Mutation analysis of the NMDAR2B (GRIN2B) gene in schizophrenia

NMDA receptor dysfunction may be involved in the pathophysiology of schizophrenia. Based on this hypothesis, we screened 48 Japanese patients with schizophrenia for mutations in the coding region of the NMDAR2B subunit gene (GRIN2B). An association study between the identified DNA sequence variants and schizophrenia was performed in 268 Japanese patients with schizophrenia and 337 Japanese control subjects. Eight single nucleotide polymorphisms were detected, all of which were synonymous. The association sample showed statistically significant excesses of homozygosity for the polymorphisms in the 3' region of the last exon in the patients with schizophrenia (P = 0.004) and higher frequency of the G allele of the 366C/G polymorphism (corrected P = 0.04) in the patients than in the controls. Although we did not detect NMDAR2B protein variants, our findings support the possibility that the GRIN2B gene or a locus in linkage disequilibrium with it may confer susceptibility to schizophrenia. Replication studies in independent samples are warranted.     

Significant association between the genetic variations in the 5' end of the N-methyl-D-aspartate receptor subunit gene GRIN1 and schizophrenia.

BACKGROUND: N-methyl-D-aspartate (NMDA) receptors play important roles in many neurophysiological processes. Evidence from previous studies indicate that NMDA receptors contribute to the pathophysiology of schizophrenia. Two NMDA receptor subunit genes, GRIN1 and GRIN2A, are both good candidate genes for schizophrenia. METHOD: We genotyped five single nucleotide polymorphisms (SNPs) in GRIN1 and two in GRIN2A in 2455 Han Chinese subjects, including population- and family-based samples, and performed case-control and transmission disequilibrium test (TDT) analyses. A microsatellite in GRIN2A was genotyped in population-based samples and a Mann-Whitney U test was performed. RESULTS: A highly significant association was detected at the 5' end of GRIN1. Analyses of single variants and multiple-locus haplotypes indicate that the association is mainly generated by rs11146020 (case-control study: p = .0000013, odds ratio = .61, 95% confidence interval .50-.74; TDT: p = .0019, T/NT = 79/123). No association was found in the GRIN2A polymorphisms. CONCLUSIONS: Our results provide support for the hypothesis that NMDA receptors are an important factor in schizophrenia. Moreover, rs11146020 is located in 5' untranslated region where several functional elements have been found. Hence, the SNP is a potential candidate in altering risk for schizophrenia and worthy of further replication and functional study.     

Polymorphisms in the promoter region of the alpha1A-adrenoceptor gene are associated with schizophrenia/schizoaffective disorder in a Spanish isolate population.

BACKGROUND: Animal models have implicated the alpha(1)-adrenergic subtypes in cognitive functions relevant to schizophrenia, but no consensus exists with regard to the status of noradrenergic receptor populations in psychiatric patients. We focused on one alpha(1)-adrenergic subtype, the alpha(1A)-adrenergic receptor, and proposed that genetic variants within the regulatory region of this gene (ADRA1A) alter the expression of this receptor, influencing susceptibility toward schizophrenia. METHODS: This study examined this proposal by testing the hypothesis that single nucleotide polymorphisms (SNPs) in the promoter region of the alpha(1A)-adrenergic gene were associated with schizophrenia by performing case-control association analysis on SNPs found in a 5' upstream region, which included the putative promoter region and 5' untranslated region. Our sample consisted of 103 schizophrenia and 14 schizoaffective disorder patients and 176 control subjects. All recruits were from a Spanish population isolate of Basque origin that is characterized by low heterogeneity, which was selected with the intent that it might facilitate the identification of disease-related polymorphisms. RESULTS: A total of eight SNPs (-9625 G/A, -7255 A/G, -6274 C/T, -4884 A/G, -4155 C/G, -2760 A/C, -1873 G/A, and -563 C/T) were confirmed at a rare allele frequency of >5%. Association with schizophrenia and schizoaffective disorder was found for the -563 C/T SNP (p = .0005 for allele and p = .007 for genotype, Bonferroni corrected) and -9625 G/A SNP (p = .02 for allele and p = .03 for genotype, Bonferroni corrected). Significant differences in the 54 haplotypes formed by these eight SNPs were also found between patients and control subjects (p = .008, Bonferroni corrected). CONCLUSIONS: Because of the strength of these results and the location of these SNPs in the regulatory region of this gene, functional studies investigating the possible influence of these SNPs on receptor expression levels in schizophrenia are warranted.     

Novel polymorphism in the gene region encoding the carboxyl-terminal intracellular domain of the NMDA receptor 2B subunit: analysis of association with schizophrenia

OBJECTIVE: N-methyl-D-aspartate (NMDA) receptor antagonists are known to produce a syndrome resembling schizophrenia, probably due to their blockade of NMDA receptors. The NMDA receptor 2B (NR2B) subunit has been identified as one of the major proteins in the postsynaptic density at glutamatergic synapses, suggesting that the carboxyl-terminal domain of the NR2B subunit may play a significant role in intracellular signal transduction. METHOD: The authors screened for genetic variations in the region of the NR2B subunit gene encoding the carboxyl-terminal intracellular domain in patients with schizophrenia and studied the association between schizophrenia and a novel polymorphism of the NR2B subunit gene. RESULTS: One silent mutation (2664C/T) was identified. No significant differences in the frequencies of 2664C/T genotypes and alleles were found between patients with schizophrenia and healthy comparison subjects. CONCLUSIONS: The findings provided no evidence of an association between schizophrenia and the 2664C/T polymorphism of the NR2B subunit gene.     

Association study of polymorphisms in the GluR6 kainate receptor gene (GRIK2) with schizophrenia

The glutamatergic dysfunction hypothesis of schizophrenia suggests genes involved in glutamatergic transmission as candidates for schizophrenia-susceptibility genes. The GluR6 kainate receptor gene GRIK2 is located on chromosome 6q16.3-q21, a schizophrenia susceptibility region, as suggested by multiple linkage studies. We examined 15 SNPs evenly distributed in the entire GRIK2 region (>700 kb) in Japanese patients with schizophrenia (n=100) and controls (n=100). Neither genotype nor allele frequency showed a significant association with the disorder. We constructed 2-SNP haplotypes from the 15 SNPs. Although we observed three long linkage disequilibrium blocks (>150 kb) within the GRIK2 region, none of the pairwise haplotypes showed a significant association with the disorder. Therefore, we conclude that GRIK2 does not play a major role in the pathogenesis of schizophrenia in the Japanese population.     

N-methyl-D-aspartate receptor NR2B subunit gene GRIN2B in schizophrenia and bipolar disorder: Polymorphisms and mRNA levels

The NR2B protein is a critical structural and functional subunit of the NMDA glutamate receptor. The glutamate neurotransmitter system has been implicated in psychosis and schizophrenia, and so we looked for genetic association and measured gene expression in human DNA and brain samples, respectively, of the GRIN2B gene that codes for the NR2B protein. We tested three genetic polymorphisms: G-200T (5'UTR), A5806C and T5988C (both 3'UTR) in 180 matched schizophrenia case-control pairs, 86 schizophrenia nuclear family trios, and 318 bipolar disorder trios (of which 158 probands had psychotic symptoms). We measured brain GRIN2B mRNA levels in schizophrenia, bipolar disorder and unaffected controls (n = 35 each). We detected genetic association between the G-200T marker and schizophrenia (p = 0.002), between T5988C and bipolar disorder (p = 0.02), and between A5806C and bipolar disorder with psychotic symptoms (p = 0.0038). The T-C-C haplotype was transmitted more frequently with bipolar disorder, but less often with schizophrenia, while the G-C-T haplotype was transmitted more often in schizophrenia. Significant differences were found in overall haplotype frequencies between schizophrenia cases and controls (p = 0.005). GRIN2B expression levels in schizophrenia, bipolar disorder and controls were not significantly different. The genetic findings suggest a role for GRIN2B in schizophrenia and bipolar disorder.     

GRIN2B近似启动子区多态性与散发性阿尔茨海默病相关性分析

目的进一步探讨中国北方汉族人群N-甲基-D-天冬氨酸受体2B(N-methyl-D-aspartate receptor 2B,NR2B)基因GRIN2B近似启动子区(-2454bp至-1269 bp)变异与散发性阿尔茨海默病(sporadic Alzheimer's disease,SAD)的关系。方法利用聚合酶链反应-限制性片段长度多态性(polymerase chain reaction-restriction fragmentlength polymorphism,PCR-RFLP)的方法对中国北方汉族362例SAD患者和334例正常对照者进行GRIN2B近似启动子多态性及载脂蛋白E(apolipoprotein,APOE)分型。结果中国北方汉族人群中GRIN2B近似启动子区除之前发现的-1447T/C(ENS10557853)和-1497G/A(rs12368476)多态性位点外,还存在-1887A/G(rs10845866)和-2315A/G(rs10845867)两个多态性位点。-1887A/G和-2315A/G多态性在SAD和正常对照组中的基因型频率(P=0.841和P=0.298)无显著性差异,等位基因频率(P=0.605和P=0.409)也无显著性差异。无论是否携带APOEε4,-1887A/G和-2315A/G多态性的基因型频率及等位基因频率也均无显著性差异。结论近似启动子区(-2454bp至-1268bp)-1887A/G和-2315A/G变异与AD发病无明显相关性。     

N-methyl-D-aspartate receptor subunit 1 regulates neurogenesis in the hippocampal dentate gyrus of schizophrenia-like mice

N-methyl-D-aspartate receptor hypofunction is the basis of pathophysiology in schizophrenia. Blocking the N-methyl-D-aspartate receptor impairs learning and memory abilities and induces pathological changes in the brain. Previous studies have paid little attention to the role of the N-methyl-D-aspartate receptor subunit 1(NR1) in neurogenesis in the hippocampus of schizophrenia. A mouse model of schizophrenia was established by intraperitoneal injection of 0.6 mg/kg MK-801, once a day, for 14 days. In N-methyl-D-aspartate-treated mice, N-methyl-D-aspartate was administered by intracerebroventricular injection in schizophrenia mice on day 15. The number of NR1-,Ki67-or BrdU-immunoreactive cells in the dentate gyrus was measured by immunofluorescence staining. Our data showed the number of NR1-immunoreactive cells increased along with the decreasing numbers of BrdU-and Ki67-immunoreactive cells in the schizophrenia groups compared with the control group. N-methyl-D-aspartate could reverse the above changes. These results indicated that NR1 can regulate neurogenesis in the hippocampal dentate gyrus of schizophrenia mice, supporting NR1 as a promising therapeutic target in the treatment of schizophrenia. This study was approved by the Experimental Animal Ethics Committee of the Ningxia Medical University,China(approval No. 2014-014) on March 6, 2014.     

Elevated GRIA1 mRNA expression in Layer II/III and V pyramidal cells of the DLPFC in schizophrenia

The functional integrity of the dorsolateral prefrontal cortex (DLPFC) is altered in schizophrenia leading to profound deficits in working memory and cognition. Growing evidence indicates that dysregulation of glutamate signaling may be a significant contributor to the pathophysiology mediating these effects; however, the contribution of NMDA and AMPA receptors in the mediation of this deficit remains unclear. The equivocality of data regarding ionotropic glutamate receptor alterations of subunit expression in the DLPFC of schizophrenics is likely reflective of subtle alterations in the cellular and molecular composition of specific neuronal populations within the region. Given previous evidence of Layer II/III and V pyramidal cell alterations in schizophrenia and the significant influence of subunit composition on NMDA and AMPA receptor function, laser capture microdissection combined with quantitative PCR was used to examine the expression of AMPA (GRIA1-4) and NMDA (GRIN1, 2A and 2B) subunit mRNA levels in Layer II/III and Layer V pyramidal cells in the DLPFC. Comparisons were made between individuals diagnosed with schizophrenia, bipolar disorder, major depressive disorder and controls (n=15/group). All subunits were expressed at detectable levels in both cell populations for all diseases as well as for the control group. Interestingly, GRIA1 mRNA was significantly increased in both cell types in the schizophrenia group compare to controls, while similar trends were observed in major depressive disorder (Layers II/III and V) and bipolar disorder (Layer V). These data suggest that increased GRIA1 subunit expression may contribute to schizophrenia pathology.     

A recently-discovered NMDA receptor gene, GRIN3B, is associated with duration mismatch negativity

The study explored associations between mismatch negativity and N-methyl-d-aspartic acid receptor subunit genes, GRIN1, GRIN2B and GRIN3B in healthy subjects and schizophrenia. Nineteen single-nucleotide polymorphisms were genotyped in 138 schizophrenia patients and 103 healthy subjects. Rs2240158 of GRIN3B was significantly associated with mismatch negativity in healthy subjects.     

Association analysis of the genetic variants of the N-methyl D-aspartate receptor subunit 2b (NR2b) and treatment-refractory schizophrenia in the Chinese

Several pieces of evidence showed that N-methyl D-aspartate (NMDA)-receptor-mediated decreases in function may be a causative factor for schizophrenia. The NMDA receptors are composed of a common glutamate receptor, an ionotropic NMDA 1 (GRIN1) subunit and one of four GRIN2 subunits (GRIN2A-GRIN2D), combined in an undetermined ratio to make up the receptor complex. In this study, we tested the hypothesis of whether the GRIN2B 366C/G and 2664C/T genetic polymorphisms are related to Chinese treatment-refractory schizophrenic patients. 193 treatment-refractory schizophrenic patients and 176 normal subjects were recruited for this study. The results demonstrated that the genotype distribution was similar between schizophrenic patients and control subjects in 366C/G (p = 0.88) and 2664C/T (p = 0.336), but we found a higher mean clozapine dosage in 2664C/C genotype patients. These results show that GRIN2B genetic variations were not a major risk factor for treatment-refractory schizophrenic patients, but may influence the effect of clozapine during treatment.     

Association study between Disrupted-in-Schizophrenia-1 (DISC1) and Japanese patients with treatment-resistant schizophrenia (TRS)

Treating the 20-30% of patients with schizophrenia whose symptoms are resistant to antipsychotic treatment, a condition known as treatment-resistant schizophrenia (TRS), can be problematic. Recently, an association between Disrupted-in-Schizophrenia-1 (DISC1), a candidate susceptibility gene for schizophrenia, and TRS was reported. Associations between three missense SNPs, rs3738401 (Q264R), rs6675281 (L607F), and rs821616 (S704C) in DISC1, especially rs3738401, showed strong significance. Thus, the main aim of our current study was to examine if the reported possible functional polymorphisms in DISC1 were related to Japanese TRS. First, DISC1 was re-investigated in 485 Japanese patients with schizophrenia and 660 healthy controls with a case-control study using four candidate SNPs, rs751229, rs3738401, rs821597, and rs821616. DISC1 was not associated with schizophrenia in the Japanese population. Second, we investigated whether these SNPs contributed to TRS in 127 inpatients with schizophrenia (35 patients; TRS and 92 patients; non-TRS). The genotypic distributions of these four SNPs were not significantly different between TRS and non-TRS in either genotypic or recessive models of minor alleles. In addition, clinical variables, such as improvement in clinical symptoms, duration of hospitalization, and total antipsychotics dose amounts, were not different among the genotypes of these SNPs. Taken together, results showed that DISC1 had no apparent degree of association with Japanese patients with schizophrenia as a candidate susceptibility gene for disease per se or TRS.     

Identification of single nucleotide polymorphisms (SNPs) and other sequence changes and estimation of nucleotide diversity in coding and flanking regions of the NMDAR1 receptor gene in schizophrenic patients

Glutamatergic dysregulation has been hypothesized to play a role in schizophrenia. The N-methyl-D-aspartate (NMDA) type of glutamate receptor especially is of interest because, in addition to binding sites for glutamate and glycine, a necessary co-agonist, this receptor also contains noncompetitive binding sites for the psychotomimetics phencyclidine (PCP), MK-801, and ketamine. PCP-induced psychosis has been a useful disease model in that both the positive as well as the negative symptomatologies seen in schizophrenia are observed. Recently, a mouse deficient in expression of the NR1 subunit gene (NMDAR1) of the heteromeric receptor has been developed and shown to display aberrant behaviors, with reduced social and sexual interactions as well as increased stereotypic motor activity. In an extensive examination of the NMDAR1 gene in our laboratory in approximately 100 chronic schizophrenic patients, 28 unique sequence changes were identified, including eight single nucleotide polymorphisms (SNPs) in the 5' untranslated region (5'UTR), six SNPs in coding regions (cSNPs), eleven intronic SNPs, two intronic deletions of 7 and 30 bp, and an intronic microinsertion/deletion. With the exception of one previously reported cSNP, all of the identified changes were novel. The frequency of polymorphisms differed significantly by ethnicity and several appeared to be in linkage disequilibrium. None of the changes appeared likely to be of functional significance, thus suggesting that changes in the genomic NMDAR1 are unlikely to contribute to the etiology of schizophrenia. Estimates of nucleotide diversity are comparable to those observed in studies of other genes.     

The NR1 N-methyl-D-aspartate subunit and brain-derived neurotrophic factor in temporal lobe epilepsy hippocampus: a comparison of patients with and without coexisting psychiatric symptoms

PURPOSE: The glutamate N-methyl-D-aspartate (NMDA) receptor and the neurotrophin brain-derived neurotrophic factor have been implicated in the pathophysiology of schizophrenia and depression. Since these psychiatric disorders are common in temporal lobe epilepsy (TLE), a comparison of TLE patients with and without coexisting psychiatric symptoms may be useful to unravel pathophysiologic mechanisms for psychosis or depression. METHODS: We used immunoautoradiography to assess the NR1 NMDA receptor subunit and brain-derived neurotrophic factor in resected TLE hippocampus. RESULTS: No changes relative to comparison controls were found for TLE patients with schizophrenia-like psychosis or depression. Increased NR1 was found in the dentate molecular layer in the dysphoria group and unmedicated depressed patients. CONCLUSIONS: An increase in NR1 protein in the dentate molecular layer suggests an upregulation of NMDA receptors in granule cells in TLE patients with dysphoria and depression. This finding is compatible with the theory that increased NMDA receptor function is involved in the pathogenesis of depression and that antidepressants may act by opposing this mechanism.     

RGS4 is not a susceptibility gene for schizophrenia in Japanese: association study in a large case-control population

The regulator of the G-protein signaling 4 (RGS4) has been implicated in the susceptibility to schizophrenia. RGS4 interacts with ErbB3 that acts as receptors for neuregulin 1 and these proteins may play a role in the pathogenesis of schizophrenia via glutamatergic dysfunction. Recently, two meta-analysis studies provided different interpretations for the genetic association between RGS4 and schizophrenia. We attempted to confirm this association in a case-control study of 1918 Japanese patients with schizophrenia and 1909 Japanese control subjects. Four widely studied single nucleotide polymorphisms (SNPs) were genotyped, and none showed association with schizophrenia. SNP 1 (rs10917670), p=0.92; SNP 4 (rs951436), p=0.91; SNP 7 (rs951439), p=0.27; and SNP 18 (rs2661319), p=0.43. A haplotype block constructed by these SNPs spans the 5' flanking region to the 5' mid-region of the RGS4 gene. Previous meta-analysis showed that both two major haplotypes of this block were risk haplotypes. The two common haplotypes were observed in the Japanese population. However, neither haplotype was significantly associated with schizophrenia. We conclude that the common haplotypes and SNPs of the RGS4 gene identified thus far are unlikely to contribute to the genetic susceptibility to schizophrenia in the Japanese population.     

Association Between the G1001C Polymorphism in the GRIN1 Gene Promoter and Schizophrenia in the Iranian Population

Schizophrenia is a complex genetic disorder to which genetic variation in the glutamatergic signaling pathways is believed to play a substantial role in the etiology of the disease. Association studies have implicated the N-methyl-d-aspartate receptor subunit gene, GRIN1, as a candidate gene for schizophrenia. In this report, we used a case control study to establish the possible association between the G1001C polymorphism in the GRIN1 gene promoter region and schizophrenia in an Iranian cohort of 200 unrelated patients and 200 controls. The allelic and genotypic frequencies of the polymorphism were determined using polymerase chain reaction restriction fragment length polymorphism. Data analysis using logistic regression and the Mantel–Haenszel chi-square test revealed a strong association between the G1001C polymorphism and schizophrenia (CG genotype: odds ratio (OR) = 2.12, 95% confidence interval (CI) 1.34–3.48, P = 0.001 and CC genotype: OR = 29.10, 95% CI 3.40–565.78, P < 0.001). Furthermore, the C allele is significantly associated with an increasing risk of schizophrenia. An erratum to this article can be found at     

Gene expression of NMDA receptor subunits in the cerebellum of elderly patients with schizophrenia

To determine if NMDA receptor alterations are present in the cerebellum in schizophrenia, we measured NMDA receptor binding and gene expression of the NMDA receptor subunits in a post-mortem study of elderly patients with schizophrenia and non-affected subjects. Furthermore, we assessed influence of genetic variation in the candidate gene neuregulin-1 (NRG1) on the expression of the NMDA receptor in an exploratory study. Post-mortem samples from the cerebellar cortex of ten schizophrenic patients were compared with nine normal subjects. We investigated NMDA receptor binding by receptor autoradiography and gene expression of the NMDA receptor subunits NR1, NR2A, NR2B, NR2C and NR2D by in situ hybridization. For the genetic study, we genotyped the NRG1 polymorphism rs35753505 (SNP8NRG221533). Additionally, we treated rats with the antipsychotics haloperidol or clozapine and assessed cerebellar NMDA receptor binding and gene expression of subunits to examine the effects of antipsychotic treatment. Gene expression of the NR2D subunit was increased in the right cerebellum of schizophrenic patients compared to controls. Individuals carrying at least one C allele of rs35753505 (SNP8NRG221533) showed decreased expression of the NR2C subunit in the right cerebellum, compared to individuals homozygous for the T allele. Correlation with medication parameters and the animal model revealed no treatment effects. In conclusion, increased NR2D expression results in a hyperexcitable NMDA receptor suggesting an adaptive effect due to receptor hypofunction. The decreased NR2C expression in NRG1 risk variant may cause a deficit in NMDA receptor function. This supports the hypothesis of an abnormal glutamatergic neurotransmission in the right cerebellum in the pathophysiology of schizophrenia.     

Deficits in sensorimotor gating and tests of social behavior in a genetic model of reduced NMDA receptor function

Reduced NMDA receptor function is hypothesized to contribute to the pathophysiology of schizophrenia. In order to model chronic and developmental NMDA receptor hypofunction, a mouse line was developed that expresses low levels of the NMDA R1 subunit (NR1) of the NMDA receptor. The present study tested the hypothesis that these NR1 hypomorphic mice would exhibit deficits in sensorimotor and conspecific interactions, analogous to deficits observed in schizophrenic patients. F1 hybrid mice homozygous for the NR1 hypomorphic mutation (NR1 -/-) were generated by crossing heterozygous mice (NR1 +/-) from C57BL/6 and 129 Sv/Ev backgrounds. To assess sensorimotor gating, mice were tested in the paradigm of prepulse inhibition of acoustic startle. The NR1 hypomorphic mice exhibited increased acoustic startle responses and also showed deficits in prepulse inhibition. Startle responses were differentially altered by predator odor exposure in the male NR1 -/- mice, in comparison to control mice. In a test of social affiliation, the wild type mice spent significantly more time investigating a novel mouse in comparison to the NR1 -/- mice. In a resident-intruder test, marked deficits were found in sex-specific aggressive behavior between the wild type and mutant mice. These data support the contention that the NR1 hypomorphic mice exhibit alterations in sensorimotor gating and typical conspecific interactions, reminiscent of behavioral disturbances associated with schizophrenia. The NR1 hypomorphic mice could represent a model system to explore novel treatment and preventative strategies for certain symptoms of schizophrenia.