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. 2018 Jan 8;13(1):e0190991. doi: 10.1371/journal.pone.0190991

Association studies of WD repeat domain 3 and chitobiosyldiphosphodolichol beta-mannosyltransferase genes with schizophrenia in a Japanese population

Momoko Kobayashi 1,#, Daisuke Jitoku 1,#, Yoshimi Iwayama 2, Naoki Yamamoto 1, Tomoko Toyota 2, Katsuaki Suzuki 3, Mitsuru Kikuchi 4, Tasuku Hashimoto 5, Nobuhisa Kanahara 5, Akeo Kurumaji 1, Takeo Yoshikawa 2, Toru Nishikawa 1,*
Editor: Weihua YUE6
PMCID: PMC5757935  PMID: 29309433

Abstract

Schizophrenia and schizophrenia-like symptoms induced by the dopamine agonists and N-methyl-D aspartate type glutamate receptor antagonists occur only after the adolescent period. Similarly, animal models of schizophrenia by these drugs are also induced after the critical period around postnatal week three. Based upon the development-dependent onsets of these psychotomimetic effects, by using a DNA microarray technique, we identified the WD repeat domain 3 (WDR3) and chitobiosyldiphosphodolichol beta-mannosyltransferase (ALG1) genes as novel candidates for schizophrenia-related molecules, whose mRNAs were up-regulated in the adult (postnatal week seven), but not in the infant (postnatal week one) rats by an indirect dopamine agonist, and phencyclidine, an antagonist of the NMDA receptor. WDR3 and other related proteins are the nuclear proteins presumably involved in various cellular activities, such as cell cycle progression, signal transduction, apoptosis, and gene regulation. ALG1 is presumed to be involved in the regulation of the protein N-glycosylation. To further elucidate the molecular pathophysiology of schizophrenia, we have evaluated the genetic association of WDR3 and ALG1 in schizophrenia. We examined 21 single nucleotide polymorphisms [SNPs; W1 (rs1812607)-W16 (rs6656360), A1 (rs8053916)-A10 (rs9673733)] from these genes using the Japanese case-control sample (1,808 schizophrenics and 2,170 matched controls). No significant genetic associations of these SNPs were identified. However, we detected a significant association of W4 (rs319471) in the female schizophrenics (allelic P = 0.003, genotypic P = 0.008). Based on a haplotype analysis, the observed haplotypes consisting of W4 (rs319471)–W5 (rs379058) also displayed a significant association in the female schizophrenics (P = 0.016). Even after correction for multiple testing, these associations remained significant. Our findings suggest that the WDR3 gene may likely be a sensitive factor in female patients with schizophrenia, and that modification of the WDR3 signaling pathway warrants further investigation as to the pathophysiology of schizophrenia.

Introduction

Schizophrenia typically develops after adolescence [1]. Methamphetamine, an indirect dopamine agonist, and phencyclidine (PCP) and ketamine, antagonists of the N-methyl-D aspartate (NMDA) type glutamate receptor, are known to cause schizophrenia-like symptoms only after the adolescent period [2–4]. Similarly, in experimental animals, the psychotomimetic effects of these drugs have also been observed after the critical period around postnatal week three [5–7]. These observations suggest that the neuron circuits and molecules in the brain related to schizophrenia might show an age-related response to these psychotomimetics. In support of this assumption, we have found that methamphetamine and PCP elicit developmental changes in the c-Fos protein expression pattern, which reflects activity modification of the cell activities in the nervous systems, in the rat neocortex across the critical period [8, 9]. Consequently, we have explored the gene transcripts that are developmentally regulated after methamphetamine and PCP administrations in the rat cerebral neocortex. Based on this series of experiments using a DNA microarray technique, we detected as candidates for this type of novel schizophrenia-related genes the WD repeat domain 3 (WDR3) and chitobiosyldiphosphodolichol beta-mannosyltransferase (ALG1), whose mRNAs were up-regulated greater in the adult (postnatal days 50) than in the infant (postnatal days 8) rats by these schizophrenomimetics. Furthermore, these genes are located in linkage regions with schizophrenia [10, 11]. WDR3, also known as DIP2 and UTP12, is broadly expressed including in the brain [12]. This protein is contained in the nuclear, nucleolus and the main component of the small 40S ribosome subunit [12–14]. It also plays an essential role in the processing of 18S rRNA [14]. However, the specific function in the brain is unexplained. On the one hand, ALG1 is presumed to be involved in the regulation of the protein N-glycosylation, having the activity to add the first mannose residue to the lipid-linked oligosaccharides [15]. Abnormal glycosylation of the glutamate transporter, which may also be regulated by N-glycosylation, was reported in the postmortem study of schizophrenia [16]. Therefore, WDR3 and ALG1 may also be associated with the susceptibility and/or pathogenesis of schizophrenia.

In the present study, we used single nucleotide polymorphisms (SNPs) in the Japanese case-control sample to implement a genetic association study of the WDR3 and ALG1 genes in schizophrenia.

Materials and methods

Subjects

We analyzed 1,808 schizophrenics (male N = 992; mean age 48.9 ± 13.7 years, female N = 816; mean age 50.9 ± 14.2 years) and 2,170 matched controls (male N = 889; mean age 39.2 ± 13.8 years, female N = 1,281; mean age 44.6 ± 14.1 years) from the Japanese population. All the case-control subjects were assembled from the Honshu area of Japan (the main island of the nation). The populations of Honshu are categorized as a single genetic cluster [17, 18]. For the same subset used in a previous study [18], the Pr (K = 1) value (specifically, number of population present in sample = 1 [19]) was greater than 0.99 [20, 21], and k (the genomic control factor [22]) was 1.074. These data showed a negligible population stratification effect in our Japanese samples [23]. All patients were diagnosed by well-trained psychiatrists based on the Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV Criteria). The control subjects were assembled from hospital staff and volunteers. Expert psychiatrists checked whether or not they have a present or past history of psychosis and a family history of mental disorder within the second degree of relationships by brief interviews. The present study was approved by the ethics committees of the Tokyo Medical and Dental University and RIKEN Brain Science Institute. All participants gave informed and written consent to participate in the study.

Gene and SNP selection and genotyping

Exploration of target genes for association analysis

Before the gene and SNP selection and genotyping, we prepared the male Wistar rats (ST strain, Clea Japan, Japan) to explore of the novel candidate genes of schizophrenia. In this study, only male rats were used in the pharmacological experiment in order to avoid changes in behavior and biochemical response to various drugs due to the onset of the female menstrual cycle. The animals were bred under a 12 hour light / dark cycle (lights on 08:00 hours) at 24.0 ± 0.5 degrees (Celsius) and had free access to food and water. The animal experiments were approved by the ethics committee of animal experiment of the Tokyo Medical and Dental University, and were strictly performed following the guidelines of the university.

To explore the novel target genes for the present association analysis, we performed a DNA microarray analysis using the GeneChip® Rat Gene 1.0 ST Array (Affymetrix, Santa Clara, CA, USA) to find in the neocortex the developmentally regulated transcripts responsive to the psychotomimetic doses (adult period) of PCP and methamphetamine across the critical period around postnatal week three. The array system interrogates 27,342 well-annotated genes with 722,254 distinct probes. A detailed explanation can be found at https://www.affymetrix.com/support/technical/datasheets/gene_1_0_st_datasheet.pdf. Data analyses have been achieved by the software, GeneSpring GX 11.0 (Agilent Technologies, Santa Clara, CA, USA).

For this screening stage, six experimental groups of rats were prepared; five saline-administered control rats at PD50; five PCP (7.5 mg/kg, s.c.)-injected rats at PD50; five methamphetamine (4.8 mg/kg, s.c.)-injected rats at PD50; five saline-administered control rats at PD8; five PCP (7.5 mg/kg, s.c.)-injected rats at PD8; and five methamphetamine (4.8 mg/kg, s.c.)-injected rats at PD8. Equal amounts of the total RNA individually isolated from the respective five animals per each treatment group were combined. The cDNA synthesis, cRNA labeling, hybridization and scanning were done according to the manufacturer’s instructions (Affymetrix).

Based upon the DNA microarray data, we finally chose WDR3 and ALG1 as the genes for the present human association study by screening the transcripts of their rat homologues that showed the development-dependent upregulation by PCP and methamphetamine injection with the log2 ratio for the PCP/control (saline) and methamphetamine/control of more than 0.263 (1.2 times the control value) at PD 50 and that less than 0.137 (1.1 times the control value) in the ratio at PD 8 [log2 ratio of the WDR3: PCP 0.595 at PD50 (151%), 0.058 at PD8 (104%), methamphetamine 0.571 at PD50 (149%), 0.009 at PD8 (101%); log2 ratio of the ALG1: PCP 0.273 at PD50 (121%), 0.128 at PD8 (109%), methamphetamine 0.265 at PD50 (120%), 0.047 at PD8 (103%)].

Selection of SNPs and genotyping

We first retrieved the region 10kb up- and down-stream of these genes that provided the correlation coefficient of r2>0.85 and minor allele frequency of MAF>0.10 from the public databases [dbSNP (build 149) of the National Center for Biotechnology (NCBI) (http://www.ncbi.nlm.nih.gov/projects/SNP/)]. We then used Carlson’s LD-Select algorithm to evaluate the selection of the SNPs [24]. Additionally, we added SNPs from the insulator regions (CTCF binding site) between the target and adjacent gene as an effective region using CTCFBSDB 2.0 (http://insulatordb.uthsc.edu/) [25].

SNP genotyping was performed by TaqMan SNP genotyping assays (Applied Biosystems, Foster City, CA, USA). We used an ABI PRISM 7900HT (Applied Biosystems) or C1000 Touch Thermal Cycler with a 384-Well Reaction Module (BIO-RAD, Hercules, CA, USA) for the Polymerase Chain Reaction (PCR), and we analyzed the fluorescent signals using the 7900HT Sequence Detection System and SDS v2.3 software (Applied Biosystems).

Statistical analyses

Fisher’s exact test using the PLINK v1.07 program was used to calculate the Hardy-Weinberg equilibrium (HWE) and the count of the alleles and genotypes in the case-control samples for association (http://zzz.bwh.harvard.edu/plink/) [26]. We calculated the P-value of the false discovery rate (FDR) using the Benjamini-Hochberg procedure as a multiple testing for deriving the observed significance to correct.

For analysis of the linkage disequilibrium (LD) test to estimate the degree of LD, we used two LD parameters, i.e., the standardized disequilibrium coefficient (D’) and r2, calculated by Haploview v4.2 (http://www.broad.mit.edu/mpg/haploview/) [27]. We computed the standardized disequilibrium coefficient based on D’ according to the method of Gabriel et al. (2002) [28]. We executed the haplotype correlated analysis for common haplotypes (frequency≧0.05), then we calculated the individual and global haplotypic P-values using UNPHASED 3.1.4 (http://www.mrc-bsu.cam.ac.uk/personal/frank/software/unphased/). The multiple testing was calculated by FDR.

Moreover, we undertook an association analysis between these genes and schizophrenia in a stratified manner according to gender and age-at-onset using Fisher’s exact test with the PLINK v1.07 program. In the age-at-onset analysis, we divided the group into two age-at-onset categories, a) <18 years old, or 18 years old and greater, and b) <16 years old, 16–25 years old, 26–35 years old, or 36 years old and greater. In schizophrenia, even if the disease has similar symptoms, the age-at-onset of the disease with different causes occasionally changes. Therefore, the latter analysis is important to eliminate the possibility of heterogeneity which is considered to be present in schizophrenia. Moreover, schizophrenia with an onset age below 18 is often classified as early-onset schizophrenia in biological and clinical studies [29].

Furthermore, we examined the interaction of these genes using the multifactor dimensionality reduction (MDR) analysis [30], available in the open-source software package (http://www.multifactordimensionalityreduction.org/). An MDR analysis was performed by the MDR 3.0.2 program, and the permutation analysis used MDRpt Version 1.0.2 beta 2 (1,000 runs) for the testing accuracy and cross-validation consistency [31]. We used the false discovery rate as a multiple testing for the chi-square P-value. Before the analysis, the specific SNPs were excluded to avoid any false evaluation, the SNP showed a low MAF (<0.05), and the SNPs displayed a high LD (r2>0.95).

The statistical power was calculated by the genetic power calculator (http://zzz.bwh.harvard.edu/gpc/cc2.html). The assumptive parameter is as follows: An additive model with the genotypic relative risk = 1.3, prevalence of disease = 0.01, risk allele frequency = 0.2, type I error rate = 0.05 and 1-type II error rate (determine N) = 0.8. These tests were used to the level such that the statistical significance was set at P<0.05.

Results

Association result

In this study, we selected 26 SNPs (16 SNPs from WDR3 and 10 SNPs from ALG1). A schematic representation of the structures of the human WDR3 and ALG1 genes and location of the SNPs are shown in Fig 1 and Table 1. The LD block structures are shown in Fig 2. Five of the WDR3 SNPs were excluded from the subsequent analysis; two SNPs due to unclear clustering by the TaqMan Assay [W3 (rs1469919) and W9 (rs6696092)], one SNP due to monomorphism [W16 (rs6656360)], and two SNPs due to significant deviations from the HWE in the controls [W11 (rs2295629) and W14 (rs3753262)]. Therefore, we examined 11 SNPs of the human WDR3 gene and 10 SNPs of the human ALG1 gene as the genetic association study of schizophrenia.

Fig 1. Genomic structure of human WDR3 and ALG1.

Fig 1

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Genomic structures and positions of the SNPs in human WDR3 (A) and ALG1 (B). Exons are denoted by boxes with untranslated regions in gray, and translated regions in white. SNPs denoted in light blue are located in the CTCF binding site, and in green are the tag SNPs (correlation coefficient: r2>0.85, minor allele frequency: MAF>0.10).

Table 1. SNP information for WDR3 and ALG1 genes.

WDR3
SNP ID rs number Major/minor Strand Location Function MAF
W1 rs1812607 C/T + 5' upstream region INS T = 0.1616/353
W2 rs965361 A/T + 5' upstream region INS T = 0.1625/355
W3 rs1469919 C/T - 5' upstream region INS T = 0.2798/611
W4 rs319471 C/T - 5' upstream region INS T = 0.1529/334
W5 rs379058 T/A + 5' upstream region INS A = 0.3608/788
W6 rs3754127 C/T + 5' upstream region tag T = 0.2807/613
W7 rs17037749 A/C + 5' upstream region INS C = 0.0412/90
W8 rs1321663 G/C + intron1 tag C = 0.0971/212
W9 rs6696092 A/G + intron3 tag G = 0.4318/943
W10 rs1321666 T/C + intron13 tag C = 0.4881/1066
W11 rs2295629 G/A + intron14 tag A = 0.1946/425
W12 rs10802003 G/C + 3' downstream region tag C = 0.0536/117
W13 rs10754369 C/T + 3' downstream region INS T = 0.0847/185
W14 rs3753262 A/T - 3' downstream region INS A = 0.3571/780
W15 rs3753261 C/T - 3' downstream region INS T = 0.0627/137
W16 rs6656360 G/A + 3' downstream region INS A = 0.0394/86
ALG1
SNP ID rs number Major/minor Strand Location Function MAF
A1 rs8053916 C/G + 5' upstream region tag G = 0.204/446
A2 rs9924614 C/T + 5' upstream region tag T = 0.254/555
A3 rs9932909 C/T + 5' upstream region tag T = 0.436/953
A4 rs3760030 C/T - 5' upstream region tag T = 0.349/762
A5 rs3760029 C/T - 5' upstream region tag T = 0.088/192
A6 rs3760027 A/G - 5' upstream region tag G = 0.272/594
A7 rs8045294 G/C + intron1 tag C = 0.467/1020
A8 rs8045473 C/G + intron1 tag G = 0.3567/779
A9 rs7195893 C/T + exon6 tag T = 0.0856/187
A10 rs9673733 C/G + 3' downstream region tag C = 0.1049/229
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INS: insulator, MAF: minor allele frequency.

Fig 2. LD block structure of WDR3 and ALG1 genes.

Fig 2

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(A) WDR3 gene consists of three, and (B) ALG1 gene consists of two haplotype blocks in schizophrenia. In the left panel, the number in the box represents D’ (×100), blank means D’ = 1. In the right panel, the number in the box represents r2 (×100).

The allelic frequency and genotypic distributions of all the experimentally genotyped SNPs are summarized in Table 2. Two ALG1 SNPs and one WDR3 SNP showed a tendency of association at the level of P <0.05 [A9 (rs7195893) and A10 (rs9673733) in the allelic tests, W8 (rs1321663) in the genotypic test]. The block-based haplotype analysis is shown in Table 3. For the haplotype analysis, the WDR3 block [W4 (rs319471)–W5 (rs379058)] and the ALG1 block [A1 (rs8053916)–A2 (rs9924614)] showed a related trend at the Global and Individual P-value, respectively. However, these allelic, genotypic, and haplotypic associations did not remain after correction for multiple testing.

Table 2. Genotyping and allele distribution of SNPs on WDR3 and ALG1 genes in schizophrenia and controls from the Japanese population.

WDR3
SNP ID Affection N HWE P Allele count MAF Allelic P (FDR P) OR (95% CI) Genotypic count Genotypic P (FDR P)
rs number
W1 CON 2,168 0.086 C T 0.216 0.446 (0.701) 1.043 (0.938–1.161) CC CT TT 0.076 (0.220)
3,400 936 1,319 762 87
rs1812607 SCZ 1,806 0.222 2,806 806 0.223 1,099 608 99
W2 CON 2,168 0.066 A T 0.215 0.430 (0.701) 1.045 (0.932–1.62) AA AT TT 0.080 (0.220)
3,402 934 1,320 762 86
rs965361 SCZ 1,808 0.277 2,810 806 0.223 1,100 610 98
W4 CON 2,170 0.914 C T 0.113 0.074 (0.413) 0.877 (0.759–1.012) CC CT TT 0.200 (0.367)
3,851 489 1,709 433 28
rs319471 SCZ 1,807 0.794 3,252 362 0.100 1,464 324 19
W5 CON 2,168 0.122 T A 0.495 0.543 (0.710) 0.972 (0.890–1.062) TT TA AA 0.681 (0.955)
2,189 2,147 534 1,121 513
rs379058 SCZ 1,808 0.541 1,851 1,765 0.488 467 917 424
W6 CON 2,169 0.428 C T 0.182 0.581 (0.710) 1.033 (0.922–1.158) CC CT TT 0.840 (0.955)
3,548 790 1,445 658 66
rs3754127 SCZ 1,807 0.354 2,938 676 0.187 1,188 562 57
W7 CON 2,169 0.530 A C 0.036 0.904 (0.904) 0.979 (0.772–1.240) AA AC CC 0.947 (0.955)
4,180 158 2,015 150 4
rs17037749 SCZ 1,808 0.286 3,487 129 0.036 1,683 121 4
W8 CON 2,169 0.088 G C 0.176 0.161 (0.590) 1.086 (0.969–1.217) GG GC CC 0.042 (0.220)
3,575 763 1,485 605 79
rs1321663 SCZ 1,807 0.248 2,934 680 0.188 1,183 568 56
W10 CON 2,167 0.697 T C 0.463 0.075 (0.413) 1.085 (0.993–1.185) TT TC CC 0.182 (0.367)
2,328 2,006 630 1,068 469
rs1321666 SCZ 1,804 0.888 1,865 1,743 0.483 480 905 419
W12 CON 2,169 0.208 G C 0.151 0.350 (0.701) 1.061 (0.939–1.198) GG GC CC 0.069 (0.220)
3,684 654 1,572 540 57
rs10802003 SCZ 1,808 0.112 3,043 573 0.158 1,271 501 36
W13 CON 2,170 0.884 C T 0.179 0.431 (0.701) 1.047 (0.934–1.174) CC CT TT 0.717 (0.955)
3,563 777 1,461 641 68
rs10754369 SCZ 1,807 0.756 2,942 672 0.186 1,195 552 60
W15 CON 2,169 0.827 C T 0.110 0.858 (0.904) 1.013 (0.881–1.166) CC CT TT 0.955 (0.955)
3,859 479 1,715 429 25
rs3753261 SCZ 1,808 0.635 3,212 404 0.112 1,424 364 20
ALG1
SNP ID Affection N HWE P Allele count MAF Allelic P (FDR P) OR (95% CI) Genotypic count Genotypic P (FDR P)
rs number
A1 CON 2,169 0.211 C G 0.311 0.149 (0.373) 0.931 (0.846–1.025) CC GC GG 0.319 (0.638)
2,987 1,351 1,041 905 223
rs8053916 SCZ 1,807 0.652 2,543 1,071 0.296 899 745 163
A2 CON 2,170 0.656 C T 0.260 0.314 (0.449) 0.948 (0.857–1.049) CC CT TT 0.587 (0.652)
3,211 1,129 1,192 827 151
rs9924614 SCZ 1,808 0.531 2,712 904 0.250 1,022 668 118
A3 CON 2,161 0.769 C T 0.179 0.554 (0.612) 0.965 (0.859–1.084) CC TC TT 0.837 (0.837)
3,550 772 1,460 630 71
rs9932909 SCZ 1,799 0.870 2,974 624 0.173 1,230 514 55
A4 CON 2,165 0.543 C T 0.229 0.612 (0.612) 1.029 (0.927–1.142) CC TC TT 0.463 (0.652)
3,337 993 1,291 755 119
rs3760030 SCZ 1,807 0.359 2,767 847 0.234 1,052 663 92
A5 CON 2,166 0.686 C T 0.158 0.079 (0.263) 0.894 (0.790–1.012) CC TC TT 0.204 (0.638)
3,647 685 1,532 583 51
rs3760029 SCZ 1,805 0.924 3,091 519 0.144 1,322 447 36
A6 CON 2,147 0.415 T C 0.138 0.399 (0.499) 1.057 (0.931–1.200) TT CT CC 0.546 (0.652)
3,701 593 1,590 521 36
rs3760027 SCZ 1,795 0.924 3,070 520 0.145 1,313 444 38
A7 CON 2,166 0.822 G C 0.393 0.298 (0.449) 0.952 (0.870–1.043) GG CG CC 0.275 (0.638)
2,630 1,702 801 1,028 337
rs8045294 SCZ 1,803 0.163 2,231 1,375 0.381 676 879 248
A8 CON 2,166 0.636 C G 0.493 0.232 (0.449) 1.056 (0.966–1.153) CC GC GG 0.476 (0.652)
2,197 2,135 551 1,095 520
rs8045473 SCZ 1,804 0.851 1,781 1,827 0.506 437 907 460
A9 CON 2,160 0.856 C T 0.137 0.018 (0.180) 0.851 (0.745–0.973) CC TC TT 0.055 (0.335)
3,728 592 1,607 514 39
rs7195893 SCZ 1,801 0.736 3,173 429 0.119 1,399 375 27
A10 CON 2,170 0.563 C G 0.182 0.037 (0.185) 0.882 (0.785–0.992) CC CG GG 0.067 (0.335)
3,552 788 1,449 654 67
rs9673733 SCZ 1,808 0.439 3,024 592 0.164 1,269 486 53
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N: number of subjects, HWE: Hardy-Weinberg equilibrium, MAF: minor allele frequency, FDR: the false discovery rate using the Benjamini-Hochberg procedure, OR: odds ratio, 95% CI: 95% confidence interval, CON: control, SCZ: schizophrenia.

Table 3. Block-based haplotype analysis of WDR3 and ALG1 genes.

WDR3
Marker Frequency OR (95% CI) P-values
SCZ CON Individual P Global P FDR P
W1 W2
C A 0.777 0.784 0.957 (0.860–1.065) 0.441
T T 0.223 0.215 1.045 (0.939–1.163) 0.415 0.506 0.506
W4 W5
C A 0.488 0.494 1.101 (0.947–1.279) 0.575
C T 0.412 0.393 0.199 (0.176–0.224) 0.086
T T 0.100 0.112 0.885 (0.766–1.022) 0.087 0.043 0.128
W6 W8 W10
C C C 0.188 0.176 1.087 (0.970–1.219) 0.153
C G C 0.108 0.105 1.031 (0.894–1.190) 0.681
C G T 0.517 0.537 0.924 (0.846–1.010) 0.079
T G C 0.186 0.182 1.028 (0.918–1.153) 0.631 0.284 0.426
ALG1
Marker Frequency OR (95% CI) P-values
SCZ CON Individual P Global P FDR P
A1 A2
C C 0.454 0.429 1.108 (1.014–1.211) 0.024
C T 0.250 0.260 0.948 (0.857–1.049) 0.301
G C 0.296 0.311 0.931 (0.846–1.025) 0.146 0.077 0.154
A4 A5 A6 A7 A8 A9
C C C G C C 0.120 0.122 0.969 (0.845–1.111) 0.661
C C C G G C 0.489 0.474 1.055 (0.962–1.156) 0.171
C T C C C T 0.113 0.127 0.867 (0.755–0.995) 0.052
T C C C C C 0.084 0.085 0.991 (0.844–1.163) 0.981
T C T C C C 0.141 0.134 1.056 (0.927–1.203) 0.384 0.504 0.504
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OR: odds ratio, 95% CI: 95% confidence interval, CON: control, SCZ: schizophrenia, FDR: the false discovery rate using the Benjamini-Hochberg procedure

For the gender-stratification analysis, the allelic and genotypic distributions of each SNP in the schizophrenic patients and controls are shown in Table 4. Among the males, all the SNPs did not show deviations from the HWE. On the other hand, among the females, WDR3 SNP W8 (rs1321663) was omitted from the analysis due to a significant deviation from the HWE in the female controls. In the female schizophrenia patients, WDR3 SNP W12 (rs10802003), ALG1 SNP A4 (rs3760030) and A7 (rs8045294) showed significant deviations from the HWE (P = 0.023, 0.026 and 0.024, respectively). We carefully interpreted the results of these 3 SNPs in the females.

Table 4. Stratification analysis of sex on WDR3 and ALG1 gene in schizophrenia and controls from Japanese population.

WDR3
SNP ID Affection N HWE P Allele count MAF Allelic P (FDR P) OR (95% CI) Genotypic count Genotypic P (FDR P)
rs number Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female
C T C T CC CT TT CC CT TT
W1 CON 887 1,281 0.211 0.272 1,374 400 2,026 536 0.225 0.209 0.529 0.099 0.951 1.135 525 324 38 794 438 49 0.242 0.082
rs1812607 SCZ 992 814 0.401 0.375 1,554 430 1,252 376 0.217 0.231 (0.845) (0.218) (0.8146–1.109) (0.978–1.318) 613 328 51 486 280 48 (0.897) (0.180)
A T A T AA AT TT AA AT TT
W2 CON 887 1,281 0.211 0.235 1,374 400 2,028 534 0.225 0.208 0.503 0.084 0.948 1.141 525 324 38 795 438 48 0.277 0.067
rs965361 SCZ 992 816 0.512 0.376 1,555 429 1,255 377 0.216 0.231 (0.845) (0.218) (0.8121–1.106) (0.983–1.325) 613 329 50 487 281 48 (0.897) (0.180)
C T C T CC CT TT CC CT TT
W4 CON 889 1,281 0.110 0.278 1,588 190 2,263 299 0.107 0.117 0.753 0.003 1.038 0.727 714 160 15 995 273 13 0.897 0.008
rs319471 SCZ 991 816 0.197 0.191 1,763 219 1,489 143 0.110 0.088 (0.845) (0.033) (0.8451–1.275) (0.589–0.897) 788 187 16 676 137 3 (0.897) (0.088)
T A T A TT TA AA TT TA AA
W5 CON 888 1,280 0.591 0.131 879 879 1,292 1,268 0.500 0.495 0.281 0.681 0.930 1.026 222 453 213 312 668 300 0.355 0.849
rs379058 SCZ 992 816 0.484 0.093 1,038 946 813 819 0.477 0.502 (0.845) (0.742) (0.8182–1.057) (0.907–1.162) 277 484 231 190 433 193 (0.897) (0.849)
C T C T CC CT TT CC CT TT
W6 CON 888 1,281 0.653 0.638 1,451 325 2,097 465 0.183 0.181 0.737 0.713 1.032 1.032 590 271 27 855 387 39 0.886 0.776
rs3754127 SCZ 991 816 0.917 0.249 1,610 372 1,328 304 0.188 0.186 (0.845) (0.742) (0.8748–1.216) (0.880–1.212) 653 304 34 535 258 23 (0.897) (0.849)
A C A C AA AC CC AA AC CC
W7 CON 889 1,280 1.000 0.419 1,716 62 2,464 96 0.035 0.038 0.717 0.742 0.923 1.065 828 60 1 1,187 90 3 0.854 0.818
rs17037749 SCZ 992 816 1.000 0.130 1,920 64 1,567 65 0.032 0.040 (0.845) (0.742) (0.6467–1.316) (0.772–1.468) 929 62 1 754 59 3 (0.897) (0.849)
G C G C GG GC CC GG GC CC
W8 CON 889 1,280 0.911 0.029 1,449 329 2,126 434 0.185 0.170 0.933 0.089 1.010 1.150 591 267 31 894 338 48 0.747 0.033
rs1321663 SCZ 992 815 0.402 0.495 1,614 370 1,320 310 0.186 0.190 (0.933) (0.218) (0.8564–1.190) (0.979–1.352) 652 310 30 531 258 26 (0.897) (0.121)
T C T C TT TC CC TT TC CC
W10 CON 888 1,279 0.590 0.311 947 829 1,381 1,177 0.467 0.460 0.452 0.087 1.051 1.117 248 451 189 382 617 280 0.622 0.127
rs1321666 SCZ 989 815 0.848 0.624 1,030 948 835 795 0.479 0.488 (0.845) (0.218) (0.9248–1.195) (0.986–1.265) 270 490 229 210 415 190 (0.897) (0.233)
G C G C GG GC CC GG GC CC
W12 CON 889 1,280 0.799 0.121 1,503 275 2,181 379 0.155 0.148 0.561 0.595 1.055 1.051 636 231 22 936 309 35 0.806 0.017
rs10802003 SCZ 992 816 0.907 0.023 1,663 321 1,380 252 0.162 0.154 (0.845) (0.742) (0.8852–1.257) (0.884–1.249) 696 271 25 575 230 11 (0.897) (0.094)
C T C T CC CT TT CC CT TT
W13 CON 889 1,281 0.114 0.292 1,456 322 2,107 455 0.181 0.178 0.768 0.459 1.028 1.064 589 278 22 872 363 46 0.300 0.257
rs10754369 SCZ 991 816 0.674 0.357 1,615 367 1,327 305 0.185 0.187 (0.845) (0.742) (0.8707–1.213) (0.907–1.250) 660 295 36 535 257 24 (0.897) (0.404)
C T C T CC CT TT CC CT TT
W15 CON 889 1,280 0.328 0.238 1,571 207 2,288 272 0.116 0.106 0.681 0.610 0.956 1.056 697 177 15 1,018 252 10 0.436 0.583
rs3753261 SCZ 992 816 0.524 1.000 1,762 222 1,450 182 0.112 0.112 (0.845) (0.742) (0.7819–1.169) (0.865–1.288) 780 202 10 644 162 10 (0.897) (0.802)
ALG1
SNP ID Affection N HWE P Allele count MAF Allelic P (FDR P) OR (95% CI) Genotypic count Genotypic P (FDR P)
rs number Male Female Male Female Male Female Male Female Male Female Male Female Male Female Male Female
C G C G CC GC GG CC GC GG
A1 CON 888 1,281 0.177 0.606 1,238 538 1,749 813 0.303 0.317 0.391 0.393 0.939 0.941 440 358 90 601 547 133 0.669 0.558
rs8053916 SCZ 992 815 0.316 0.741 1,409 575 1,134 496 0.290 0.304 (0.495) (0.650) (0.816–1.080) (0.823–1.076) 507 395 90 392 350 73 (0.768) (0.697)
C T C T CC CT TT CC CT TT
A2 CON 889 1,281 0.732 0.825 1,302 476 1,909 653 0.268 0.255 0.264 0.636 0.919 0.965 479 344 66 713 483 85 0.403 0.482
rs9924614 SCZ 992 816 0.556 0.110 1,485 499 1,227 405 0.252 0.248 (0.495) (0.752) (0.794–1.064) (0.836–1.114) 552 381 59 470 287 59 (0.768) (0.697)
C T C T CC TC TT CC TC TT
A3 CON 882 1,279 0.907 0.573 1,456 308 2,094 464 0.175 0.181 1 0.455 0.999 0.939 600 256 26 860 374 45 0.687 0.415
rs9932909 SCZ 989 810 0.270 0.388 1,633 345 1,341 279 0.174 0.172 (1) (0.650) (0.843–1.183) (0.797–1.106) 679 275 35 551 239 20 (0.768) (0.697)
C T C T CC TC TT CC TC TT
A4 CON 884 1,281 0.344 1.000 1,360 408 1,977 585 0.231 0.228 0.337 0.677 1.077 0.967 528 304 52 763 451 67 0.623 0.155
rs3760030 SCZ 991 816 0.492 0.026 1,498 484 1,269 363 0.244 0.222 (0.495) (0.752) (0.926–1.252) (0.833–1.122) 570 358 63 482 305 29 (0.768) (0.610)
C T C T CC TC TT CC TC TT
A5 CON 889 1,277 0.535 1.000 1,493 285 2,154 400 0.160 0.157 0.158 0.289 0.876 0.909 624 245 20 908 338 31 0.315 0.567
rs3760029 SCZ 991 814 1.000 0.888 1,698 284 1,393 235 0.143 0.144 (0.495) (0.650) (0.733–1.047) (0.763–1.082) 727 244 20 595 203 16 (0.768) (0.697)
T C T C TT CT CC TT CT CC
A6 CON 880 1,267 0.888 0.475 1,514 246 2,187 347 0.140 0.137 0.307 1.000 1.101 0.997 650 214 16 940 307 20 0.391 0.918
rs3760027 SCZ 982 813 0.266 0.296 1,666 298 1,404 222 0.152 0.137 (0.495) (1) (0.917–1.321) (0.831–1.195) 711 244 27 602 200 11 (0.768) (0.918)
G C G C GG CG CC GG CG CC
A7 CON 888 1,278 0.727 0.517 1,066 710 1,564 992 0.400 0.388 0.593 0.240 0.963 0.925 317 432 139 484 596 198 0.796 0.043
rs8045294 SCZ 989 814 0.894 0.024 1,205 773 1,026 602 0.391 0.370 (0.659) (0.650) (0.845–1.098) (0.814–1.052) 368 469 152 308 410 96 (0.796) (0.430)
C G C G CC GC GG CC GC GG
A8 CON 888 1,278 0.503 0.240 908 868 1,267 1,289 0.489 0.504 0.396 0.358 1.059 0.943 237 434 217 303 661 314 0.691 0.627
rs8045473 SCZ 991 813 0.446 0.233 985 997 830 796 0.503 0.490 (0.495) (0.650) (0.932–1.204) (0.832–1.068) 251 483 257 203 424 186 (0.768) (0.697)
C T C T CC TC TT CC TC TT
A9 CON 885 1,275 0.673 0.905 1,524 246 2,204 346 0.139 0.136 0.056 0.157 0.829 0.872 654 216 15 953 298 24 0.121 0.364
rs7195893 SCZ 987 814 0.648 0.870 1,741 233 1,432 196 0.118 0.120 (0.495) (0.650) (0.684–1.005) (0.723–1.052) 769 203 15 630 172 12 (0.768) (0.697)
C G C G CC CG GG CC CG GG
A10 CON 889 1,281 0.911 0.397 1,453 325 2,099 463 0.183 0.181 0.196 0.094 0.892 0.867 594 265 30 855 389 37 0.363 0.183
rs9673733 SCZ 992 816 0.423 0.795 1,654 330 1,370 262 0.166 0.161 (0.495) (0.650) (0.754–1.056) (0.734–1.024) 693 268 31 576 218 22 (0.768) (0.610)
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N: number of subjects, HWE: Hardy-Weinberg equilibrium, MAF: minor allele frequency, FDR: the false discovery rate using the Benjamini-Hochberg procedure, OR: odds ratio, 95% CI: 95% confidence interval, CON: control, SCZ: schizophrenia

As shown in Table 4, among the females, WDR3 SNP W4 (rs319471) exhibited a significant allelic association in the female schizophrenic patients compared to the female controls [the C allele is overrepresented in the patients; P = 0.003; odds ratio (OR), 95% confidence interval (95% CI) = 1.38, 1.12–1.70]. This association remained even after correction for multiple testing (P = 0.033). WDR3 SNP W4 (rs319471), W12 (rs10802003) and ALG1 SNP A7 (rs8045294) also displayed a tendency to genotypic association in the female subjects with schizophrenia compared to the female controls, however, it was not significant after multiple testing (Table 4).

As displayed in Table 5, in the haplotype analysis, the block range from W4 (rs319471) to W5 (rs379058) showed a significant association in the female subjects with schizophrenia compared to the female controls (global haplotypic P = 0.016), even after correcting for the multiple testing; T [W4 (rs319471)]–T [W5 (rs379058)] is overrepresented in the controls (P = 0.003; OR, 95% CI = 0.731, 0.592–0.901). We did not observe such an association in the male schizophrenics compared to the male controls.

Table 5. Sex stratified block-based haplotype analysis of WDR3 and ALG1 genes.

(A) WDR3
Male
Marker Frequency OR (95% CI) P-values
SCZ CON Individual P Global P FDR P
W1 W2
C A 0.783 0.774 1.053 (0.903–1.229) 0.507
T T 0.216 0.225 0.949 (0.813–1.108) 0.509 0.801 0.870
W4 W5
C A 0.477 0.493 0.933 (0.821–1.061) 0.297
C T 0.413 0.400 1.051 (0.923–1.198) 0.399
T T 0.111 0.105 1.055 (0.858–1.298) 0.648 0.253 0.759
W6 W8 W10
C C C 0.187 0.186 1.009 (0.855–1.189) 0.920
C G C 0.105 0.099 1.069 (0.865–1.322) 0.537
C G T 0.521 0.533 0.953 (0.838–1.084) 0.462
T G C 0.187 0.183 1.031 (0.874–1.216) 0.717 0.870 0.870
Female
Marker Frequency OR (95% CI) P-values
SCZ CON Individual P Global P FDR P
W1 W2
C A 0.769 0.791 0.878 (0.756–1.019) 0.096
T T 0.231 0.208 1.139 (0.981–1.323) 0.085 0.087 0.131
W4 W5
C A 0.502 0.495 1.028 (0.908–1.164) 0.660
C T 0.411 0.389 1.094 (0.964–1.242) 0.152
T T 0.088 0.116 0.731 (0.592–0.901) 0.003 0.016 0.048
W10 W12
C C 0.155 0.148 1.054 (0.886–1.253) 0.554
C G 0.333 0.312 1.101 (0.964–1.257) 0.157
T G 0.512 0.540 0.895 (0.790–1.014) 0.081 0.213 0.213
(B) ALG1
Male
Marker Frequency OR (95% CI) P-values
SCZ CON Individual P Global P FDR P
A1 A2
C C 0.459 0.430 1.125 (0.989–1.280) 0.074
C T 0.252 0.268 0.92 (0.795–1.065) 0.265
G C 0.290 0.303 0.939 (0.816–1.080) 0.379 0.198 0.395
A4 A5 A6 A7 A8
C C C G C 0.114 0.121 0.924 (0.756–1.129) 0.445
C C C G G 0.489 0.477 1.044 (0.917–1.189) 0.520
C T C C C 0.145 0.159 0.89 (0.743–1.065) 0.203
T C C C C 0.087 0.088 0.992 (0.790–1.247) 0.946
T C T C C 0.150 0.136 1.114 (0.926–1.340) 0.255 0.523 0.523
Female
Marker Frequency OR (95% CI) P-values
SCZ CON Individual P Global P FDR P
A1 A2
C C 0.448 0.428 1.085 (0.957–1.230) 0.202
C T 0.248 0.255 0.963 (0.835–1.112) 0.610
G C 0.304 0.317 0.941 (0.823–1.076) 0.375 0.437 0.720
A4 A5 A6 A7 A8 A9
C C C G C C 0.126 0.124 1.008 (0.834–1.219) 0.939
C C C G G C 0.496 0.475 1.074 (0.944–1.222) 0.288
C T C C C T 0.114 0.126 0.882 (0.726–1.072) 0.207
T C C C C C 0.083 0.084 0.978 (0.779–1.228) 0.853
T C T C C C 0.131 0.133 0.975 (0.809–1.174) 0.791 0.720 0.720
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OR: odds ratio, 95% CI: 95% confidence interval, CON: control, SCZ: schizophrenia, FDR: the false discovery rate using the Benjamini-Hochberg procedure

Based on the age-at-onset stratification analysis, three of the WDR3 SNPs [W4 (rs319471), W8 (rs1321663) and W12 (rs10802003)] and four of the ALG1 SNPs [A1 (rs8053916), A5 (rs3760029), A6 (rs3760027) and A9 (rs7195893)] displayed a tendency to correlation with the different onset age groups of schizophrenia, although it was not significant after multiple testing (S1 Table).

By classifying the onset age groups of the male and female (S2 Table), five of the WDR3 SNPs [W1 (rs1812607), W2 (rs965361), W4 (rs319471), W12 (rs10802003) and W13 (rs10754369)] exhibited a tendency to correlation in several of the onset age groups, although did not remain significant after multiple testing. In addition, these SNPs have a commonality that the onset-aged between the 26 and 35 year groups in the male and female schizophrenics. In the case sample, three of the WDR3 SNPs [W4 (rs319471), W7 (rs17037749) and W13 (rs10754369)] showed a slight deviation from the HWE in the specific onset age groups of the males and females (16–25 years old in the males: P = 0.010, over 36 years old in the females: P = 0.040, over 36 years old in the females: P = 0.022, respectively). Three of the ALG1 SNPs [A1 (rs8053916), A4 (rs3760030) and A7 (rs8045294)], although not significant after multiple testing, showed a tendency to correlation. Moreover, in the case sample, A4 (rs3760030) showed a slight deviation from the HWE in specific groups (26–35 years old in the females; P = 0.006). We further cautiously interpreted the results of the SNPs deviating from the HWE in the males and females.

Gene-gene interaction analysis

Based on the MDR analysis, five of the WDR3 SNPs were excluded for the same reasons as for the case-control Fisher’s exact test: W3 (rs1469919), W9 (rs6696092), W11 (rs2295629), W14 (rs3753262) and W16 (rs6656360). The LD block which consisted of W1 (rs1812607) -W2 (rs965361) showed a high LD (r2>0.95). Therefore, we searched the tag SNP for avoid any false evaluation. Using the HaploView program to examine the tag SNP, W1 (rs1812607) was detected. Therefore, SNP W2 (rs965361) was omitted. For the sex stratified analysis, the same six SNPs were excluded due to same reasons in the males. In the females, WDR3 SNP W8 (rs1321663) was additionally excluded for the same reasons as for the case-control Fisher’s exact test. Therefore, 10 ALG1 SNPs and 10 WDR3 SNPs were analyzed in all the samples of the case-controls and male case-control samples. For the females, 10 ALG1 SNPs and 9 WDR3 SNPs were analyzed.

The testing accuracy (TA) represents the average value of the sensitivity and specificity. A TA of 0.55 and greater means that the MDR model is typically statistically significant. The best P-value was the combination of ALG1 SNP A9 (rs7195893) and WDR3 SNP W10 (rs1321666) in the female schizophrenia (P = 0.047), but the TA of this model was less than 0.55 (TA = 0.543). The chi-square P-value supported this result (P = 0.208). Therefore, it was not enough to indicate the interaction of these genes (Table 6).

Table 6. The MDR analysis for the best determined model.

Total
model TA CVC Permutation P χ2 P FDR P
TA CVC
WD-08 (rs1321663) 0.499 4/10 0.868 0.947 0.001 0.978 0.978
AL-10 (rs9673733), WD-10 (rs1321666) 0.478 2/10 0.999–1.000 0.999–1.000 0.759 0.384 0.978
AL-01 (rs8053916), AL-08 (rs8045473), WD-08 (rs1321663) 0.506 4/10 0.665 0.947 0.062 0.803 0.978
AL-02 (rs9924614), AL-07 (rs8045294), AL-10 (rs9673733), WD-10 (rs1321666) 0.501 2/10 0.834 0.999–1.000 0.001 0.973 0.978
Male
model TA CVC Permutation P χ2 P FDR P
TA CVC
AL-09 (rs7195893) 0.498 7/10 0.886 0.621 0.003 0.960 0.960
WD-04 (rs319471), WD-12 (rs10802003) 0.518 9/10 0.453 0.349 0.262 0.609 0.960
AL-01 (rs8053916), AL-09 (rs7195893), WD-13 (rs10754369) 0.490 2/10 0.964–0.965 0.999–1.000 0.077 0.781 0.960
AL-01 (rs8053916), AL-03 (rs9932909), AL-08 (rs8045473), WD-08 (rs1321663) 0.488 3/10 0.972–0.973 0.990 0.106 0.745 0.960
Female
model TA CVC Permutation P χ2 P FDR P
TA CVC
WD-04 (rs319471) 0.511 8/10 0.623 0.493 0.123 0.726 0.726
AL-09 (rs7195893), WD-10 (rs1321666) 0.543 10/10 0.047 0.212 1.584 0.208 0.726
AL-10 (rs9673733), WD-05 (rs379058), WD-10 (rs1321666) 0.516 2/10 0.516 0.999–1.000 0.215 0.643 0.726
AL-02 (rs9924614), AL-07 (rs8045294), WD-05 (rs379058), WD-10 (rs1321666) 0.516 4/10 0.519–0.520 0.962 0.207 0.649 0.726
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TA: testing accuracy, CVC: cross-validation consistency, FDR: the false discovery rate using the Benjamini-Hochberg procedure.

Power estimation

The power analysis showed a 99.09% power in the genotypic test and a 99.64% power in the allelic test for the case-control statistics in our sample. Based on the stratified analysis according to sex, the powers of the female and male groups were 85.48% and 82.51% in the genotypic test and 91.3% and 89.02% in the allelic test, respectively. The other stratified groups consisting of the classified age at onset are shown in Table 7.

Table 7. Power estimation of case-control sample and classified groups.

Total
N Genotypic Allelic
SCZ CON Power (%) N (80%) Power (%) N (80%)
Under 17 264 2170 53.92 468 64.42 381
Over 18 1426 98.1 719 99.18 589
Under 15 107 26.15 434 33.78 353
16–25 918 93.63 610 96.72 498
26–35 461 75.49 511 83.79 417
Over 36 204 44.41 455 54.67 371
Male
N Genotypic Allelic
SCZ CON Power (%) N (80%) Power (%) N (80%)
Under 15 50 889 14.18 437 18.26 356
16–25 519 67.46 685 76.89 560
26–35 252 45.65 544 55.94 444
Over 36 94 22.22 460 28.87 375
Female
N Genotypic Allelic
SCZ CON Power (%) N (80%) Power (%) N (80%)
Under 15 57 1281 15.73 431 20.36 351
16–25 399 64.79 558 74.62 455
26–35 209 42.68 488 52.81 397
Over 36 110 25.88 451 33.46 367
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N: number of subjects, N (80%): Number that reaches 80% detection power, CON: control, SCZ: schizophrenia

Discussion

This is the first genetic study of the WDR3 and ALG1 genes in schizophrenia to the best of our knowledge. We detected related signals between the WDR3 genes and female schizophrenic patients. In the allelic tests, W4 (rs319471) indicated a significant association with schizophrenia among the female schizophrenia patients. In our block-based haplotype analysis, the block range from W4 (rs319471) to W5 (rs379058) exhibited a significant association in the female schizophrenics. In these analyses, no association was detected in the male or the group of all subjects. Indeed, gender differences related to schizophrenia have been widely known [32]. For example, the clinical observations showed that male patients were inclined to have earlier onset and a more severe course than female patients. In addition, male schizophrenics have more negative symptoms and cognitive deficits, while female schizophrenics show more affective symptoms [33]. For the molecular biological approaches, several genes that have sex-specific genetic associations with schizophrenia were reported such as Disrupted in Schizophrenia 1 (DISC1), reelin (RELN), D-amino acid oxidase (DAO) and synapse-associated protein 97/discs, large homolog 1 of Drosophila (DLG1) in previous studies [34–37]. Therefore, the female specific association revealed in the WDR3 gene might be involved in the molecular basis of the schizophrenic pathology.

WDR3 SNP W4 (rs319471), which is located in the CTCF binding site of the 5’ upstream of the WDR3 gene, showed a significant association with female schizophrenics. This study focused on the CTCF binding site to select the SNPs as the gene expression control by the insulator function. This function is well known to enhancer-blocking activity and as a barrier to chromosomal position effects [38]. Consequently, the polymorphism of this site might be linked to the insulator function/dysfunction of the WDR3 and flanking cluster genes. We searched the sequence that contains 50 base pairs up- and down-stream of W4 (rs319471) at CTCFBSDB 2.0; a database for CTCF binding sites and genome organization (http://insulatordb.uthsc.edu/) [25, 39]. As a result, only when the SNP consists of the C allele does the sequence (ATCACTGCC) closely conform to the CTCF consensus. It might influence the expression level. We searched the expression quantitative trait loci (eQTLs) using the Brain eQTL Almanac (http://www.braineac.org/) to investigate whether W4 (rs319471) affects the expression of WDR3 in the females. The change in the expression level was reported in a multitude of genes, however, there is no significant report on the expression level of WDR3 in the database. Although we have to carefully consider that the database does not categorize the data by sex. To estimate the difference in the expression level in the female schizophrenics, the data by sex are needed. If it formed a CTCF consensus, the possibility is considered that the minor allele frequency is lower than in the healthy control at the female W4 (rs319471, minor allele: T), thus it is possible that the CTCF-binding activity is higher in schizophrenia. Moreover, based on a block-based haplotype analysis, the block consisting of W4 (rs319471) showed a significant correlation in female schizophrenia. It may support the fact that W4 (rs319471) is located in the disease susceptibility region. Actually, the influence of the CTCF-binding activity in the brain was reported. The CTCF-deficient neuron showed defects in the dendritic arborization and spine density during brain development [40]. Additionally, a decline in the cohesin function in the brain leads to a defective synapse development and anxiety-related behavior [41]. This means that the CTCF-binding activity has relevance to functional neural development and neuronal diversity. Accordingly, W4 (rs319471) has the possibilities involved in the pathophysiology of schizophrenia via the chromatin conformational changes.

The ALG1 SNPs showed only a statistically-weak correlation, however, two SNPs [A4 (rs3760030) and A7 (rs8045294)] that showed a tendency of association with female schizophrenia were reported as the eQTLs [42, 43]. Furthermore, the chromosome 16p13 region, ALG1 located, was reported to have copy number variations associated with schizophrenia [44]. The ALG1 gene did not show a strong correlation with schizophrenia in this study, however, the SNPs that showed a trend associated with a specific onset-age groups were observed. This may suggest that the SNPs or there genomic region affects the onset age of schizophrenia.

As for the age-at-onset analysis, there was no statistically significant association. This may be explained by the low statistical power in our stratified age-at-onset groups (<80%). Therefore, a larger sample size group needs to be studied to use the age-at-onset analysis.

In conclusion, our present associations study demonstrated that the WDR3 gene is selectively related to female schizophrenia. These results indicated that the WDR3 gene may be a susceptibility factor in female subjects with schizophrenia, and that regulation of the WDR3 signaling pathway ensures further research from the aspect of the pathophysiology of schizophrenia.

Further study is required to elucidate the gender-dependent correlation between the WDR3 gene and schizophrenia using different ethnic populations and larger sample sizes.

Supporting information

S1 Table. Stratification analysis of onset-age groups on WDR3 and ALG1 genes in schizophrenia and controls from Japanese population.

N: number of subjects, HWE: Hardy-Weinberg equilibrium, MAF: minor allele frequency, FDR: the false discovery rate using the Benjamini-Hochberg procedure, OR: odds ratio, 95% CI: 95% confidence interval, CON: control, SCZ: schizophrenia.

(PDF)

Click here for additional data file. (224.9KB, pdf)
S2 Table. Stratification analysis of onset-age groups by sex on WDR3 and ALG1 genes in schizophrenia and controls from Japanese population.

N: number of subjects, HWE: Hardy-Weinberg equilibrium, MAF: minor allele frequency, FDR: the false discovery rate using the Benjamini-Hochberg procedure, OR: odds ratio, 95% CI: 95% confidence interval, CON: control, SCZ: schizophrenia.

(PDF)

Click here for additional data file. (265.2KB, pdf)

Acknowledgments

We are sincerely thankful to the patients and healthy volunteers who participated in this study.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

Toru Nishikawa received the grant entitled “Strategic Research Program for Brain Sciences” funded by the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and Daisuke Jitoku received the grant named “Grant-in-Aid for Scientific Research (C) (No. 24791202)” funded by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Stratification analysis of onset-age groups on WDR3 and ALG1 genes in schizophrenia and controls from Japanese population.

N: number of subjects, HWE: Hardy-Weinberg equilibrium, MAF: minor allele frequency, FDR: the false discovery rate using the Benjamini-Hochberg procedure, OR: odds ratio, 95% CI: 95% confidence interval, CON: control, SCZ: schizophrenia.

(PDF)

Click here for additional data file. (224.9KB, pdf)
S2 Table. Stratification analysis of onset-age groups by sex on WDR3 and ALG1 genes in schizophrenia and controls from Japanese population.

N: number of subjects, HWE: Hardy-Weinberg equilibrium, MAF: minor allele frequency, FDR: the false discovery rate using the Benjamini-Hochberg procedure, OR: odds ratio, 95% CI: 95% confidence interval, CON: control, SCZ: schizophrenia.

(PDF)

Click here for additional data file. (265.2KB, pdf)

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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