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ZNF804A has been implicated in susceptibility to schizophrenia by several genome-wide association studies (GWAS), follow-up association studies and meta-analyses. However, the biological functions of ZNF804A are not entirely understood. To identify the genes that are affected by ZNF804A, we manipulated the expression of the ZNF804A protein in HEK293 human embryonic kidney cell lines and performed a cDNA microarray analysis followed by qPCR. We found that ZNF804A-overexpression up-regulated four genes (ANKRD1, INHBE, PIK3AP1, and DDIT3) and down-regulated three genes (CLIC2, MGAM, and BIRC3). Furthermore, we confirmed that the expression of ANKRD1, PIK3AP1, INHBE and DDIT3 at the protein level was significantly increased by ZNF804A-overexpression. This is the first report to identify genes whose protein expressions are regulated by ZNF804A. ANKRD1, PIK3AP1, INHBE and DDIT3 are related to transforming growth factor-β (TGF-β) signaling, which plays a crucial role in cell growth and differentiation. On the other hand, recent studies have reported that TGF-β signaling is associated with schizophrenia. These results provide basis for a more progressive investigation of ZNF804A contributions to the susceptibility or pathophysiology of schizophrenia.

The chitinase 3-like 1 (CHI3L1) gene, a cellular survival factor against several environmental and psychosocial stresses, has been sown to be more highly expressed in the hippocampus and prefrontal cortex of patients with schizophrenia than unaffected individuals. We recently reported a significant association between schizophrenia and SNP rs4950928, which is located in the promoter region of the CHI3L1 gene, in a Japanese population. The G-allele at this SNP in the gene has been associated with higher transcriptional activity in a luciferase reporter assay and with higher mRNA levels in the peripheral blood cells of patients with schizophrenia. We investigated the impact of the CHI3L1 polymorphism rs4950928 on serum YKL-40 levels, the protein product of CHI3L1. We found that individuals with the G-allele, who were more prevalent among patients with schizophrenia, had significantly higher serum YKL-40 levels (p=0.043). Personality traits are considered to be an important aspect of schizophrenia primarily because they may influence symptoms and social functioning. Personality trait analyses using the temperament and character inventory (TCI) indicated that schizophrenic patients have a unique personality profile that appears to be present across cultures. We hypothesized that higher serum YKL-40 levels are associated with personality trait in patients with schizophrenia. Thus, we next examined the impact of the risk CHI3L1 polymorphism on personality traits using the TCI. We found that individuals with the G-allele had significantly higher self-transcendence scores (p=0.0054). These findings suggest possible associations between the SNP in the CHI3L1 gene, the risk for schizophrenia, and higher serum YKL-40 levels and personality traits in a Japanese population.

Genome-wide association and follow-up studies have reported an association between schizophrenia and rs12807809 of the NRGN gene on chromosome 11q24.2. We investigated the association of five linkage disequilibrium-tagging SNPs and haplotypes that cover the NRGN gene with schizophrenia in a Japanese sample of 2,019 schizophrenia patients and 2,574 controls to determine whether rs12807809 is the most strongly associated variant for schizophrenia in the vicinity of the NRGN gene. We found that the rs12807809–rs12278912 haplotype of the NRGN gene was associated with schizophrenia (global P = 0.0042). The frequencies of the TG and TA haplotypes of rs12807809–rs12278912 in patients were higher (OR = 1.14, P = 0.0019) and lower (OR = 0.85, P = 0.0053), respectively, than in the controls. We did not detect any evidence of association of schizophrenia with any SNPs; however, two nominal associations of rs12278912 (OR = 1.10, P = 0.057) and rs2075713 (OR = 1.10, P = 0.057) were observed. Furthermore, we detected an association between the rs12807809–rs12278912 haplotype and NRGN expression in immortalized lymphoblasts derived from 45 HapMap JPT subjects (z = 2.69, P = 0.007) and confirmed the association in immortalized lymphoblasts derived from 42 patients with schizophrenia and 44 healthy controls (z = 3.09, P = 0.002). The expression of the high-risk TG haplotype was significantly lower than the protective TA haplotype. The expression was lower in patients with schizophrenia than in controls; however, this difference was not statistically significant. This study provides further evidence of the association of the NRGN gene with schizophrenia, and our results suggest that there is a link between the TG haplotype of rs12807809–rs12278912, decreased expression of NRGN and risk of developing schizophrenia. © 2012 Wiley Periodicals, Inc.

Brain-derived neurotrophic factor (BDNF) plays a critical role in activity-dependent neuroplasticity underlying learning and memory in the hippocampus. Recent human studies have indicated that a common single nucleotide polymorphism of the BDNF gene, the Val66Met polymorphism, has impact on episodic memory, hippocampal morphology and memory-related hippocampal activity measured by functional magnetic resonance imaging (fMRI). However, two issues remain to be clarified: (1) whether the genotype effect of this polymorphism on memory-related brain activity is allele dose dependent and (2) whether the effect of this polymorphism in Asian population is the same as effects observed in Caucasian sample. To clarify these issues, we studied the relationship of the Val66Met polymorphism genotype and hippocampal activity during episodic memory task using fMRI in healthy 58 biologically unrelated Japanese. Although there was no genotype effect on episodic memory function obtained by behavioral assessments, fMRI measurements revealed a significantly negative correlation between the dose of Met-BDNF allele and encoding related brain activity in the bilateral hippocampi and right parahippocampal gyrus. There was no genotype effect on retrieval related brain activity. These data indicated a genetic mechanism for normal variation in human memory and suggest effects of BDNF signaling on hippocampal function in humans.

Human neuropsin (NP) (hNP) has been implicated in the progressive change of cognitive abilities during primate evolution. The hNP gene maps to chromosome 19q13, a region reportedly linked to schizophrenia and bipolar disorder. Therefore, hNP is a functional and positional candidate gene for association with schizophrenia, mood disorders, and cognitive ability. Polymorphism screening was performed for the entire hNP gene. The core promoter region was determined and whether or not transcriptional activity alters in an allele-dependent manner was examined by using the dual-luciferase system. Allelic and genotypic distributions of five single-nucleotide polymorphisms (SNPs) were compared between patients with schizophrenia (n=439), major depression (n=409), bipolar disorder (n=207), and controls (n=727). A possible association of the hNP genotype with memory index (assessed with Wechsler Memory Scale, revised, WMS-R) and intelligence quotient (IQ assessed with Wechsler Adult Intelligence Scale, revised; WAIS-R) was examined in healthy controls (n=166). A total of 28 SNPs, including nine novel SNPs, were identified. No significant effects on transcriptional activity were observed for SNPs in the promoter region. A significant allelic association was found between several SNPs and bipolar disorder (for SNP23 at the 3′ regulatory region; odds ratio 1.48, 95% confidential interval 1.16–1.88, P=0.0015). However, such an association was not detected for schizophrenia or depression. Significant differences were observed between SNP23 and attention/concentration sub-scale score of WMS-R (P=0.016) and verbal IQ (P

It is well known that genetic factors contribute to the susceptibility for schizophrenia. Recent advances in the molecular genetics of schizophrenia strongly suggest several susceptibility genes (e.g. dysbindin, neuregulin-1, DISC1, COMT, G72, RGS4 and Akt1). We discuss the evidence and biology of these genes. As glutamate transmission is especially implicated in these genes, neurobiological basis of schizophrenia might be elucidated by investigation of functional interactions between susceptibility genes for schizophrenia and the glutamatergic system.

Background Although the pathogenesis of bipolar disorder remains unclear, heritable factors have been shown to be involved. The breakpoint cluster region (BCR) gene is located on chromosome 22q11, one of the most significant susceptibility loci in bipolar disorder linkage studies. The BCR gene encodes a Rho GTPase activating protein, which is known to play important roles in neurite growth and axonal guidance. Methods We examined patients with bipolar disorder (n = 171), major depressive disorder (n = 329) and controls (n = 351) in Japanese ethnicity for genetic association using eleven single nucleotide polymorphisms (SNPs), including a missense one (A2387G; N796S), in the genomic region of BCR. Results Significant allelic associations with bipolar disorder were observed for three SNPs, and associations with bipolar II disorder were observed in ten SNPs including N796S SNP (bipolar disorder, p = .0054; bipolar II disorder p = .0014). There was a significant association with major depression in six SNPs. S796 allele carriers were in excess in bipolar II patients (p = .0046, odds ratio = 3.1, 95% CI 1.53–8.76). Furthermore, we found a stronger evidence for association with bipolar II disorder in a multi-marker haplotype analysis (p = .0002). Conclusions Our results suggest that genetic variations in the BCR gene could confer susceptibility to bipolar disorder and major depressive disorder.

The RS447 megasatellite DNA, which maps to human chromosome 4p16.1, is a highly polymorphic conserved tandem repetitive sequence containing a functional deubiquitinating enzyme gene, USP17. To characterize the hypervariability seen in RS447 fully, we have conducted a pedigree analysis of RS447 transmission by high-resolution pulsed-field gel electrophoresis. We have identified 44 distinct alleles in 74 unrelated chromosomes containing 20-103 copies of the 4.7-kb RS447 unit. Five of 60 parent-to-offspring transmissions clearly show changes in copy number, indicating a high frequency (approximately 8.3%) of meiotic instability. Evidence for somatic mosaicism has also been observed. Searches of the database have revealed the presence of minor RS447 sequences mapping to chromosome 8p23, raising the possibility of a rearrangement or transposition of RS447 within the human genome. These results suggest that the unstable nature of RS447 megasatellite DNA gives rise to its hypervariability and may contribute to the structural dynamics of this repetitive DNA in the genome.

A susceptibility gene for schizophrenia, dysbindin, is a component of BLOC-1, which interacts with the adaptor protein (AP)-3 complex. As a direct interaction between dysbindin and AP-3 complex was reported, we examined a possible association between 16 SNPs in the AP3 complex genes and schizophrenia using 432 cases and 656 controls. Nominal association between rs6688 in the AP3M1 gene and schizophrenia (chi(2)=6.33, P=0.012, odds ratio=0.80) was no longer positive after correction for multiple testing (corrected P=0.192). The present results suggest that AP3 complex genes might not play a major role in the pathogenesis of schizophrenia in this population.

A variety of evidence suggests brain-derived neurotrophic factor (BDNF) as a candidate gene for schizophrenia, and several genetic studies have shown a significant association between the disease and certain SNPs within BDNF (specifically, Val66Met and C270T). According to a recent study, the functional microsatellite marker BDNF-LCPR (BDNF-linked complex polymorphic region), which affects the expression level of BDNF, is associated with bipolar disorder. The goals of our current study were to 1) evaluate the quality of HapMap-based linkage disequilibrium (LD) tagging of BDNF-LCPR, 2) examine whether these tagging SNPs are associated with schizophrenia in a Japanese population, and 3) conduct a meta-analysis of the two most extensively studied polymorphisms: Val66Met and C270T. We genotyped eight tagging SNPs, including Val66Met and C270T. Our LD evaluation showed that BDNF-LCPR could be represented by these tagging SNPs in controls (with 73.5% allelic coverage). However, the functional A1 allele was not captured due to its low minor allele frequency (2.2%). In a case-control study (1117 schizophrenics and 1102 controls), no association was found in single-marker or multimarker analysis. Moreover, in a meta-analysis, the Val66Met polymorphism was not associated with schizophrenia, whereas C270T showed a trend for association in a fixed model (p=0.036), but not in a random model (p=0.053). From these findings, we conclude that if BDNF is indeed associated with schizophrenia, the A1 allele in BDNF-LCPR would be the most promising candidate. Further LD evaluation, as well as an association study in which BDNF-LCPR is genotyped directly, would be required for a more conclusive result.

Neuronal apoptosis inhibitory protein (NAIP) has been shown to inhibit apoptosis in vitro and in vivo with an expression which is regulated in a variety of cells and tissues and may be modulated by a variety of external stimuli. To understand the molecular basis of the transcriptional regulation of the NAIP gene, we have analyzed the 5′-flanking region and transcription of the human NAIP gene. The functional promoter and silencer elements were identified by luciferase reporter constructs in transient transfection experiments using four different human cells. Although the location of the functional elements were shared among the different cells used, the activities for the NAIP promoter varied. Further, cell type-specific protein binding activities were observed by an electrophoretic mobility shift assay (EMSA). EMSA analysis with specific antibodies and DNA sequence analysis identified the POU domain transcription factor Brn-2 as a candidate transcriptional regulator of the NAIP gene. The DNA sequence of the promoter region of the ΨNAIP gene, a copy gene for NAIP , was nearly identical to that of the NAIP gene, indicating a common regulatory mechanism for transcription of the NAIP and ΨNAIP genes. Indeed, the transcript of the ΨNAIP gene was identified. These results provided the first evidence for the functional promoter and candidate transcriptional factor for the NAIP gene and transcription of the ΨNAIP gene.

We have recently identified a tandem repetitive DNA sequence that we designated the RS447 megasatellite. In this study, we describe a functional novel deubiquitinating enzyme (USP17, 60 kDa) gene that is intronless and encoded by the RS447 repeating unit. Northern blot analysis in conjunction with 5' and 3' rapid amplification of cDNA ends confirmed the presence of poly(A)(+) containing RS447 RNA in normal cells. We also identified a functional promoter sequence as well as an open reading frame within every RS447 repeat. When USP17 was expressed in Escherichia coli, it exhibited deubiquitinating activity in vivo. An antibody against USP17 detected USP17 protein in human cells. Our results indicate that the RS447 repeating unit on this megasatellite repeat codes for and actively expresses a functional deubiquitinating enzyme. Although it is expressed ubiquitously in human tissues, USP17 exhibited a unique expression pattern in that its complementary strand is transcribed as an antisense transcript that may modulate the level of USP17 expression in the human brain.

We previously isolated a novel 4.7-kb RS447 sequence, which tandemly repeated approximately 50–70 copies and resided on human chromosome 4p15 (M. Kogi et al., 1997, Genomics 42: 278–283). Another tandem array (or arrays) of several RS447 copies was hereby identified on the distal part of chromosome 8p. To analyze copy-number polymorphisms of the RS447 repeats, genomic DNA samples of eight nonkindred Japanese were subjected to pulsed-field gel electrophoresis. The copy numbers of the RS447 tandem arrays on 4p15 varied drastically from allele to allele and ranged from approximately 34 to 94 copies. All eight Japanese subjects were apparently heterozygous for the RS447 copy number, and 12 copy-number-different alleles have been at least clearly distinguished. The RS447 tandem repeats were thus found to be hypervariable and highly polymorphic in a human population. The RS447 sequences, however, do not appear to be either “selfish” or “junk” DNA. The unit size and sequence of RS447 were found to be very similar between members in the human genome. The unit size of 4746 bp comprises a putative open reading frame of 1590 bp. The RS447 sequence was well conserved in all the tested mammalian species. The head-to-tail tandem repetitive structure in the RS447 homologs was also confirmed in those species. The RS447 sequence is, therefore, considered to consist of a new class of tandemly repeated satellite DNA elements in the mammalian genome, which may thus be called “megasatellite DNA.”

BACKGROUND: Hypofunction of the glutamate N-Methyl-d-aspartate (NMDA) receptor has been implicated in the pathophysiology of schizophrenia. p250GAP is a brain-enriched NMDA receptor-interacting RhoGAP. p250GAP is involved in spine morphology, and spine morphology has been shown to be altered in the post-mortem brains of patients with schizophrenia. Schizotypal personality disorder has a strong familial relationship with schizophrenia. Several susceptibility genes for schizophrenia have been related to schizotypal traits. METHODS: We first investigated the association of eight linkage disequilibrium-tagging single-nucleotide polymorphisms (SNPs) that cover the p250GAP gene with schizophrenia in a Japanese sample of 431 schizophrenia patients and 572 controls. We then investigated the impact of the risk genetic variant in the p250GAP gene on schizotypal personality traits in 180 healthy subjects using the Schizotypal Personality Questionnaire. RESULTS: We found a significant difference in genotype frequency between the patients and the controls in rs2298599 (χ(2) = 17.6, p = 0.00015). The minor A/A genotype frequency of rs2298599 was higher in the patients (18%) than in the controls (9%) (χ(2) = 15.5, p = 0.000083). Moreover, we found that subjects with the rs2298599 risk A/A genotype, compared with G allele carriers, had higher scores of schizotypal traits (F(1,178) = 4.08, p = 0.045), particularly the interpersonal factor (F(1,178) = 5.85, p = 0.017). DISCUSSION: These results suggest that a genetic variation in the p250GAP gene might increase susceptibility not only for schizophrenia but also for schizotypal personality traits. We concluded that the p250GAP gene might be a new candidate gene for susceptibility to schizophrenia.

BACKGROUND: The rs12807809 single-nucleotide polymorphism in NRGN is a genetic risk variant with genome-wide significance for schizophrenia. The frequency of the T allele of rs12807809 is higher in individuals with schizophrenia than in those without the disorder. Reduced immunoreactivity of NRGN, which is expressed exclusively in the brain, has been observed in Brodmann areas (BA) 9 and 32 of the prefrontal cortex in postmortem brains from patients with schizophrenia compared with those in controls. METHODS: Genotype effects of rs12807809 were investigated on gray matter (GM) and white matter (WM) volumes using magnetic resonance imaging (MRI) with a voxel-based morphometry (VBM) technique in a sample of 99 Japanese patients with schizophrenia and 263 healthy controls. RESULTS: Although significant genotype-diagnosis interaction either on GM or WM volume was not observed, there was a trend of genotype-diagnosis interaction on GM volume in the left anterior cingulate cortex (ACC). Thus, the effects of NRGN genotype on GM volume of patients with schizophrenia and healthy controls were separately investigated. In patients with schizophrenia, carriers of the risk T allele had a smaller GM volume in the left ACC (BA32) than did carriers of the non-risk C allele. Significant genotype effect on other regions of the GM or WM was not observed for either the patients or controls. CONCLUSIONS: Our findings suggest that the genome-wide associated genetic risk variant in the NRGN gene may be related to a small GM volume in the ACC in the left hemisphere in patients with schizophrenia.

Genetic variation in dysbindin (DTNBP1: dystrobrevin-binding protein 1) has recently been shown to be associated with schizophrenia. The dysbindin gene is located at chromosome 6p22.3, one of the most promising susceptibility loci in schizophrenia linkage studies. We attempted to replicate this association in a Japanese sample of 670 patients with schizophrenia and 588 controls. We found a nominally significant association with schizophrenia for four single nucleotide polymorphisms and stronger evidence for association in a multi-marker haplotype analysis (P = 0.00028). We then explored functions of dysbindin protein in primary cortical neuronal culture. Overexpression of dysbindin induced the expression of two pre-synaptic proteins, SNAP25 and synapsin I, and increased extracellular basal glutamate levels and release of glutamate evoked by high potassium. Conversely, knockdown of endogenous dysbindin protein by small interfering RNA (siRNA) resulted in the reduction of pre-synaptic protein expression and glutamate release, suggesting that dysbindin might influence exocytotic glutamate release via upregulation of the molecules in pre-synaptic machinery. The overexpression of dysbindin increased phosphorylation of Akt protein and protected cortical neurons against neuronal death due to serum deprivation and these effects were blocked by LY294002, a phosphatidylinositol 3-kinase (PI3-kinase) inhibitor. SiRNA-mediated silencing of dysbindin protein diminished Akt phosphorylation and facilitated neuronal death induced by serum deprivation, suggesting that dysbindin promotes neuronal viability through PI3-kinase-Akt signaling. Genetic variants associated with impairments of these functions of dysbindin could play an important role in the pathogenesis of schizophrenia.