTM1 (null type of GSTM1: GSTM1*0) and GSTT1 (null type of GSTT1: GSTT1*0) results in the absence of the respective enzyme activities. Polymorphism of GSTM3 appears as a 3 bp deletion in intron 6 (GSTM3*A as a wild-type and GSTM3*B with the deletion). Polymorphisms with an amino acid substitution are found in GSTO1 (Ala140Asp and Thr217Asn), GSTP1 (Ile105val), and GSTT2 (Met139Ile), and those of GSTO1 and GSTP1 have been shown to decrease the activity of the enzyme [Watson et al., 1998; Tanaka-Kagawa et al., 2003]. Increased incidence of the null-type GSTM1 gene (GSTM1*0) in schizophrenia patients has been reported in a Japanese and a Korean population [Harada et al., 2001; Pae et al., 2004]. Glutathione peroxidase 1 (GPX1), which belongs to a family of selenium-dependent peroxidases, protects cells by eliminating hydrogen peroxides and a wide range of organic peroxides by using GSH as a reducing substrate [Schweizer et al., 2004]. Lower levels of glutathione peroxidase have been reported in schizophrenic patients and schizophrenic patients with tardive dyskinesia [Ranjekar et al., 2003; Yao et al., 2006; Zhang et al., 2007]. One functional polymorphism of the GPX1 gene is a substitution at codon 198 (Pro198Leu), and the leucine allele is less responsive to added selenium than it is with the proline allele [Hu et al., 2005]. Human glutamate cysteine ligase (GCL) is a rate-limiting enzyme for GSH synthesis that plays a crucial role in antioxidant defense mechanisms in most mammalian cells [Meister and Anderson, 1983], and GCL modifier (GCLM) is one of two subunits composing GCL [Huang et al., 1993]. Nakamura et al.  found a functional polymorphism (ss60197536: 588C/T) in the 50 flanking region of the GCLM gene, and the minor 588T allele of the polymorphism suppressed oxidant-induced up-regulation of GCLM gene expression and was associated with lower plasma GSH levels [Nakamura et al., 2002]. Furthermore, it has been demonstrated that a polymorphism (A/G: rs2301022) in intron 1 of the GCLM gene was associated with schizophrenia in a Danish population [Tosic et al., 2006]. Considering the role of GSH and its metabolism in oxidative stress, we considered that it would be of great interest to study the association between GST-related genes and schizophrenia. Interestingly, these GSH-related genes are located in the genomic region conferring associated with susceptibility to schizophrenia: the genes of GSTM1 and M3 are on chromosome 1p13.3, GSTP1 is on 11q13, GSTT1 and T2 are on 22q11.2, GPX1 is on 3p21.3 and GCLM is on 1p21 [Mulcrone et al., 1995; Shaw et al., 1998; Lewis et al., 2003; Arinami et al., 2005]. The aim of the present study was, therefore, to evaluate the influence of the genetic variance of the GSH-related genes (GSTM1, GSTP1, GSTO1, GSTT1, GSTT2, GPX1, and GCLM) as a risk factor for schizophrenia in a Japanese population. 87 MATERIALS AND METHODS Subjects The research was performed after the study was approved by the Ethics Committee of Chiba University Graduate School of Medicine. Genomic DNA was extracted from blood samples after obtaining written informed consent. The subjects were 220 healthy controls (102 males and 118 females; mean age, 32.9 16.7 years) with no past history of psychotic disorders or drug dependence and 214 schizophrenic patients (106 males and 108 females; mean age, 51.8 14.8). The average age of onset for the 214 patients was 24.9 years (SD 8.3). Gender and geographical origin of both groups were matched, but due to the substantial numbers of chronic inpatients with the long mean duration of illness (26.7 14.7 years), the mean age between controls and patient groups were statistically different (unpaired t-test, P < 0.01). Each patient met the DSM-IV criteria for schizophrenia [American Psychiatric Association, 1994] and had no other psychiatric disorders. In order to compare the distributions of gene polymorphisms amongst schizophrenia subtypes, the patient group was subdivided according to DSM-IV criteria into a disorganized-type (n ¼ 19, 9 females; mean age, 49.1 15.3 years old; onset, 19.8 5.8 years old), catatonic-type (n ¼ 19, 10 females; mean age, 53.3 16.0 years old; onset, 25.3 5.6 years old), paranoid-type (n ¼ 75, 42 females; mean age, 48.8 14.9 years old; onset, 25.7 8.5 years old), residual-type (n ¼ 83, 40 females; mean age, 55.3 13.1 years old; onset, 24.1 7.9 years old), and undifferentiated-type (n ¼ 18, 7 females; mean age, 43.9 18.3 years old; onset, 27.1 9.2 years old) group of patients. Genotyping GSTM1 and GSTT1. Genotyping was performed by PCR coamplified with the b-globin gene as a positive control. PCR conditions were the same as reported previously [Sreelekha et al., 2001]. The primers used for GSTM1, GSTT1, and b-globin are shown in Table I, and the amplified PCR product sizes were 220, 450, and 268 bp, respectively. Negative controls (containing the PCR mixture without DNA template) were also used to test for contamination. Following the genotyping, subjects were categorized as having either the positive (those with þ/þ and þ/null genotypes) or null genotype (those with null/null genotype). GSTM3, GSTO1, GSTP1, GPX1. Genotyping was performed by PCR-restriction fragment length polymorphism (RFLP) analysis, and the PCR conditions of GSTO1, GSTP1, and GPX1 were the same as in previous reports [Li et al., 2003; Hashimoto et al., 2005; Matsuzawa et al., 2005]. The primers for each gene are shown in Table II. The PCR product of GSTM3 was 400 bp in length. Digestion was performed with the restriction enzyme EarI (New England Biolabs, Inc., Beverly, MA), and the bands indicate the presence of 220, 93, and 87 bp fragments (GSTM3*A/*A; although, the 93 and 87 bp fragments are usually seen as a single band), 307, 220, 93, and 87 bp fragments (*A/*B), and 307 and 93 bp fragments (*B/*B). The PCR product of GSTO1 Ala140Asp was 455 bp in length. Digestion was performed with Cac8I (New England Biolabs, Inc.), and the bands are visible with 243, 145, and 67 bp fragments (C/C, although, the 67 bp fragment is usually not visible on the gels), 388, 243, 145, and 67 bp fragments (C/A) and 388 and 67 bp 88 AMERICAN JOURNAL OF MEDICAL GENETICS PART B TABLE I. PCR Primers for GSTs, GPX1, and GCLM Genes Primer sequences Gene GSTP1 GSTM1 GSTM3 GSTT1 GSTO1 (Ala140Asp) GSTO1 (Thr217Asn) GPX1 b-globin Forward (50 –30 ) GTAGTTTGCCCAAGGTCAAG CGCCATCTTGTGCTACATTGCCCG TGCCCTGATTAACTACAAAGATGA TCCAGGAGGCCCATGAGGTCA TGTGCCCCTACGGTA GGAGACTCTGTGATGTCATCC TGTGCCCCTACGGTA CAACTTCATCCACGTTCACC Reverse (50 –30 ) AGCCACCTGAGGGGTAAG TTCTGGATTGTAGCAGATCA GGGAGCCTGTGAGTGTTTTTAT TTCTGCTTTATGGTGGGGTCT AAGTGACTTGGAAAGTGGGAA CATAGCTTTATTGCTGACTCCTG CCAAATGACAATGACACAGG GAAGAGCCAAGGACAGGTAC Primer or probe sequences Reverse primer (50 –30 ) or probe2 (50 –30 ) Gene Forward primer (50 –30 ) or probe1 (50 –30 ) TaqMan 50 -exonclease allelic discrimination assay was used for genotyping GSTT2 and GCLM genes GSTT2 GAGAAGGTGGAACGCAACAG TTGTCCTCCAGCCATTGCA Probe1: VIC-CTGCCATGGACCAGG-MGB Probe2: FAM-CTGCCATAGACCAGG-MGB GCLM 588 ACAGCGCGAGGCAGACA TGCTTCTGAGAACGAAAACTACGT Probe1: VIC-TCTCCCGGCGTTCA-MGB Probe2: FAM-TCTCCCACGTTCA- MGB rs2301022 CAGAGTCACACACCACAGTTTGTA GTTTTATCCTACTGTTATGAAGCACCCTAA Probe1: VIC-CAAAGGACTAATTCTGG-MGB Probe2: FAM-CAAAGGACTAGTTCTGG-MGB fragments (A/A). The PCR product of GSTO1 Thr217Asn was 248 bp in length. Because the distribution of this polymorphism had not yet been reported, we preliminarily surveyed the minor allele frequencies in 32 control samples by direct sequencing. The sequencing reaction was performed on an ABI 310 genetic analyzer (PE Biosystems, Foster City, CA) following the manufacturer’s protocol. Because none of the samples had the minor allele, we did not perform further genotyping. The PCR product of GSTP1 gene was 433 bp in length. Digestion was performed with BsmA1 (New England Biolabs, Inc.), and the bands are visible with 328 and 105 bp (A/A), 328, 222, 106, and 105 bp (A/G), and 222, 106, and 105 bp (G/G). PCR product of GPX1 is 337 bp. Digestion was performed with ApaI (New England Biolabs, Inc.), and the bands are visible with 337 bp fragment (C/C), 337, 258, and 79 bp fragments (C/T, although, the 79 bp fragment is typically not visible) and 258 and 79 bp fragment (T/T). All of the DNA fragments were separated on a 2% agarose gel stained with ethidium bromide. GSTT2 and GCLM. Genotyping was performed by TaqMan 50 exonuclease allelic discrimination assay in accord with the manufacturer’s protocol (Applied Biosystems, Foster city, CA). The primers and probes used for these genes are shown in Table I. Statistical Analyses The differences between groups were evaluated by Fisher’s exact test. Hardy–Weinberg equilibrium was tested using the Chi-square goodness-of-fit test. The odds ratio and 95% confidence intervals (CIs) between two variables were calculated as an estimate of risk. Multiple regression analysis was performed to evaluate the relation between the schizophrenic patients’ age of onset and the genetic polymorphisms. For the regression analysis, age of onset was used as the dependent variable, and the genotypes were used as the inde- pendent variables. The analysis was performed with SPSS software (SPSS version 12.0J, Tokyo, Japan). Values of P < 0.05 were considered to indicate statistical significance in all analyses. RESULTS The frequencies of alleles and genotypes in schizophrenic patients and controls are shown in Table II. All of the genotype distributions in both schizophrenic patients and controls were in Hardy–Weinberg equilibrium (c2, P > 0.05, df ¼ 1). As shown in Table II, the overall distributions of genotypes and alleles of each gene were not significantly different between schizophrenic patients and controls. In the multiple regression analysis, none of the genotypes showed significant association with age of onset of schizophrenia. No gender difference was found in the distributions of any of the genes. The data for GSTM3 are not shown because the GSTM3*A/*B type was seen in only one sample and none of the samples had the GSTM3*B/*B type, and thus further analysis was not executed for GSTM3. In the analysis of the gene distributions among schizophrenia subtypes, our study revealed that the null type of the GSTM1 gene was significantly more frequent among patients with residual-type schizophrenia than among control subjects (P ¼ 0.037, odds ratio ¼ 1.790, 95% CI ¼ 1.003–1.986; Table III). For the other genes, however, no significant difference was found between any of the schizophrenia subtypes and the controls (data not shown). From the perspective of the functional deficits resulting from a gene polymorphism, those subjects with any of the high-risk genotypes GSTM1*0, GSTT1*0, or GSTP1 (Ile/Val and Val/Val type) are considered to have lesser overall GST activity. Similarly, those with GPX1 (Leu/Pro and Pro/Pro) or GCLM 588 (C/T and T/T) are considered to have lower amounts of the corresponding MATSUZAWA ET AL. 89 TABLE II. Genotype and Allele Distributions of Controls and Schizophrenia Patients n Positive Null GSTM1 Ct Pt 220 214 101 85 P ¼ 0.208 45.9% 39.7% 119 129 54.1% 60.3% GSTT1 Ct Pt 220 214 140 127 P ¼ 0.375 63.6% 59.1% 80 88 36.4% 40.9% Genotype GSTT2 (Met139Ile) Ct Pt n 220 214 GG 157 155 GA 55 25.9% 50 24.4% P ¼ 0.884 71.4% 72.4% Allele AA 8 9 3.6% 4.2% G 369 360 Genotype GSTO1 (Ala140Asp) Ct Pt n 220 214 CC 175 165 CA 41 19.0% 45 21.4% P ¼ 0.875 79.5% 77.1% n 220 214 AA 162 154 AG 54 25.0% 55 26.3% P ¼ 0.891 73.6% 72.0% AA 4 4 1.8% 1.9% C 391 375 n 220 214 CC 189 180 CT 29 13.3% 31 14.7% P ¼ 0.798 85.9% 84.1% GG 4 5 1.8% 2.3% A 378 363 n 220 214 CC 148 153 67.3% 71.5% TT 2 3 0.9% 1.4% C 407 391 n 220 214 GG 131 126 59.5% 58.9% CT 63 29.9% 52 25.4% P ¼ 0.579 GA 73 35.8% 76 37.6% P ¼ 0.729 G 85.9% 62 84.8% 65 P ¼ 0.701 14.1% 15.2% T 92.5% 33 91.4% 37 P ¼ 0.618 7.5% 8.6% Allele TT 9 9 4.1% 4.2% C 359 358 Genotype GCLM rs2301022 Ct Pt 11.1% 12.4% Allele Genotype GCLM 588 ss60197536 Ct Pt A 83.9% 49 87.6% 53 P ¼ 0.599 Allele Genotype GPX1 (Pro198Leu) Ct Pt 16.1% 15.9% Allele Genotype GSTP1 (Ile105Val) Ct Pt A 83.9% 71 84.1% 68 P ¼ 0.924 T 81.6% 81 83.6% 70 P ¼ 0.474 18.4% 16.4% Allele AA 16 12 7.3% 5.6% G 335 328 A 76.1% 105 76.6% 100 P ¼ 0.873 23.9% 23.4% 90 AMERICAN JOURNAL OF MEDICAL GENETICS PART B TABLE III. Distribution of GSTM1 Genotype in Schizophrenia Patients Divided by Subtypes and Controls Subjects Controls Disorganized type Catatonic type Paranoid type Residual type Undifferentiated type n 220 19 19 75 83 18 Positive 101 6 9 35 27 9 45.9% 31.6% 47.4% 46.7% 32.5% 50.0% Null 119 13 10 40 56 9 54.1% 68.4% 52.6% 53.3% 67.4% 50.0% P-value Odds ratio (95% CI) 0.336 1.00 1.00 0.037 0.808 1.839 (0.674–5.014) 0.943 (0.369–2.411) 0.970 (0.574–1.640) 1.760 (1.003–1.986) 0.849 (0.325–2.219) P values are shown versus controls. CI; confidence interval. The bold type is used because the residual type showed significant difference (P < 0.05). enzyme activities than those with the high-risk genotypes. Therefore, we stratified patients and controls according to the number of these high-risk genotypes for the combination analysis of the GSTs, and GPX1 and GCLM. As shown in Table IV, the frequency of highrisk genotypes was significantly increased among residual-type schizophrenics compared to controls (P ¼ 0.005). The calculated odds ratio of residual-type schizophrenia patients with any risk genotypes of those gene reached 5.91 (95% CI ¼ 1.375–25.38) with those who had no risk genotypes as a reference. DISCUSSION In the present study, we examined the association between the polymorphisms of GSTM1, GSTP1, GSTO1, GSTT1, GSTT2, GPX1, and GCLM and schizophrenia in a Japanese population. To the best of our knowledge, this is the first study to investigate these six gene polymorphisms simultaneously in the same subjects. There have been several reports which analyzed the allele frequencies of GSTM1, GSTT2, GSTP1, and GPX1 in Japanese samples. The minor allele frequencies of our control samples were in good accord with previous reports [Harada et al., 2001; Nakazato et al., 2003; Yoshimura et al., 2003; Ichimura et al., 2004; Hashimoto et al., 2005]. Case–control association studies between GSTM1 [Harada et al., 2001; Pae et al., 2004], GSTP1 [Pae et al., 2003; Shinkai et al., 2005], GPX1 [Shinkai et al., 2004], and GCLM [Tosic et al., 2006] and schizophrenia have been reported. Our study did not show that the distribution of GSTM1 genotype was significantly different between normal controls and schizo- phrenic patients as a whole (Table II). However, the null type of the GSTM1 gene was significantly more frequent in residual-type schizophrenia patients than control subjects (P ¼ 0.037, odds ratio ¼ 1.790, 95% CI ¼ 1.003–1.986; Table III). Harada et al.  reported that the null type of GSTM1 was more frequent among schizophrenic patients as a whole and also among those with disorganized-type schizophrenia than in normal controls. The reason for this discrepancy might be the small sample size of their study (87 schizophrenics and 176 controls). Considering the small sample size of both studies, a lager sample size for each subtype of schizophrenia should be needed to obtain the sufficient statistic power to exclude type I error. In addition, the results we found in the residual-type schizophrenics would be rendered as insignificant with applying post hoc analysis for multiple testing. However, it should be noted that both our study and that of Harada et al.  indicated that the distribution of the null-type GSTM1 gene was significantly more frequent in the subtypes of schizophrenia that are dominantly characterized by negative and cognitive symptoms according to DSM-IV criteria. Moreover, Pae et al.  reported a positive association between the GSTM1 null type and schizophrenia in a Korean population (P ¼ 0.014, odds ratio ¼ 1.93, 95% CI ¼ 1.115–3.351) but provided no information as to subtypes. Several lines of evidence suggest that the heterogenerous pathophysiological basis underlies the schizophrenic individuals with different clinical features [Kirkpatrick et al., 2001], and especially those who with persistent negative symptoms should be distinguished [Buchanan, 2007]. Further study with a large sample size would verify the difference in the effect of polymorphisms of TABLE IV. Combination Analysis of GSTs, GPX1, and GCLM-588 Genotypes Number of high-risk genotypesa Subjects (n) Controls (220) Schizophrenia (214) Disorganized type (20) Catatonic type (19) Paranoid type (75) Residual type (83) Undifferentiated type (17) a 0 28 (12.7%) 22 (10.3%) 2 (10.0%) 0 (0%) 13 (17.3%) 2 (2.4%) 0 (0%) 1 110 (50.0%) 119 (55.6%) 8 (40.0%) 13 (68.4%) 35 (46.7%) 55 (66.3%) 9 (52.9%) 2 78 (35.5%) 66 (30.8%) 8 (40.0%) 6 (31.6%) 25 (33.3%) 23 (27.7%) 8 (47.1%) 3 4 (1.8%) 7 (3.3%) 2 (10.0%) 0 (0%) 2 (2.7%) 3 (3.6%) 0 (0%) Those with any high-risk genotype of GSTs (GSTM1*0, GSTT1*0, GSTP1 (Ile/Val or Val/Val type)), GPX1 (Leu/Pro or Pro/Pro type), or GCLM-588 (C/T or T/T type). The bold type is used because the residual type showed significant difference (P < 0.05). P-value 0.428 0.188 0.293 0.438 0.005 0.441 MATSUZAWA ET AL. GSTM1 among schizophrenia subtypes. Taken together, these results suggest that the deletion of GSTM1 is likely to play a role in the pathogenesis of a subtype of schizophrenia in Asian populations, including Japanese and Korean populations. Our samples did not show any significant difference between GSTP1 polymorphism (Ile105Val) and schizophrenia, consistent with a previous report demonstrating a negative association between GSTP1 Ile105Val and schizophrenia in a Korean population [Pae et al., 2003]. From the viewpoint of the role of oxidative stress in the development of tardive dyskinesia, Shinkai et al.  reported no association between GSTP1 Ile105Val polymorphism and chronic treatment-refractory schizophrenia with and without tardive dyskinesia. Taken together, these findings suggest that GSTP1 Ile105Val polymorphism is unlikely to contribute to the genetic susceptibility in the pathogenesis of schizophrenia in Japanese and Korean populations. The GSTT1 null type was more frequent in the schizophrenic patients (40.9%) than in the controls (36.4%), but the difference did not reach the level of statistical significance (P ¼ 0.375). In addition, patients with residual-type schizophrenia showed a greater tendency toward increased frequency of the GSTT1 null type (47.0%, P ¼ 0.113, data not shown) compared to the controls. Very recently, Saadat et al.  reported that the GSTT1 null type (GSTT1*0) was less frequent in schizophrenic patients (17.8%, odds ratio ¼ 0.42, 95% CI ¼ 0.28–0.63, P < 0.001) than in controls (33.9%), suggesting that it was associated with a reduced risk of developing schizophrenia in the Iranian population. The reason underlying this discrepancy is unknown, since the frequencies (Japanese ¼ 36.4%, Iranian ¼ 33.9%) of this polymorphism in the Japanese and Iranian populations were almost the same. Factors other than ethnicity may have contributed to this discrepancy. As is other polymorphisms of GSTs, the genetic risk of the GSTT1 null type has been investigated from the perspective of the association with a number of cancers, and the effect of the GSTT1 null type as a genetic risk for cancer has varied among studies [Raimondi et al., 2006]. The present study is the first to investigate the association of the polymorphisms of GSTO1 Ala140Asp and GSTT2 Met139Ile with schizophrenia. Several studies have reported a positive association between GSTO1 Ala140Asp and neurological diseases such as Alzheimer’s disease and Parkinson’s disease [Li et al., 2003; Kolsch et al., 2004], but our study showed no association between this polymorphism and schizophrenia. It has been reported that the polymorphism encoding the GSTO1 Thr217Asn substitution had a considerable effect on the GSTO1 activity [Tanaka-Kagawa et al., 2003]. We therefore investigated the frequency of this polymorphism in the present study, but no substitution was detected in our samples. This implies that the substitution, if present at all, must be extremely rare in the Japanese population, which is concordant with previous studies [Marnell et al., 2003; Whitbread et al., 2003]. As to GSTT2, no association with schizophrenia was shown. Whether the GSTT2 Met139Ile polymorphism affects the function of the protein remains unknown, but our study suggests that this SNP might not contribute to the risk of schizophrenia in the Japanese population. Therefore, GSTO1 and GSTT2 are unlikely to be associated with susceptibility to schizophrenia in the Japanese population. 91 In our study, neither the genotype nor the allele distributions of the GPX1 Pro198Leu polymorphism were significantly different between schizophrenia patients and normal controls (Table II). This finding was consistent with a previous association study on the same polymorphism performed with 113 nuclear families that had a proband with schizophrenia, and which found no association between the SNP and patients [Shinkai et al., 2004]. Although these two studies did not provide any evidence of an association between the GPX1 gene and schizophrenia, several studies have reported that the glutathione peroxidase activity was altered in schizophrenic patients. Postmortem brain sample studies revealed that the glutathione peroxidase level was significantly lower in the caudate region of schizophrenic brains than in the caudate region of control brains [Yao et al., 2006], and the levels of erythrocytes’ plasma membrane glutathione peroxidase as well as other antioxidant enzymes (superoxide dismutase and catalase) were significantly lower in schizophrenic patients than in controls [Ranjekar et al., 2003]. Furthermore, in a study by Zhang et al. , schizophrenic patients with tardive dyskinesia showed lower plasma glutathione peroxidase levels but higher malondialdehide levels, and malondialdehide levels were positively correlated with the severity of symptoms, suggesting that oxidative stress is involved in the pathophysiology of schizophrenia with tardive dyskinesia. In addition, Buckman et al.  reported a strong negative correlation between the glutathione peroxidase activity of platelets and erythrocytes and morphological changes in the brains of schizophrenic patients as measured by CT scan. Taking these results into consideration, there might be other genetic grounds besides Pro198Leu polymorphism causing the altered GPX1 activity in schizophrenia. Recently, Tosic et al.  reported the intriguing result that the expression level of the GCLM gene was lower in the fibroblasts of schizophrenic patients than in those of controls, and that a significant relation was observed between either of two SNPs (ss60197536 (588) and rs2301122) in the GCLM gene and schizophrenia in Swiss and Danish populations. In regard to the Japanese population, our study suggests that neither GCLM polymorphism (ss60197536 and rs2301122) is associated with susceptibility to the pathogenesis of schizophrenia. Although the reasons underlying this discrepancy are currently unclear, one possibility is that there is an ethnic difference in the two SNPs of the GCLM gene that engendered different results in the two studies (Japanese vs. Swiss and Danish; see below and Table V). In contrast, it has been reported that GCLM knockout mice show an increased sensitivity to oxidative stress and a decreased level of GSH [Yang et al., 2002]. It is thus likely that GCLM may be involved in the pathophysiology of schizophrenia, since GCLM may play a role in the decreased GSH levels in CSF and in the medial prefrontal cortex of schizophrenia [Do et al., 2000]. Although the individual effects of the GSTT1, GSTP1, GPX1, and GCLM genes did not alter the risk for schizophrenia, the combination analysis revealed that the residual type of schizophrenia has significantly more risk genotypes of GSTs, GPX1, or GCLM (Table IV). The odds ratio of residual-type schizophrenia with any risk genotypes of those gene showed 5.91 (95% CI ¼ 1.375–25.38) with those who had no risk genotypes as a reference. Accumulating evidence shows that the proteins including GSTs, GPX1, and Caucasians African-Americansc P* b 51.8%, 48.2% (n ¼ 220) 74.4%, 25.6% (n ¼ 79) x 2 ¼ 19.5, df ¼ 2; P < 0.0001 47.7%, 40.9%, 11.4%b (n ¼ 220) 31.6%, 50.6%, 17.7% (n ¼ 79) x 2 ¼ 59.6, df ¼ 4; P < 0.0001 80.5%, 19.5% (n ¼ 79) x2 ¼ 9.23, df ¼ 2; P ¼ 0.011 73.6%, 23.4%b (n ¼ 220) d x 2 ¼ 31.2, df ¼ 2; P < 0.0001 73.1%, 23.7%, 3.1% (n ¼ 350) b x 2 ¼ 61.8, df ¼ 2; P < 0.0001 43.6%, 45.9%, 10.5% (n ¼ 220) e x 2 ¼ 63.6, df ¼ 2; P < 0.0001 54.4%, 36.1%, 9.5% (n ¼ 377) f x2 ¼ 1.37, df ¼ 2; P ¼ 0.485 70.7%, 26.7%, 2.6% (n ¼ 348) f x2 ¼ 12.2, df ¼ 2; P ¼ 0.002 45.1%, 42.0%, 12.9% (n ¼ 348) GCLM in the GSH metabolism play substantial role in maintaining the antioxidant properties of GSH, and thus the cells with alternated those protein function might be more susceptible to oxygen free radicals when exposed [Dringen, 2000a; Klivenyi et al., 2000; Smeyne et al., 2007]. The small sample size for each subtype of schizophrenia patient was a limitation of our study. Pae et al. [2003, 2004] referred to ethnic differences of the distributions of GSTM1 and GSTP1 polymorphisms. Table V summarizes the ethnic differences in the distributions in GSTM1, GSTP1, GSTT1, GSTM3, GSTO1, GPX1, and GCLM [Risch et al., 2001; Gilliland et al., 2002; Ravn-Haren et al., 2006; Tosic et al., 2006; Wahner et al., 2007]. As can be seen, there were significant ethnic differences in each genetic distribution. Further genetic association studies of these GSH-related genes using other ethnic population (e.g., Caucasians, African-Americans) would be interest to study the role of oxidative stress in the pathogenesis of schizophrenia. Some limitations in the present study should be considered. First, sample size is small. Larger sample size for each subtypes of schizophrenia should be needed to obtain the sufficient statistic power to exclude type I error. In addition, the results we found in the residual-type schizophrenics would be rendered as insignificant with applying post hoc analysis for multiple testing. However, the heterogenerous pathophysiological basis could underlie the schizophrenic individuals with different clinical features [Kirkpatrick et al., 2001], and especially those with persistent negative symptoms should be distinguished [Buchanan, 2007]. Further study with larger sample size would verify the difference in the effect of polymorphisms of GSH-related genes among schizophrenia subtypes. Second, due to the substantial numbers of chronic inpatients with the long duration of illness (mean 26.7 14.7 years) of schizophrenia subjects, the mean age between controls and patient groups were statistically different (unpaired t-test, P < 0.01). Although, most of the young control subjects were medical students and they are confirmed not to develop schizophrenia so far, this factor might decrease the power of our study. Japanesea 45.9%, 54.1% (n ¼ 220) 73.6%, 25.0%, 1.8% (n ¼ 220) 63.6%, 36.4% (n ¼ 220) 99.0%, 1.0%, 0.0% (n ¼ 220) 79.5%, 19.0%, 1.8% (n ¼ 220) 85.9%, 13.3%, 0.9% (n ¼ 220) 67.3%, 29.9%, 4.1% (n ¼ 220) 59.5%, 35.8%, 7.3% (n ¼ 220) CONCLUSION *Chi-square test. a Our data. b Wahner et al. . c Gilliland et al. . d Risch et al. . e Ravn-Haren et al. . f Tosic et al. . The bold type means significant difference (P < 0.05). Gene GSTM1: positive, null type GSTP1: Ile105/Val Ile/Ile, Ile/Val, Val/Val GSTT1: positive, null type GSTM3 A/B: A/A, A/B, B/B GSTO1 Ala140Asp: Ala/Ala, Ala/Asp, Asp/Asp GPX1 Pro198Leu: Pro/Pro, Pro/Leu, Leu/Leu GCLM ss60197536: C/C, C/T, T/T GCLM rs2301022: G/G, G/A, A/A Ethnicity AMERICAN JOURNAL OF MEDICAL GENETICS PART B TABLE V. Summarized Ethnic Differences of Distributions in GSTM1, GSTP1, GSTT1, GSTM3, GSTO1, GPX1, and GCLM 92 In conclusion, the present study suggests that the GSTM1 null type might increase the risk for the residual-type of schizophrenia, and that other GSH-related genes, including GSTM1, GSTT1, GSTP1, GPX1, and GCLM, might have no major genetic effects on the pathogenesis of schizophrenia in the Japanese population. The present results do not, however, exclude the possibility that oxidative stress plays a role in the pathophysiology of schizophrenia. In addition, the present findings suggest that the ethnic difference might underlie in the antioxidant system in which those GSHrelated enzymes involved. ACKNOWLEDGMENTS This study was supported in part by grants from the Minister of Education, Culture, Sports, Science, and Technology of Japan (to K.H.). 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