Association analysis of the DISC1 gene with schizophrenia in the Japanese population and DISC1 immunoreactivity in the postmortem brain

Neuroscience Research 77 (2013) 222–227

Contents lists available at ScienceDirect

Neuroscience Research journal homepage: www.elsevier.com/locate/neures

Association analysis of the DISC1 gene with schizophrenia in the Japanese population and DISC1 immunoreactivity in the postmortem brain Woraphat Ratta-apha a , Akitoyo Hishimoto a,∗ , Kentaro Mouri a , Kyoichi Shiroiwa a , Toru Sasada a , Masakuni Yoshida a , Irwan Supriyanto a , Yasuhiro Ueno b , Migiwa Asano c , Osamu Shirakawa d , Hideru Togashi e , Yoshimi Takai e , Ichiro Sora a a

Department of Psychiatry, Kobe University Graduate School of Medicine, Kobe, Japan Department of Legal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan c Department of Legal Medicine, Ehime University Graduate School of Medicine, Ehime, Japan d Department of Neuropsychiatry, Kinki University School of Medicine, Osaka, Japan e Division of Molecular and Cellular Biology, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kobe, Japan b

a r t i c l e

i n f o

Article history: Received 12 April 2013 Received in revised form 24 August 2013 Accepted 26 August 2013 Available online 4 September 2013 Keywords: DISC1 Ser704Cys SNP Schizophrenia Postmortem brain Immunoreactivity

a b s t r a c t The Disrupted-in-Schizophrenia 1 (DISC1) gene plays a role in the regulation of neural development. Previous evidence from genetic association and biological studies implicates the DISC1 gene as having a role in the pathophysiology of schizophrenia. In the present study, we explored the association between DISC1 missense mutation rs821616 (Ser704Cys) single nucleotide polymorphism (SNP) and four other SNPs (rs1772702, rs1754603, rs821621, rs821624) in the related haplotype block and schizophrenia in the Japanese population. We could not find a significant association of selected SNPs with schizophrenia after correction for multiple testing. We performed a meta-analysis of the Ser704Cys variant in schizophrenia using data from the present study and five previous Japanese population studies, and found no association with schizophrenia. We also examined DISC1 immunoreactivity in postmortem prefrontal cortex specimens of schizophrenia patients compared to control samples. The immunoreactivity revealed a significant decrease of DISC1 protein expression in the schizophrenia samples after ruling out potential confounding factors. However, the Ser704Cys variant did not show effects on DISC1 immunoreactivity. These results provide evidence that this functional genetic variation of DISC1 do not underlie the pathophysiology of schizophrenia in the Japanese population. © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

1. Introduction Schizophrenia is a severe and debilitating mental disorder with a prevalence of approximately 1%, and it has a pathogenesis that involves many factors such as genetic, environmental factors and psychological etiologies (Owen et al., 2002). The Disrupted-in-Schizophrenia 1 (DISC1) gene was originally identified in a Scottish family with a high incidence of psychiatric disorders. In this family, a balanced translocation between

Abbreviations: DISC1, Disrupted-in-Schizophrenia 1; SNP, single nucleotide polymorphism; HWE, Hardy–Weinberg equilibrium; LD, linkage disequilibrium; DLPFC, dorsolateral prefrontal cortex. ∗ Corresponding author at: Department of Psychiatry, Faculty of Medical Science, Kobe University Graduate School of Medicine, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Tel.: +81 78 382 6065; fax: +81 78 382 6079. E-mail address: [email protected] (A. Hishimoto).

chromosomes 1 and 11 that disrupts the DISC1 locus appeared to co-segregate with schizophrenia, major depression and bipolar disorder (Blackwood et al., 2001; Millar et al., 2000; St Clair et al., 1990). DISC1 transcripts are highly expressed during embryonic neurogenesis and puberty (Austin et al., 2004; Schurov et al., 2004), and plays progenitor cell proliferation, assembly of centrosome and microtubule networks, and synaptic signaling (Porteous et al., 2011; Wang and Brandon, 2011). Several linkage and association studies have pointed to DISC1 as a candidate for susceptibility to psychiatric disorders especially schizophrenia (Callicott et al., 2005; Hennah et al., 2003). Sawa and Snyder hypothesized that the chromosomal abnormality could lead to a carboxy-terminal-truncated DISC1 protein, or degradation of the protein, or both (Sawa and Snyder, 2005). Kamiya et al. showed that the putative C-terminal-truncated protein could act as a dominant-negative (Kamiya et al., 2005). The results from the expression study of lymphoblastoid cell lines from patients in a balanced translocation family by Millar et al.

0168-0102/$ – see front matter © 2013 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. http://dx.doi.org/10.1016/j.neures.2013.08.010

W. Ratta-apha et al. / Neuroscience Research 77 (2013) 222–227

showed that patients with the breakpoint had lower expression and could not identify the truncated protein (Millar et al., 2005). The dominant-negative protein or the haplo-insufficiency leads to an overall loss of DISC1 function. The present data regarding DISC1 expression in schizophrenic patients are limited. Postmortem studies have given mixed results. There were no difference in expression of DISC1 in total brain homogenates (Sawamura et al., 2005) and dorsolateral prefrontal cortex (DLPFC) (Rastogi et al., 2009). However, Lipska et al. reported a trend toward increased expression (approximately 20%) in the hippocampus of schizophrenia patients (Lipska et al., 2006). One of the most common missense variants of DISC1, which in exon 11 has a serine-to-cysteine substitution at position 704 (Ser704Cys SNP; rs821616), has been reported to be associated with schizophrenia in Asian population, at least in the Chinese Han population (Qu et al., 2007; Song et al., 2008). However, the Ser704Cys variant was not associated with schizophrenia (Hotta et al., 2011; Kinoshita et al., 2012; Zhang et al., 2005) in Japanese populations. This genetic variant has been reported to show many phenotypes: (1) reduction of gray matter volume of the cingular cortex, cingular gyrus and posterior gyrus and reduction of white matter integrity in the posterior prefrontal cortex in carriers of the Cys704 allele (Hashimoto et al., 2006) and (2) altered hippocampal structure and function, and altered engagement of hippocampus in carriers of the Ser704 allele (Callicott et al., 2005). The present study was designed to examine whether there is a relationship of DISC1 gene polymorphisms and schizophrenia in the Japanese population. We performed an association study of the DISC1 gene and schizophrenia subjects and a meta-analysis of the missense variant Ser704Cys in a Japanese population. Furthermore, we investigated the potential role of DISC1 in schizophrenia by examining the immunoreactivity of DISC1 in the prefrontal cortex of postmortem brains of schizophrenia subjects and nonpsychiatric control subjects. 2. Materials and methods 2.1. Subjects The association study used samples from two different groups. The schizophrenic subjects consisted of 503 unrelated patients (mean age ± SD = 54.6 ± 14.0); 243 males and 260 females. The patients were diagnosed by at least two psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders 4th edition (DSM-IV) for schizophrenia on the basis of unstructured interviews and reviews of their medical records at each hospital. None of the schizophrenic subjects had a history of substance abuse or organic mental disorders. The control subjects consisted of 511 healthy subjects (mean age ± SD = 54.6 ± 14.0); 225 males and 286 females. None of the control subjects were related to other subjects in the control group and none manifested psychiatric problems in an unstructured interview conducted by two psychiatrists using the DSM-IV criteria. Control subjects with a history and/or familial history of psychiatric disorders and/or suicidal behaviors were excluded from this study. For the immunoreactivity study using postmortem prefrontal cortex, brain samples were obtained from 24 control samples and 14 schizophrenic patients. Demographic data, the postmortem interval and the pH of the brain are shown in Supplementary Table 1. The diagnosis of schizophrenia was made by the attending psychiatrists before the death of the patients, and then three psychiatrists verified the diagnosis by reviewing the clinical records of each patient according to the DSM-IV. This study was conducted with the approval of the ethical committee for genetic studies of the Kobe University Graduate School of Medicine. Informed consent was obtained from the genotyping

223

participants and from the close relatives of the subjects used for postmortem brain studies. 2.2. DISC1 SNPs selection and genotyping Initially we selected the rs821616 SNP (Ser704Cys) of the DISC1 gene for our studies based on its involvement in schizophrenia that was shown by previous studies (Callicott et al., 2005; Hashimoto et al., 2006; Qu et al., 2007). After the common rs821616 SNP showed nominally significantly associated with schizophrenia, we confirmed the positive result of the rs821616 SNP by selecting the rs821621 SNP, which has a perfect linkage disequilibrium with the rs821616 SNP (r2 and D = 1) for genotyping. Then, to detect a genetic association of a haplotype block including the rs821616 SNP with schizophrenia, we selected more SNPs from the same haplotype block by using the criterion of an r2 threshold > 0.8 in ‘pair-wise tagging only’ mode, rs1772702, rs1754603, and rs821624 SNPs (tagging SNPs) were selected by the tagger program within the Haploview software [http://broad.mit.edu/mpg/tagger] (Supplementary Figure 1). For determination of genotype, DNA was extracted from peripheral blood samples and stored at −40 ◦ C until use. All of the five SNPs were genotyped with TaqMan probe assays. We selected the pre-designed TaqMan SNP genotyping assays from the Applied Biosystems database. Each reaction for the TaqMan probe assays was performed in a final volume of 5 ␮L with the Universal Master Mix (Applied Biosystems, Foster City, CA, USA), and genotyping was performed with a 7500 Real Time PCR System (Applied Biosystems). 2.3. Meta-analysis of the DISC1 Ser704Cys (rs821616) variant We performed PubMed searches using the keywords “DISC1”, “schizophrenia”, “Ser704Cys”, “Japanese” and “association study”. We included articles examining any association study of the Ser704Cys variant with the schizophrenia in a Japanese population and added the data from our present study. Meta-analyses with the fixed-effects model and random-effects model were performed using the comprehensive meta-analysis version 2.0 software (Biostat Inc., http://www.meta-analysis.com/). 2.4. Immunoreactivity study Brain samples from DLPFC were prepared as described previously (Lin et al., 1999). Autopsied brains were obtained from the Department of Legal Medicine, Kobe University Graduate School of Medicine. Brains were removed and stored at −80 ◦ C. To obtain tissue blocks, the brain samples were moved to −20 ◦ C, then the right DLPFC was dissected into coronal blocks of approximately 1 cm thick on dry ice. The dissected brain samples were stored at −80 ◦ C until use. Approximately 300–500 ml of each sample was divided, homogenized in RIPA Buffer and protease inhibitor cocktail (Roche, Germany). The samples were sonicated, and protein concentration was measured by the Lowry method. Protein concentration was adjusted to 1 mg/␮l with RIPA buffer, and an aliquot of the sample was suspended in SDS buffer containing 2-mercaptoethanol. For immunoblotting, equal amounts of protein (5–10 ␮L/lane) for each sample were loaded into 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred from the gels to polyvinylidene difluoride (PVDF) membranes by blotting in Tris/Glycine buffer at 4 ◦ C for 180 min. The blotted membranes were blocked with skim milk and incubated with primary antibodies [anti-DISC1, 1:500 (Life Technologies, CA, USA) and anti-actin, clone 4 monoclonal antibody, 1:5000 (Millipore, CA, USA)] at 4 ◦ C overnight. Membranes were washed in TBS-T buffer, incubated for 1 h with horseradish peroxidase (HRP) conjugated antibodies [1:1000 anti-mouse IgG (Amersham Bioscience,

224

W. Ratta-apha et al. / Neuroscience Research 77 (2013) 222–227

2 = 6.46, allelic p = 0.011 and rs821621; genotypic p = 0.019, 2 = 5.471, allelic p = 0.019). However, the differences of these two SNPs did not withstand correction for multiple comparisons (Table 1). In the haplotype analysis, the schizophrenia groups showed nominally significant associations between two to three markers [rs1772702-rs1754603-rs821616] in the LD block (Supplementary Table 2). Although the combinations including Ser704Cys SNP had a relatively high significance, these significances were disappeared after correction of multiple tests (data not shown).

Little Chalfont, UK) or anti-rabbit IgG (Jackson ImmunoResearch Laboratories, PA, USA)], and then washed with TBS-T buffer. Protein bands were detected by enhanced chemiluminescence. Densitometry of the DISC1 protein bands was determined by luminous image analyzer LAS4000-mini and MultiGauge image analysis software (FUJIFILM Life Science, Tokyo, Japan) and ImageJ Software (http://rsbweb.nih.gov/ij/). 2.5. Data analysis The data were analyzed with SPSS software, version 16.0 (SPSS Inc., Chicago, IL). For the genotyping study, the Haploview software program version 4.2 (http://www.broad.mit.edu/mpg/haploview/) was used to determine the Hardy–Weinberg equilibrium (HWE), linkage disequilibrium (LD), and allelic/haplotype frequencies, as well as the association between SNPs of the controls and the schizophrenia group. Permutation tests based on 10,000 replications were performed to calculate the corrected allelic p value using Haploview software. Study power analysis was performed with the PS v2.1.31 software (Dupont and Plummer, 1998). Genotype-based association was tested with the Cochran–Armitage test for trend. Chi-squared test was used to calculate global p value in haplotype analysis. In the immunoreactivity study, the variables’ assumption of normality was tested. If the variables met the assumption of normality, Student’s t-test was used to analyze the difference between groups. The Pearson’s correlation coefficient was used to analyze the correlation of variables. The comparison of DISC1 immunoreactivity between control and schizophrenia group were tested by analysis of covariance (ANCOVA). The statistical significance was set at p value <0.05.

3.2. Meta-analysis of the DISC1 Ser704Cys (rs821616) variant Four association studies examining Japanese populations were found in a literature search (Hashimoto et al., 2006; Hotta et al., 2011; Kinoshita et al., 2012; Zhang et al., 2005). For each of these published studies and the present study, the study population and the genotype counts for both the schizophrenia and control groups are shown in Supplementary Table 3. All studies were independent and the reported frequencies were in HWE. Significant heterogeneity among the studies was detected in the pool data (Q = 15.953, df = 4, p = 0.003); therefore, we performed both a fixed-effects model and a random-effects model for the analysis. The result showed no significant association in the allele frequencies (number of T alleles in schizophrenia = 708, total number = 1651, random-effects model: pool OR = 0.97, 95% CI = 0.78–1.21, Z-value = −0.268, p value = 0.789) (Fig. 1). 3.3. DISC1 protein immunoreactivity, correlation of protein expression, and the effect of Ser704Cys variant on immunoreactivity in postmortem prefrontal cortex specimens

3. Results

Postmortem samples were used from a control group and a schizophrenic group. These groups were in normal distribution by using the Kolmogorov–Smirnov (KS) statistic. There were no differences in age (t = −0.372, df = 36, p = 0.712) and postmortem interval (t = −0.429, df = 36, p = 0.670) between the two groups. However, the measured pH of the postmortem brains was higher in the schizophrenia group than in the control group (t = −2.26, df = 36, p = 0.030) (Supplementary Table 4). We measured the DISC1 immunoreactivity, which was normalized by ␤-actin, between 14 postmortem brain samples from schizophrenic patients and 24 postmortem control brain

3.1. Association of the DISC1 gene with psychiatric disorders The genotype distribution was in HWE for all of the SNPs in the schizophrenia and control groups (except rs1754603 SNP in the schizophrenic group) (Table 1). These five selected SNPs were in LD with each other (D = 0.82–0.97) (Supplementary Figure 2). Genotypic distribution and allelic frequency of the rs821616 and rs821621 SNPs were significantly different between the schizophrenia and control groups (rs821616; genotypic p = 0.010,

Table 1 Association between Disrupted-in-Schizophrenia-I (DISC1) gene single nucleotide polymorphism with schizophrenia. SNP ID positiona

Phenb

Minor allele MAF

c

Genotype distributiond

Alleled

p value e

f

Power

OR (95% CI)

g

Allele

M/M

M/m

m/m

M

m

HWE

Genotype

Allelle

rs1772702 230205022

SCZ CON

0.228 0.242

C

297 295

164 180

30 33

758 770

224 246

0.300 0.493

0.470

0.460 (0.946)

0.082

1.08 (0.88–1.33)

rs1754603 230205245

SCZ CON

0.121 0.142

A

384 377

93 116

13 14

861 870

119 144

0.032 0.227

0.192

0.175 (0.630)

0.165

1.20 (0.93–1.55)

rs821616 230211221

SCZ CON

0.100 0.137

A

398 376

88 125

5 7

884 877

98 139

1.000 0.473

0.010*

0.011* (0.052)

0.443

1.43 (1.09–1.88)

rs821621 230212687

SCZ CON

0.099 0.133

T

393 376

84 120

6 7

870 872

96 134

0.659 0.615

0.019*

0.019* (0.092)

0.383

1.39 (1.05–1.84)

rs821624 230213717

SCZ CON

0.391 0.409

G

188 177

223 246

81 85

599 600

385 416

0.315 1.000

0.412

0.407 (0.920)

0.089

1.08 (0.90–1.29)

a b c d e f g

SNP ID: single nucleotide polymorphism identification numbers and positions (available at http://www.ncbi.nlm.nih.gov/sites/). Phen: phenotype – SCZ, schizophrenia; CON, control. MAF: minor allele frequency. M: major allele; m: minor allele. HWE: Hardy–Weinberg equilibrium. Genotypic p values were tested with Cochran–Armitage test for trend. Allelic p values, were tested with Chi square.

W. Ratta-apha et al. / Neuroscience Research 77 (2013) 222–227

225

Fig. 1. Meta-analysis and forest plot of the Ser704Cys polymorphism in schizophrenia: Includes data from previously published Ser704Cys association studies of schizophrenia in Japanese population.

samples. As shown in Fig. 2A, the protein bands of DISC1 and ␤-actin were located at 91 kilodalton (kDa) and 43 kDa, respectively. After correction by ␤-actin, the DISC1 immunoreactivity of the schizophrenic subjects was significantly less than that of the control subjects (t = 3.920, df = 36, p < 0.001) (Fig. 2B). DISC1 immunoreactivity was not correlated with age (Pearson r = 0.048, p = 0.773), brain pH (Pearson r = −0.221, p = 0.182), and postmortem interval (Pearson r = 0.044, p = 0.792). In the schizophrenia group, the duration of illness was not correlated with DISC1 immunoreactivity (Pearson r = −0.406, p = 0.215). To exclude effects of pH, one-way ANCOVA that took pH into account as a covariate was conducted. The DISC1 immunoreactivity corrected by ␤-actin of the schizophrenia groups was still lower than that of the control group [F(1, 35) = 12.557, p = 0.001]. To rule out potential confounding factors, we used one-way ANCOVA that took pH, PMI, age, and gender into account as a covariate in subsequent analyses. In this analysis, the DISC1 immunoreactivity corrected by ␤-actin of the schizophrenia groups was again lower than that of the control group [F(1, 32) = 7.816, p = 0.009]. Brain samples were also genotyped. Two-way ANCOVA was used for analyzing the effect of Ser704Cys variant on DISC1 immunoreactivity in postmortem brain. The level of DISC1 immunoreactivity was set as the dependent variable, while pH, PMI, age, and gender were taken as covariates to rule out potential confounding factors. In the first analysis, the phenotype (control versus schizophrenia) and genotype (AA, AT, TT) were set as

Fig. 2. DISC1 immunoreactivity in postmortem brain samples. (A) Representative immunoblots of DISC1 and ␤-actin for two subjects of each group (control group and schizophrenia group). The bands of DISC1 and ␤-actin are located at 91 kDa and 43 kDa. (B) Comparison of DISC1 immunoreactivity between schizophrenia and control postmortem brain samples. respectively.

independent variables, and DISC1 immunoreactivity did not differ between the schizophrenia and control samples [F(1, 29) = 0.002, p = 0.964]. When the phenotype and allele (Ser-allele or Cys-allele carrier) were set as the independent variables, the result also showed no difference of DISC1 immunoreactivity between two groups [F(1, 30) = 0.016, p = 0.900]. We then analyzed only the schizophrenia group comparing the difference of immunoreactivity between the Ser-allele and the Cys-allele carrier group by taking all the confounding factors as covariates as before; the result also showed no difference of DISC1 immunoreactivity between two groups [F(1, 8) = 4.961, p = 0.057]. 4. Discussion The present study provides two types of experiments that examine the relationship of the DISC1 gene and schizophrenia. For the association study, the nominal significant association of the genotypic distribution and allelic frequency of rs821616 SNP and its perfect linkage disequilibrium SNP (rs821621 SNP) were observed between the two groups, although the significance did not withstand after correction for multiple comparisons. As the previous investigation by Schumacher et al. reported the significant association of DISC1 with schizophrenia in females (Schumacher et al., 2009), we applied gender-based analyses for the association. Inconsistent with the previous findings, the gender-based analyses showed the significant association of the genotypic distribution and allelic frequency of rs821616, rs821621, and rs1754603 SNPs between the male schizophrenia and control groups (Supplementary Table 1). As these findings might be type I error due to multiple comparisons, the nominal positive findings should be carefully interpreted. In the present meta-analysis, the heterogeneity between the five studies was detected (Hashimoto et al., 2006; Hotta et al., 2011; Kinoshita et al., 2012; Zhang et al., 2005). It may be due to the limited number of the studies and the variation in study outcomes between studies. Allelic frequency was quite different between the five studies. This present study showed lower T allelic frequency in the schizophrenia group compared to the other previous studies (9.98% vs. 10.32–14.85%), while the allelic frequency in the control group was the same. However, the finding is consistent with results from previous meta-analyses that have not identified DISC1 as a prominent hit for schizophrenia (Kinoshita et al., 2012; Mathieson et al., 2012). According to our study, the Ser704Cys variants also did not show significant effects on DISC1 protein levels in postmortem brains. There are a number of nonsynonymous changes that have

226

W. Ratta-apha et al. / Neuroscience Research 77 (2013) 222–227

been reported in the DISC1 gene, but their impacts on DISC1 function or protein expression are not yet clear (Chubb et al., 2008). Identifying and characterizing the causative variants or alleles that may alter the function of the DISC1 should be a focus of further studies. For the immunoreactivity study, the DLPFC region was selected because of its relation to cognitive dysfunction of schizophrenia, whereas previous studies focused on the hippocampus and orbitofrontal cortex where the DISC1 protein is predominately expressed (Lipska et al., 2006; Millar et al., 2000; Sawamura et al., 2005). We found significant decreases of DISC1 expression in schizophrenic subjects, which supported previous findings with experiments performed in cell lines of schizophrenic patients in a balanced translocation family (Millar et al., 2005); lower levels of DISC1 might be related to the pathogenesis of schizophrenia. The number of postmortem brain samples we used was quite small compared to that of previous studies, but, to our best of knowledge, the existing data of DISC1 immunoreactivity examined in Japanese postmortem brain samples is limited. Because many factors [e.g., pre-mortem factors (metabolic state of the diseased, presence of a toxic substance or drug, hypoxia) and treatment with antipsychotics] affect protein stability and expression of genes in brain tissues, our findings should be also carefully interpreted. In addition, Nakata et al. and the Weinberger laboratory reported that DISC1 has many isoforms including many still unknown isoforms. All of these patterns of DISC1 protein bands did not fully detected by western blotting (Nakata et al., 2009). DISC1 is relatively showed low signal expression and high background levels in brain and difficult to detect by available antibodies (Nakata et al., 2009). Brandon and Sawa suggested to use reliable pairs of antibodies against DISC1 to detect DISC1 protein in brain samples in immuno-precipitation western blot for further improvement of researches (Brandon and Sawa, 2011). Several limitations in the present study should be considered. (1) The number of subjects in the association study, which according to the sample power data, was still a small sample size. (2) We did not have completed clinical background information. So we cannot examine an association study of a genetic link between DISC1 and other behavioral endophenotypes such as memory and anhedonia. In the immunoreactivity study, the data also uncompleted, and it was not clear that the psychotropic medications and other factors might affect the expression or not. (3) This study investigated only the common Ser704Cys variant and neighboring SNPs; therefore, the expression and function of additional variants should be examined in schizophrenia. (4) The immunoreactivity of DISC1 was examined only in prefrontal cortex specimens. The other areas of the brain that are involved in the pathophysiology of schizophrenia should also be examined. (5) The use of postmortem brain tissue for performing immunoreactivity and measuring the quantification of expression can be complicated by various confounding factors and the quality may have been affected by degradation during the postmortem interval. Moreover, the quality of commercially available DISC1 antibodies should be aware and follow the recommended guideline as we stated in the discussion. Next, the studies of DISC1 expression in postmortem brain samples with well-controlled confounding factors should be performed to reveal the role of DISC1 in schizophrenic brain. Furthermore, the future researches using multiple mouse models for DISC1, human cell culture-based and in vivo models are needed for further understanding functions and related pathways of DISC1 (Porteous et al., 2011). In conclusion, the present study showed that DISC1 immunoreactivity in the postmortem brain was significantly lower in a schizophrenia group than in a control group, while we cannot find an association between the DISC1 gene and schizophrenia, including with meta-analysis of the missense mutation rs821616

(Ser704Cys) SNP. These results provide evidence that the functional genetic variation of DISC1, Ser704Cys, does not underlie the pathophysiology of schizophrenia in the Japanese population. Because the cause of reduction in expression of DISC1 in schizophrenia is unclear, further expression studies of DISC1 and association studies with a larger sample size will better reveal the role of DISC1 in schizophrenia. DISC1 is one of challenging targets for the exploration of new strategies for understanding pathophysiology and treatment of schizophrenia. Acknowledgements This work was supported in part by research grants from the Ministry of Education, Culture, Sports, Science and Technology (MECSS) in Japan. We thank Ms. Y. Nagashima for technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neures. 2013.08.010. References Austin, C.P., Ky, B., Ma, L., Morris, J.A., Shughrue, P.J., 2004. Expression of Disruptedin-Schizophrenia-1, a schizophrenia-associated gene, is prominent in the mouse hippocampus throughout brain development. Neuroscience 124, 3–10. Blackwood, D.H., Fordyce, A., Walker, M.T., St Clair, D.M., Porteous, D.J., Muir, W.J., 2001. Schizophrenia and affective disorders—cosegregation with a translocation at chromosome 1q42 that directly disrupts brain-expressed genes: clinical and P300 findings in a family. Am. J. Hum. Genet. 69, 428–433. Brandon, N.J., Sawa, A., 2011. Linking neurodevelopmental and synaptic theories of mental illness through DISC1. Nat. Rev. Neurosci. 12, 707–722. Callicott, J.H., Straub, R.E., Pezawas, L., Egan, M.F., Mattay, V.S., Hariri, A.R., Verchinski, B.A., Meyer-Lindenberg, A., Balkissoon, R., Kolachana, B., Goldberg, T.E., Weinberger, D.R., 2005. Variation in DISC1 affects hippocampal structure and function and increases risk for schizophrenia. Proc. Natl. Acad. Sci. U. S. A. 102, 8627–8632. Chubb, J.E., Bradshaw, N.J., Soares, D.C., Porteous, D.J., Millar, J.K., 2008. The DISC locus in psychiatric illness. Mol. Psychiatry 13, 36–64. Dupont, W.D., Plummer Jr., W.D., 1998. Power and sample size calculations for studies involving linear regression. Control. Clin. Trials 19, 589–601. Hashimoto, R., Numakawa, T., Ohnishi, T., Kumamaru, E., Yagasaki, Y., Ishimoto, T., Mori, T., Nemoto, K., Adachi, N., Izumi, A., Chiba, S., Noguchi, H., Suzuki, T., Iwata, N., Ozaki, N., Taguchi, T., Kamiya, A., Kosuga, A., Tatsumi, M., Kamijima, K., Weinberger, D.R., Sawa, A., Kunugi, H., 2006. Impact of the DISC1 Ser704Cys polymorphism on risk for major depression, brain morphology and ERK signaling. Hum. Mol. Genet. 15, 3024–3033. Hennah, W., Varilo, T., Kestila, M., Paunio, T., Arajarvi, R., Haukka, J., Parker, A., Martin, R., Levitzky, S., Partonen, T., Meyer, J., Lonnqvist, J., Peltonen, L., Ekelund, J., 2003. Haplotype transmission analysis provides evidence of association for DISC1 to schizophrenia and suggests sex-dependent effects. Hum. Mol. Genet. 12, 3151–3159. Hotta, Y., Ohnuma, T., Hanzawa, R., Shibata, N., Maeshima, H., Baba, H., Hatano, T., Takebayashi, Y., Kitazawa, M., Higa, M., Suzuki, T., Arai, H., 2011. Association study between Disrupted-in-Schizophrenia-1 (DISC1) and Japanese patients with treatment-resistant schizophrenia (TRS). Prog. Neuropsychopharmacol. Biol. Psychiatry 35, 636–639. Kamiya, A., Kubo, K., Tomoda, T., Takaki, M., Youn, R., Ozeki, Y., Sawamura, N., Park, U., Kudo, C., Okawa, M., Ross, C.A., Hatten, M.E., Nakajima, K., Sawa, A., 2005. A schizophrenia-associated mutation of DISC1 perturbs cerebral cortex development. Nat. Cell Biol. 7, 1167–1178. Kinoshita, M., Numata, S., Tajima, A., Ohi, K., Hashimoto, R., Shimodera, S., Imoto, I., Itakura, M., Takeda, M., Ohmori, T., 2012. Meta-analysis of association studies between DISC1 missense variants and schizophrenia in the Japanese population. Schizophr. Res. 141, 271–273. Lin, X.H., Kitamura, N., Hashimoto, T., Shirakawa, O., Maeda, K., 1999. Opposite changes in phosphoinositide-specific phospholipase C immunoreactivity in the left prefrontal and superior temporal cortex of patients with chronic schizophrenia. Biol. Psychiatry 46, 1665–1671. Lipska, B.K., Peters, T., Hyde, T.M., Halim, N., Horowitz, C., Mitkus, S., Weickert, C.S., Matsumoto, M., Sawa, A., Straub, R.E., Vakkalanka, R., Herman, M.M., Weinberger, D.R., Kleinman, J.E., 2006. Expression of DISC1 binding partners is reduced in schizophrenia and associated with DISC1 SNPs. Hum. Mol. Genet. 15, 1245–1258. Mathieson, I., Munafo, M.R., Flint, J., 2012. Meta-analysis indicates that common variants at the DISC1 locus are not associated with schizophrenia. Mol. Psychiatry 17, 634–641.

W. Ratta-apha et al. / Neuroscience Research 77 (2013) 222–227 Millar, J.K., Pickard, B.S., Mackie, S., James, R., Christie, S., Buchanan, S.R., Malloy, M.P., Chubb, J.E., Huston, E., Baillie, G.S., Thomson, P.A., Hill, E.V., Brandon, N.J., Rain, J.C., Camargo, L.M., Whiting, P.J., Houslay, M.D., Blackwood, D.H., Muir, W.J., Porteous, D.J., 2005. DISC1 and PDE4B are interacting genetic factors in schizophrenia that regulate cAMP signaling. Science 310, 1187–1191. Millar, J.K., Wilson-Annan, J.C., Anderson, S., Christie, S., Taylor, M.S., Semple, C.A., Devon, R.S., St Clair, D.M., Muir, W.J., Blackwood, D.H., Porteous, D.J., 2000. Disruption of two novel genes by a translocation co-segregating with schizophrenia. Hum. Mol. Genet. 9, 1415–1423. Nakata, K., Lipska, B.K., Hyde, T.M., Ye, T., Newburn, E.N., Morita, Y., Vakkalanka, R., Barenboim, M., Sei, Y., Weinberger, D.R., Kleinman, J.E., 2009. DISC1 splice variants are upregulated in schizophrenia and associated with risk polymorphisms. Proc. Natl. Acad. Sci. U. S. A. 106, 15873–15878. Owen, M.J., Donovan, O., Gottesman II, M.C., 2002. Schizophrenia. In: Owen, M.J., Gottesman, I.I. (Eds.), Psychiatric Genetics and Genomics. Oxford University Press, Oxford, England, pp. 247–266. Porteous, D.J., Millar, J.K., Brandon, N.J., Sawa, A., 2011. DISC1 at 10: connecting psychiatric genetics and neuroscience. Trends Mol. Med. 17, 699–706. Qu, M., Tang, F., Yue, W., Ruan, Y., Lu, T., Liu, Z., Zhang, H., Han, Y., Zhang, D., Wang, F., 2007. Positive association of the Disrupted-in-Schizophrenia-1 gene (DISC1) with schizophrenia in the Chinese Han population. Am. J. Med. Genet. B: Neuropsychiatr. Genet. 144B, 266–270. Rastogi, A., Zai, C., Likhodi, O., Kennedy, J.L., Wong, A.H., 2009. Genetic association and post-mortem brain mRNA analysis of DISC1 and related genes in schizophrenia. Schizophr. Res. 114, 39–49.

227

Sawa, A., Snyder, S.H., 2005. Genetics. Two genes link two distinct psychoses. Science 310, 1128–1129. Sawamura, N., Sawamura-Yamamoto, T., Ozeki, Y., Ross, C.A., Sawa, A., 2005. A form of DISC1 enriched in nucleus: altered subcellular distribution in orbitofrontal cortex in psychosis and substance/alcohol abuse. Proc. Natl. Acad. Sci. U. S. A. 102, 1187–1192. Schumacher, J., Laje, G., Abou Jamra, R., Becker, T., Muhleisen, T.W., Vasilescu, C., Mattheisen, M., Herms, S., Hoffmann, P., Hillmer, A.M., Georgi, A., Herold, C., Schulze, T.G., Propping, P., Rietschel, M., McMahon, F.J., Nothen, M.M., Cichon, S., 2009. The DISC locus and schizophrenia: evidence from an association study in a central European sample and from a meta-analysis across different European populations. Hum. Mol. Genet. 18, 2719–2727. Schurov, I.L., Handford, E.J., Brandon, N.J., Whiting, P.J., 2004. Expression of disrupted in schizophrenia 1 (DISC1) protein in the adult and developing mouse brain indicates its role in neurodevelopment. Mol. Psychiatry 9, 1100–1110. Song, W., Li, W., Feng, J., Heston, L.L., Scaringe, W.A., Sommer, S.S., 2008. Identification of high risk DISC1 structural variants with a 2% attributable risk for schizophrenia. Biochem. Biophys. Res. Commun. 367, 700–706. St Clair, D., Blackwood, D., Muir, W., Carothers, A., Walker, M., Spowart, G., Gosden, C., Evans, H.J., 1990. Association within a family of a balanced autosomal translocation with major mental illness. Lancet 336, 13–16. Wang, Q., Brandon, N.J., 2011. Regulation of the cytoskeleton by Disrupted-inschizophrenia 1 (DISC1). Mol. Cell. Neurosci. 48, 359–364. Zhang, X., Tochigi, M., Ohashi, J., Maeda, K., Kato, T., Okazaki, Y., Kato, N., Tokunaga, K., Sawa, A., Sasaki, T., 2005. Association study of the DISC1/TRAX locus with schizophrenia in a Japanese population. Schizophr. Res. 79, 175–180.