Common variants at 1p36 are associated with superior frontal gyrus volume

Abstract

The superior frontal gyrus (SFG), an area of the brain frequently found to have reduced gray matter in patients with schizophrenia, is involved in self-awareness and emotion, which are impaired in schizophrenia. However, no genome-wide association studies of SFG volume have investigated in patients with schizophrenia. To identify single-nucleotide polymorphisms (SNPs) associated with SFG volumes, we demonstrated a genome-wide association study (GWAS) of gray matter volumes in the right or left SFG of 158 patients with schizophrenia and 378 healthy subjects. We attempted to bioinformatically ascertain the potential effects of the top hit polymorphism on the expression levels of genes at the genome-wide region. We found associations between five variants on 1p36.12 and the right SFG volume at a widely used benchmark for genome-wide significance (P<5.0 × 10 ^-8 ). The strongest association was observed at rs4654899, an intronic SNP in the eukaryotic translation initiation factor 4 gamma, 3 (EIF4G3) gene on 1p36.12 (P=7.5 × 10^-9 ). No SNP with genome-wide significance was found in the volume of the left SFG (P>5.0 × 10^-8 ); however, the rs4654899 polymorphism was identified as the locus with the second strongest association with the volume of the left SFG (P=1.5 × 10^-6 ). In silico analyses revealed a proxy SNP of rs4654899 had effect on gene expression of two genes, HP1BP3 lying 3[variant prime] to EIF4G3 (P=7.8 × 10^-6 ) and CAPN14 at 2p (P=6.3 × 10^-6 ), which are expressed in moderate-to-high levels throughout the adult human SFG. These results contribute to understand genetic architecture of a brain structure possibly linked to the pathophysiology of schizophrenia.

OPEN

Citation: Transl Psychiatry (2014) 4, e472; doi:http://dx.doi.org/10.1038/tp.2014.110

Web End =10.1038/tp.2014.110 2014 Macmillan Publishers Limited All rights reserved 2158-3188/14

http://www.nature.com/tp

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R Hashimoto1,2,13, M Ikeda3,13, F Yamashita4, K Ohi2, H Yamamori2,5, Y Yasuda2, M Fujimoto2, M Fukunaga6, K Nemoto7, T Takahashi8, M Tochigi9, T Onitsuka10, H Yamasue9, K Matsuo11, T Iidaka12, N Iwata3, M Suzuki8, M Takeda1,2, K Kasai9 and N Ozaki12

The superior frontal gyrus (SFG), an area of the brain frequently found to have reduced gray matter in patients with schizophrenia, is involved in self-awareness and emotion, which are impaired in schizophrenia. However, no genome-wide association studies of SFG volume have investigated in patients with schizophrenia. To identify single-nucleotide polymorphisms (SNPs) associated with SFG volumes, we demonstrated a genome-wide association study (GWAS) of gray matter volumes in the right or left SFG of 158 patients with schizophrenia and 378 healthy subjects. We attempted to bioinformatically ascertain the potential effects of the top hit polymorphism on the expression levels of genes at the genome-wide region. We found associations between ve variants on 1p36.12 and the right SFG volume at a widely used benchmark for genome-wide signicance (Po5.0 108). The strongest association was observed at rs4654899, an intronic SNP in the eukaryotic translation initiation factor 4 gamma, 3 (EIF4G3) gene on 1p36.12 (P = 7.5 109). No SNP with genome-wide signicance was found in the volume of the left SFG (P45.0 108); however, the rs4654899 polymorphism was identied as the locus with the second strongest association with the volume of the left SFG (P = 1.5 106). In silico analyses revealed a proxy SNP of rs4654899 had effect on gene expression of two genes, HP1BP3 lying 3 to

EIF4G3 (P = 7.8 106) and CAPN14 at 2p (P = 6.3 106), which are expressed in moderate-to-high levels throughout the adult human SFG. These results contribute to understand genetic architecture of a brain structure possibly linked to the pathophysiology

Translational Psychiatry (2014) 4, e472; doi:http://dx.doi.org/10.1038/tp.2014.110

Web End =10.1038/tp.2014.110 ; published online 21 October 2014

INTRODUCTION

Schizophrenia is a common and complex psychiatric disorder with a lifetime risk of approximately 1%. This disorder has a strong genetic component; the estimated heritability is 81%.1 Multiple genetic variants that have a small effect have been implicated in the pathogenesis of schizophrenia.2 A genome-wide association study (GWAS) of single-nucleotide polymorphisms (SNPs) that accesses tens of thousands of DNA samples from patients and controls can be a powerful tool for identifying common risk factors for complex diseases, such as schizophrenia. GWASs on schizophrenia have identied several genome-wide signicant associated variants.3,4 Subsequently, GWASs on neurobiological quantitative traits as intermediate phenotypes that possibly reect the underlying genetic vulnerability better than diagnostic categorization, such as schizophrenia,5,6 have been performed to minimize the clinical and genetic heterogeneity in studies of schizophrenia.7

The superior frontal gyrus (SFG) of the brain is frequently found to have reduced gray matter in individuals with rst-episode schizophrenia and neuroleptic naive schizophrenia, as well as

chronic patients with schizophrenia.8,9 The SFG is involved in self-awareness and emotion.10,11 Self-awareness is the cognitive ability to differentiate between self and non-self cues and is necessary to understand the behavior of other humans. Disturbance in self-awareness linked to social cognition is a core feature of schizophrenia.12 Emotional disturbances, including meaningless laughter, are often observed in patients with schizophrenia. Meaningless laughter was also observed in unaffected siblings of schizophrenia, thus indicating its heritability.13 In addition, laughter can be elicited by electrical stimulation of the SFG. Gray matter volumes of bilateral SFG have a strong genetic component, with an estimated heritability of 7680%.14 As there is considerable inter-individual variation in the degree of reduced volume of the SFG, it appears that genetic inuences have a role in determining the degree of volume reduction of the SFG in schizophrenia. Although GWASs of bilateral hippocampal volume have recently been reported,15,16 no study has investigated other brain areas in patients with schizophrenia. To identify an SNP related to SFG volumes, we conducted a GWAS of gray matter volumes in the right or left SFG of patients with schizophrenia and healthy subjects.

1Molecular Research Center for Children's Mental Development, United Graduate School of Child Development, Osaka University, Suita, Osaka, Japan; 2Department of Psychiatry, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; 3Department of Psychiatry, Fujita Health University School of Medicine, Toyoake, Aichi, Japan; 4Division of Ultrahigh Field MRI, Institute for Biomedical Sciences, Iwate Medical University, Yahaba, Iwate, Japan; 5Department of Molecular Neuropsychiatry, Osaka University Graduate School of Medicine, Suita, Osaka, Japan; 6Biofunctional Imaging, Immunology Frontier Research Center, Osaka University, Suita, Osaka, Japan; 7Department of Neuropsychiatry, Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan; 8Department of Neuropsychiatry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan; 9Department of Neuropsychiatry, Graduate School of Medicine, University of Tokyo, Tokyo, Japan; 10Department of Neuropsychiatry, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 11Division of Neuropsychiatry, Department of Neuroscience, Yamaguchi University Graduate School of Medicine, Yamaguchi, Japan and 12Department of Psychiatry, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan. Correspondence: Professor R Hashimoto, Molecular Research Center for Children's Mental Development, United Graduate School of Child Development, Osaka University, D3, 2-2, Yamadaoka, Suita, Osaka 5650871, Japan. E-mail: mailto:[email protected]

Web End [email protected]

13These authors contributed equally to this work.

Received 3 June 2014; revised 4 August 2014; accepted 31 August 2014

ORIGINAL ARTICLE

Common variants at 1p36 are associated with superior frontal gyrus volume

of schizophrenia.

GWA study of superior frontal gyrus R Hashimoto et al

2

MATERIALS AND METHODS Subjects

We selected 281 patients with schizophrenia (52.0% males, 146 males and 135 females; mean age 36.0 12.4 years) and 413 healthy controls (49.6% males, 205 males and 208 females; mean age 36.4 12.8 years) for a GWAS of schizophrenia-related phenotypes, such as structural brain morphology, neurocognitive function and neurophysiological assessments.1719 All of

the subjects were biologically unrelated, there were no rst- or second-degree relatives, and all were of Japanese descent.20,21 The subjects were

excluded if they had neurological or medical conditions that could potentially affect the central nervous system, such as atypical headaches, head trauma with loss of consciousness, chronic lung disease, kidney disease, chronic hepatic disease, thyroid disease, active cancer, cerebrovascular disease, epilepsy, seizures, substance-related disorders or mental retardation. Patients with schizophrenia were recruited from the Osaka University Hospital. Each patient had been diagnosed by at least two trained psychiatrists according to the criteria from the DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition) based on the Structured Clinical Interview for DSM-IV. Current symptoms of schizophrenia were evaluated using the positive and negative syndrome scale. Controls were recruited through local advertisements at Osaka University. The healthy subjects were evaluated using the non-patient version of the Structured Clinical Interview for DSM-IV to exclude individuals who had current or past contact with psychiatric services or who had received psychiatric medications.

Superior frontal volumes obtained from the magnetic resonance imaging data were assessed in 158 patients with schizophrenia and 378 healthy subjects. Detailed demographic information is shown in Supplementary Table S1. Mean age and handedness did not differ signicantly between the cases and controls (P40.50); however, the gender ratio, years of education and estimated premorbid intelligence quotient differed signicantly between the cases and controls (Po0.05).

The ratio of male was higher in patients with schizophrenia compared with the controls. The years of education and estimated premorbid intelligence quotient were signicantly lower in patients with schizophrenia compared with the controls. When the genotype groups in the top ve SNPs with genome-wide signicance of the right SFG volume were compared within the patient and control groups, we found no differences across the demographic variables, except for the gender ratio in the controls (rs6700718, rs1354792, rs10218584, and rs6702110; Po0.05). Written informed consent was obtained from all the subjects after the procedures had been fully explained. This study was performed in accordance with the World Medical Associations Declaration of Helsinki and was approved by the Research Ethical Committee of Osaka University.

Magnetic resonance imaging procedure and extraction of SFG volumesAll magnetic resonance imaging data were obtained using a 1.5-T GE Signa

EXCITE system (Tokyo, Japan). A three-dimensional volumetric acquisition of a T1-weighted gradient echo sequence produced a gapless series of 124 sagittal sections using a spoiled gradient-recalled acquisition in the steady state (SPGR) sequence (TE/TR, 4.2/12.6 ms; ip angle, 15; acquisition matrix, 256 256; 1NEX, FOV, 24 24 cm; slice thickness, 1.4 mm). We screened all scans and found no gross abnormalities, such as infarcts, hemorrhages or brain tumors, in any of the subjects. Each image was visually examined to eliminate any images with motion or metal artifacts, and the anterior commissureposterior commissure line was adjusted.22

MR images were processed with the VBM8 toolbox (http://dbm.neuro.uni-jena.de/vbm/download/

Web End =http://dbm.neuro.uni http://dbm.neuro.uni-jena.de/vbm/download/

Web End =jena.de/vbm/download/ ) implemented for SPM8 (Wellcome Department of Imaging Neuroscience, University College London, UK, http://www.fil.ion.ucl.ac.uk/spm

Web End =http://www.l.ion. http://www.fil.ion.ucl.ac.uk/spm

Web End =ucl.ac.uk/spm ) running in MATLAB (The Mathworks, Natick, MA, USA) for tissue segmentation and anatomical normalization, as described elsewhere.2325 The voxel values of the normalized gray matter images were modulated according to the nonlinear component of the transformation, which resulted in approximating brain-size-adjusted gray matter volumes while preserving local volume changes.26 Gray matter volumes of the bilateral SFG were then calculated by using the maximum probabilistic atlas using 20 hand-labeled images (Supplementary Figure S1).27,28

SNP selection and SNP genotypingGenotyping was performed using the Affymetrix Genome-Wide Human

SNP Array 6.0 (Affymetrix, Santa Clara, CA, USA), according to the manufacturers protocol. The genotypes were called from the CEL les

using Birdseed v2 for the 6.0 chip implemented in the Genotyping Console software (Affymetrix). We then applied the following quality control (QC) criteria to exclude samples: (i) arrays with low QC (o0.4) according to

Birdseed v2 (N = 0), (ii) samples for which o95% of the genotypes were called (N = 0) and (iii) samples in the same family according to

(40.4,

N = 0). Next, we excluded SNPs that: (i) had low call rates (o0.95), (ii) were duplicated, (iii) were localized to sex chromosomes, (iv) deviated from

HardyWeinberg equilibrium in the controls (Po0.0001) or (v) had low minor allele frequencies o0.05. After all of these exclusions, 517 946 SNPs that underwent QC remained for experimental analysis.

To test for the existence of a genetic structure in the data, we performed a principal component analysis using EIGENSTRAT 3.0 software.29 Ten

eigenvectors were calculated. Genotype information from the JPT (Japanese in Tokyo, Japan), CHB (Han Chinese in Beijing, China), CEU (Utah residents with ancestors from northern and western Europe) and YRI (Yoruba in Ibadan, Nigeria) in HapMap phase III was compared with our data set to check for population stratication (Supplementary Figure S2).

Statistical analysesStatistical analyses of the demographic variables were performed using

PASW Statistics 18.0 software (SPSS Japan, Tokyo, Japan). Differences in the clinical characteristics between patients and controls were analyzed using 2 tests for the categorical variables and the MannWhitney U-test for the continuous variables. Multiple linear regression analysis was performed to compare the gray matter volumes in the right and left SFG regions among genotypes (the number of major alleles; 0, 1 or 2) using PLINK 1.07 software. Diagnosis, age and gender were included as covariates. QuantileQuantile is listed in Supplementary Figure S3.

RESULTSWe observed associations between ve variants (rs4654899, rs6702110, rs6700718, rs10218584 and rs1354792) on 1p36.12 and the right SFG volume at a widely used benchmark for genome-wide signicance (Po5.0 108, r2 among SNPs 40.8;

Figure 1). The strongest association was observed at rs4654899, an intronic SNP in the eukaryotic translation initiation factor 4 gamma, 3 (EIF4G3) gene on 1p36.12 (P = 7.5 109; Figure 2). No SNP with genome-wide signicance was found in the volume of the left SFG; however, the rs4654899 polymorphism was identied as the locus with the second strongest association with the volume of the left SFG (P = 1.5 106; Figure 1). The top 10 and top 200 markers on each SFG are shown in Tables 1 and 2 and Supplementary Tables S2 and S3. Post hoc analyses separately assessed in patients and controls also revealed reduced but signicant associations (Tables 1 and 2 and Supplementary Tables S2 and S3). Genotype effects of rs4654899 on gray matter volume of right superior frontal gyrus were found in patients with schizophrenia and controls (Figure 3). We attempted to bioinformatically ascertain the potential effects of the rs4654899 polymorphism on the expression levels of genes at the genome-wide region by using the mRNA by SNP Browser 1.0.1 database (http://www.sph.umich.edu/csg/liang/asthma/

Web End =http://www.sph.umich.edu/csg/liang/asthma/). Signicant effects of the rs3767248 proxy SNP for rs4654899 (r2 = 1.0) were identied

Figure 1. Manhattan plots derived from the multiple linear regression analysis of the bilateral superior frontal volumes. The blue line indicates a P-value of 1.0E 05. The red line indicates a P-value of5.0E 08.

Translational Psychiatry (2014), 1 5 2014 Macmillan Publishers Limited

GWA study of superior frontal gyrus R Hashimoto et al

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in the expressions of the heterochromatin protein 1, binding protein 3 (HP1BP3) gene (P = 7.8 106), which lies 3 to EIF4G3, as a cis-acting effect (o200 kb), and the calpain 14 (CAPN14) gene (P = 6.3 106), as a trans-acting effect (4200 kb; Supplementary Table S4). Both HP1BP3 and CAPN14 are expressed in moderate-to-high levels throughout the adult human SFG (Supplementary Figures S4 and Supplementary Figure S5), as visualized in the Allen

Institute Human Brain Atlas Explorer 2 software (http://human.brain-map.org/static/brainexplorer

Web End =http://human. http://human.brain-map.org/static/brainexplorer

Web End =brain-map.org/static/brainexplorer ).

DISCUSSIONTo date, it remained unclear whether there were genetic variants strongly related to SFG volume in patients with schizophrenia and healthy subjects. This study is the rst GWAS to identify the SNPs associated with the SFG, which have an important role in schizophrenia-related social functions and is reduced in patients with schizophrenia. We revealed that there were associations at the genome-wide signicant level between SFG and genetic variants of the EIF4G3 gene on 1p36.12. Individuals with minor A-allele of the most signicant variant rs4654899 had smaller right SFG volumes compared with those with major C-allele in both patients and controls. Bioinformatical data indicate that the rs3767248 proxy SNP for rs4654899 has important roles in the expression of the HP1BP3 and CAPN14 genes, which are expressed in human adult SFG. The HP1BP3 and CAPN14 gene expressions of the minor G-allele of the rs3767248 polymorphism were signicantly lower than those of the major A-allele. However, whether the expression levels of these genes in the brains or serums of patients with schizophrenia are lower or higher than those in healthy subjects is unknown. Further study is needed to investigate the difference of the expressions between patients and controls.

Figure 2. The strongest association with the right superior frontal gyrus was found for rs4654899. P-values ( log10) are shown in regions peripheral to rs4654899 ( 750 kb).

Table 1. TOP 10 SNPs for the right superior frontal gyrus

Rank SNP Chr Bp m M MAF Combined subjects Schizophrenia Controls Closest gene

N P N P N P

1 rs4654899 1 21410231 A C 0.33 509 969.5 7.52E 09 153 1164 3.71E 04 356 935.9 1.86E 06 EIF4G3 2 rs6702110 1 21515906 G A 0.32 526 965.3 1.07E 08 155 1255 1.79E 04 371 902.3 4.34E 06 EIF4G3 3 rs6700718 1 21299363 A C 0.33 537 895.1 2.89E 08 159 1110 4.68E 04 378 847.7 6.81E 06 EIF4G3 4 rs10218584 1 21474480 G C 0.33 537 891.8 3.21E 08 159 1110 4.68E 04 378 842.7 7.68E 06 EIF4G3 5 rs1354792 1 21391875 C T 0.33 535 892.7 3.46E 08 157 1114 5.35E 04 378 847.7 6.81E 06 EIF4G3 6 rs6703227 1 21374810 C T 0.33 537 879.3 5.28E 08 159 1056 9.55E 04 378 847.7 6.81E 06 EIF4G3 7 rs1609558 1 21525228 C T 0.30 533 899.8 5.36E 08 158 1031 1.18E 03 375 870.9 7.67E 06 EIF4G3 8 rs12402486 1 21447935 A G 0.34 527 880.9 5.58E 08 159 1110 4.68E 04 368 830 1.20E 05 EIF4G3 9 rs2874367 1 21324491 A C 0.33 531 874.5 7.19E 08 157 1092 6.24E 04 374 829 1.25E 05 EIF4G3 10 rs6945071 7 26122423 G A 0.16 537 1035 8.46E 07 159 1120 3.25E 03 378 983.2 1.02E 04 NFE2L3

Abbreviations: Chr, chromosome; Bp, nucleotide location; m, minor allele; M, major allele; MAF, minor allele frequency; SNP, single-nucleotide polymorphism. Genome-wide signicant P-values are shown as bold font and are underlined.

Table 2. TOP 10 SNPs for the left superior frontal gyrus

Rank SNP Chr Bp m M MAF Combined subjects Schizophrenia Controls Closest gene

N P N P N P

1 rs4574391 4 27212223 C T 0.25 533 880 7.63E 07 158 693 4.27E 02 375 965 3.51E 06 STIM22 rs4654899 1 21410231 A C 0.33 509 787 1.51E 06 153 691 3.81E 02 356 863 4.22E 06 EIF4G33 rs2046701 4 27211040 C A 0.25 532 826 2.22E 06 158 697 3.87E 02 374 887 1.28E 05 STIM24 rs1609558 1 21525228 C T 0.3 533 763 2.33E 06 158 524 1.11E 01 375 885 1.67E 06 EIF4G35 rs6702110 1 21515906 G A 0.32 526 769 3.37E 06 155 714 3.90E 02 371 834 9.21E 06 EIF4G36 rs10218584 1 21474480 G C 0.33 537 704 8.32E 06 159 566 8.48E 02 378 791 1.05E 05 EIF4G37 rs1354792 1 21391875 C T 0.33 535 704 8.69E 06 157 625 5.92E 02 378 776 1.59E 05 EIF4G38 rs2623384 3 99064220 G A 0.39 525 724 9.15E 06 157 894 6.79E 03 368 642 5.92E 04 COL8A1 9 rs2292343 17 45455670 C G 0.34 524 721 9.74E 06 155 836 1.22E 02 369 666 3.45E 04 EFCAB13 10 rs3883317 17 45484111 A G 0.34 534 703 1.03E 05 158 743 2.70E 02 376 675 1.72E 04 EFCAB13

Abbreviations: Chr, chromosome; Bp, nucleotide location; m, minor allele; M, major allele; MAF, minor allele frequency; SNP, single-nucleotide polymorphism.

2014 Macmillan Publishers Limited Translational Psychiatry (2014), 1 5

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Figure 3. Impact of the rs4654899 genotype of the EIF4G3 gene on the right superior frontal gyrus. Each column shows relative gray matter volumes of the right superior frontal gyrus. Error bars represent the standard error.

To our knowledge, no study has reported associations between these genes and schizophrenia, although the chromosomal region (1p36.12) related to the risk of schizophrenia has been reported.30

The exact functions of these two genes are unknown; however, HP1BP3 is predicted to bind to DNA and have a role in nucleosome assembly. CAPN14, which belongs to the calpain large subunit family, is a cytosolic calcium-activated cysteine protease involved in a variety of cellular processes, including apoptosis, cell division, modulation of integrincytoskeletal interactions and synaptic plasticity.

In this study, we examined the effects of genotypes on SFG volumes in a combined sample of patients and controls, and found similar effects of genotypes in patients and controls. Susceptibility genes for schizophrenia do not directly encode for their clinical syndrome/behaviors. The syndrome/behaviors observed in schizophrenia are produced by intermediate steps that occur between genes and syndrome/behaviors; and intermediate steps, such as changes of brain volumes, underlie the syndrome/behavior of schizophrenia. The intermediate pheno-types are located on the pathogenesis path, and are likely associated with a more basic and proximal etiological process rather than pathogenesis of disease itself.5,6 Therefore, each genetic variant is related to controls as well as patients, and accumulations of each genetic variant could contribute to pathogenesis of schizophrenia through intermediate steps.

To date, although abnormal brain lateralization in schizophrenia causing a failure of left hemisphere dominance has been reported,31 there is no evidence of SFG lateralization in schizophrenia. In addition, there is no report for developmental/ functional differences between the right and left SFG. We found genome-wide signicant variants related to right SFG volumes, whereas these variants were not related to left SFG volumes at genome-wide signicant level. The difference of signicance between right and left SFG was due to a difference of genotype effects in patients (for example, rs4654899, right: P = 3.71 104,

left: P = 3.81 102) but not in controls (right: P = 1.86 106, left: P = 4.22 106). As it has been reported that gray matter volume decits were more extensive in individuals with rst-episode schizophrenia and neuroleptic naive than that of their neuroleptic-treated counterparts in left SFG,9 confounding factors, such as duration of antipsychotic treatment or dose of antipsychotics, might affect our results.

In this study, we provide new insights into the genetic architecture of a brain structure closely linked to schizophrenia. It is still unclear whether and to what extent the effects of the

genetic variant on SFG volumes observed here might be associated with an increased risk for schizophrenia. We suggest that the variant may have a role in the impairments of self-awareness and emotion noted in patients with schizophrenia through volumetric vulnerability of the SFG.

There were several limitations to this study. We recruited a relatively large sample with an only Japanese ethnicity to avoid population stratication. However, the existence of a false-positive association cannot be excluded as an explanation for our results. Further investigations of other samples with much larger sample sizes and/or with different ethnicities are needed to conrm our ndings. It is unclear whether our results are directly/indirectly linked to the rs4654899 SNP, to other SNPs in high linkage disequilibrium with this SNP or to interactions between this SNP and other SNPs. To determine whether rs4654899 is the most strongly associated variant for SFG volume in the chromosomal region, an extensive search such as sequencing for other functional variants at this locus could provide further information underlying the genomic mechanism for this variant.

In conclusion, we found that genetic variants of the EIF4G3 gene could be associated with structural vulnerability of the SFG. Further replication studies are necessary to conrm our ndings. Identication of causal variants and the functional effects of these genes may help to reveal additional genetic variables involved in the neurodevelopment and pathogenesis of schizophrenia.

CONFLICT OF INTEREST

The authors declare no conict of interest.

ACKNOWLEDGMENTS

We thank all the individuals who participated in this study. This work was supported by research grants from the Japanese Ministry of Health, Labor and Welfare (H22-seishin-ippan-001); KAKENHI, 22390225-Grant-in-Aid for Scientic Research (B), 23659565-Grant-in-Aid for Challenging Exploratory Research and Grant-in-Aid for Scientic Research on Innovative Areas (Comprehensive Brain Science Network) from the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) and the Japan Foundation for Neuroscience and Mental Health.

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