Minori Koga

Brain Cannabinoid CB2 Receptor in Schizophrenia

BACKGROUND Neural endocannabinoid function appears to be involved in schizophrenia. Two endocannabinoid receptors, CB1 and CB2, are found in the brain and elsewhere in the body. We investigated roles of CB2 in schizophrenia. MATERIALS AND METHODS An association study was performed between tag single nucleotide polymorphisms (SNPs) in the CNR2 gene encoding the CB2 receptor and schizophrenia in two independent case-control populations. Allelic differences of associated SNPs were analyzed in human postmortem brain tissues and in cultured cells. Prepulse inhibition and locomotor activity in C57BL/6JJmsSlc mice with CB2 receptor antagonist AM630 administration was examined. RESULTS The analysis in the first population revealed nominally significant associations between schizophrenia and two SNPs, and the associations were replicated in the second population. The R63 allele of rs2501432 (R63Q) (p = .001), the C allele of rs12744386 (p = .005) and the haplotype of the R63-C allele (p = 5 x 10(-6)) were significantly increased among 1920 patients with schizophrenia compared with 1920 control subjects in the combined population. A significantly lower response to CB2 ligands in cultured CHO cells transfected with the R63 allele compared with those with Q63, and significantly lower CB2 receptor mRNA and protein levels found in human brain with the CC and CT genotypes of rs12744386 compared with TT genotype were observed. AM630 exacerbated MK-801- or methamphetamine-induced disturbance of prepulse inhibition and hyperactivity in C57BL/6JJmsSlc mice. CONCLUSIONS These findings indicate an increased risk of schizophrenia for people with low CB2 receptor function.

Pathway-based association analysis of genome-wide screening data suggest that genes associated with the γ-aminobutyric acid receptor signaling pathway are involved in neuroleptic-induced, treatment-resistant tardive dyskinesia

Objective Neuroleptic-induced tardive dyskinesia (TD) is an involuntary movement disorder that develops in patients who have undergone long-term treatment with antipsychotic medications, and its etiology is unclear. In this study, a genome-wide association screening was done to identify the pathway(s) in which genetic variations influence susceptibility to neuroleptic-induced TD. Methods Screening with Sentrix Human-1 Genotyping BeadChip (Illumina, San Diego, California, USA) was done for 50 Japanese schizophrenia patients with treatment-resistant TD and 50 Japanese schizophrenia patients without TD. A total of 40 573 single nucleotide polymorphisms that were not in linkage disequilibrium with each other and were located in the exonic and intronic regions of 13 307 genes were analyzed. After gene-based corrections, P values for allelic associations were subjected to canonical pathway-based analyses with Ingenuity Pathway Analysis software (Ingenuity Systems, Inc., Redwood City, California, USA). Results Eight genes (ABAT, ALDH9A1, GABRA3, GABRA4, GABRB2, GABRAG3, GPHN, and SLC6A11) contained polymorphisms with gene-based corrected allelic P values of less than 0.05. They were aggregated significantly in 33 genes belonging to the &ggr;-aminobutyric acid (GABA) receptor signaling pathway (P=0.00007, corrected P=0.01). Associations were replicated in an independent sample of 36 patients with TD and 136 patients without TD for polymorphisms in SLC6A11 (GABA transporter 3) (P=0.0004 in the total sample), GABRB2 (&bgr;-2 subunit of GABA-A receptor) (P=0.00007 in the total sample), and GABRG3 (&ggr;-3 subunit of GABA-A receptor) (P=0.0006 in the total sample). Conclusion The results suggest that the GABA receptor signaling pathway may be involved in genetic susceptibility to treatment-resistant TD, at least in a subgroup of Japanese patients with schizophrenia. The present results suggest that benzodiazepines may be considered as possible treatment option for TD.

Role of STAT4 polymorphisms in systemic lupus erythematosus in a Japanese population: a case-control association study of the STAT1-STAT4 region

IntroductionRecent studies identified STAT4 (signal transducers and activators of transcription-4) as a susceptibility gene for systemic lupus erythematosus (SLE). STAT1 is encoded adjacently to STAT4 on 2q32.2-q32.3, upregulated in peripheral blood mononuclear cells from SLE patients, and functionally relevant to SLE. This study was conducted to test whether STAT4 is associated with SLE in a Japanese population also, to identify the risk haplotype, and to examine the potential genetic contribution of STAT1. To accomplish these aims, we carried out a comprehensive association analysis of 52 tag single nucleotide polymorphisms (SNPs) encompassing the STAT1-STAT4 region.MethodsIn the first screening, 52 tag SNPs were selected based on HapMap Phase II JPT (Japanese in Tokyo, Japan) data, and case-control association analysis was carried out on 105 Japanese female patients with SLE and 102 female controls. For associated SNPs, additional cases and controls were genotyped and association was analyzed using 308 SLE patients and 306 controls. Estimation of haplotype frequencies and an association study using the permutation test were performed with Haploview version 4.0 software. Population attributable risk percentage was estimated to compare the epidemiological significance of the risk genotype among populations.ResultsIn the first screening, rs7574865, rs11889341, and rs10168266 in STAT4 were most significantly associated (P < 0.01). Significant association was not observed for STAT1. Subsequent association studies of the three SNPs using 308 SLE patients and 306 controls confirmed a strong association of the rs7574865T allele (SLE patients: 46.3%, controls: 33.5%, P = 4.9 × 10-6, odds ratio 1.71) as well as TTT haplotype (rs10168266/rs11889341/rs7574865) (P = 1.5 × 10-6). The association was stronger in subgroups of SLE with nephritis and anti-double-stranded DNA antibodies. Population attributable risk percentage was estimated to be higher in the Japanese population (40.2%) than in Americans of European descent (19.5%).ConclusionsThe same STAT4 risk allele is associated with SLE in Caucasian and Japanese populations. Evidence for a role of STAT1 in genetic susceptibility to SLE was not detected. The contribution of STAT4 for the genetic background of SLE may be greater in the Japanese population than in Americans of European descent.

Identification of Novel Candidate Genes for Treatment Response to Risperidone and Susceptibility for Schizophrenia: Integrated Analysis Among Pharmacogenomics, Mouse Expression, and Genetic Case-Control Association Approaches

BACKGROUND Pharmacogenomic approaches based on genomewide sets of single nucleotide polymorphisms (SNPs) are now feasible and offer the potential to uncover variants that influence drug response. METHODS To detect potential predictor gene variants for risperidone response in schizophrenic subjects, we performed a convergent analysis based on 1) a genomewide (100K SNP) SNP pharmacogenetic study of risperidone response and 2) a global transcriptome study of genes with mRNA levels influenced by risperidone exposure in mouse prefrontal cortex. RESULTS Fourteen genes were highlighted as of potential relevance to risperidone activity in both studies: ATP2B2, HS3ST2, UNC5C, BAG3, PDE7B, PAICS, PTGFRN, NR3C2, ZBTB20, ST6GAL2, PIP5K1B, EPHA6, KCNH5, and AJAP1. The SNPs related to these genes that were associated in the pharmacogenetic study were further assessed for evidence for association with schizophrenia in up to three case-control series comprising 1564 cases and 3862 controls in total (Japanese [JPN] 1st and 2nd samples and UK sample). Of 14 SNPs tested, one (rs9389370) in PDE7B showed significant evidence for association with schizophrenia in a discovery sample (p(allele) = .026 in JPN_1st, two-tailed). This finding replicated in a joint analysis of two independent case-control samples (p(JPN_2nd+UK) = .008, one-tailed, uncorrected) and in all combined data sets (p(all) = .0014, two-tailed, uncorrected and p(all) = .018, two-tailed, Bonferroni correction). CONCLUSIONS We identified novel candidate genes for treatment response to risperidone and provide evidence that one of these additionally may confer susceptibility to schizophrenia. Specifically, PDE7B is an attractive candidate gene, although evidence from integrated methodology, including pharmacogenomics, pharmacotranscriptomic, and case-control association approaches.

Involvement of SMARCA2/BRM in the SWI/SNF chromatin-remodeling complex in schizophrenia

Chromatin remodeling may play a role in the neurobiology of schizophrenia and the process, therefore, may be considered as a therapeutic target. The SMARCA2 gene encodes BRM in the SWI/SNF chromatin-remodeling complex, and associations of single nucleotide polymorphisms (SNPs) to schizophrenia were found in two linkage disequilibrium blocks in the SMARCA2 gene after screening of 11 883 SNPs (rs2296212; overall allelic P = 5.8 x 10(-5)) and subsequent screening of 22 genes involved in chromatin remodeling (rs3793490; overall allelic P = 2.0 x 10(-6)) in a Japanese population. A risk allele of a missense polymorphism (rs2296212) induced a lower nuclear localization efficiency of BRM, and risk alleles of intronic polymorphisms (rs3763627 and rs3793490) were associated with low SMARCA2 expression levels in the postmortem prefrontal cortex. A significant correlation in the fold changes of gene expression from schizophrenic prefrontal cortex (from the Stanley Medical Research Institute online genomics database) was seen with suppression of SMARCA2 in transfected human cells by specific siRNA, and of orthologous genes in the prefrontal cortex of Smarca2 knockout mice. Smarca2 knockout mice showed impaired social interaction and prepulse inhibition. Psychotogenic drugs lowered Smarca2 expression while antipsychotic drugs increased it in the mouse brain. These findings support the existence of a role for BRM in the pathophysiology of schizophrenia.

Genome-wide association study identifies a potent locus associated with human opioid sensitivity

Opioids, such as morphine and fentanyl, are widely used as effective analgesics for the treatment of acute and chronic pain. In addition, the opioid system has a key role in the rewarding effects of morphine, ethanol, cocaine and various other drugs. Although opioid sensitivity is well known to vary widely among individual subjects, several candidate genetic polymorphisms reported so far are not sufficient for fully understanding the wide range of interindividual differences in human opioid sensitivity. By conducting a multistage genome-wide association study (GWAS) in healthy subjects, we found that genetic polymorphisms within a linkage disequilibrium block that spans 2q33.3–2q34 were strongly associated with the requirements for postoperative opioid analgesics after painful cosmetic surgery. The C allele of the best candidate single-nucleotide polymorphism (SNP), rs2952768, was associated with more analgesic requirements, and consistent results were obtained in patients who underwent abdominal surgery. In addition, carriers of the C allele in this SNP exhibited less vulnerability to severe drug dependence in patients with methamphetamine dependence, alcohol dependence, and eating disorders and a lower ‘Reward Dependence’ score on a personality questionnaire in healthy subjects. Furthermore, the C/C genotype of this SNP was significantly associated with the elevated expression of a neighboring gene, CREB1. These results show that SNPs in this locus are the most potent genetic factors associated with human opioid sensitivity known to date, affecting both the efficacy of opioid analgesics and liability to severe substance dependence. Our findings provide valuable information for the personalized treatment of pain and drug dependence.

A polymorphism of the metabotropic glutamate receptor mGluR7 (GRM7) gene is associated with schizophrenia

INTRODUCTION Glutamate dysfunction has been implicated in the pathophysiology of schizophrenia. The metabotropic glutamate receptors (mGluRs) are G-protein-coupled receptors. GRM7, the gene that encodes mGluR7, is expressed in many regions of the human central nervous system. The GRM7 gene is located on human chromosome 3p26, which has been suggested by linkage analysis to contain a susceptibility locus for schizophrenia. METHODS We screened for mutations in all exons, exon/intron junctions, and promoter regions of the GRM7 gene in Japanese patients with schizophrenia and evaluated associations between the detected polymorphisms and schizophrenia. We examined the influence of one polymorphism associated with schizophrenia on the expression of GRM7 by dual-luciferase assay in transfected cells. RESULTS Twenty-five polymorphisms/mutations were detected in GRM7. Case-control analysis revealed a potential association of a synonymous polymorphism (371T/C, rs3749380) in exon 1 with schizophrenia in our case-control study of 2293 Japanese patients with schizophrenia and 2382 Japanese control subjects (allelic p=0.009). Dual-luciferase assay revealed suppression of transcription activity by exon 1 containing this polymorphism and a statistically significant difference in the promoter activity between the T and C alleles. CONCLUSIONS Our results support the possible association of a GRM7 gene polymorphism with genetic susceptibility to schizophrenia.

Monoallelic and unequal allelic expression of the HTR2A gene in human brain and peripheral lymphocytes.

BACKGROUND The 102T/C polymorphism of the serotonin 2A receptor (HTR2A) gene is reported to be associated with schizophrenia and other diseases and phenotypes. Altered HTR2A expression has been found in relation to several neuropsychiatric conditions, including depression and schizophrenia. Studies of expression of HTR2A messenger RNA (mRNA) and protein in postmortem brains suggest that the 102C allele might be less transcriptionally active than the T allele. However, equal expression of both alleles has also been reported. METHODS We performed primer extension assays to measure relative expression of allele-specific HTR2A transcripts in mRNAs isolated from the prefrontal cortex of 31 individuals with schizophrenia and from peripheral lymphocytes (PBLs) of 31 healthy individuals heterozygous for 102T/C. We also examined the allele transmission pattern of HTR2A in PBLs of nine families. RESULTS Analyses of DNA and mRNA revealed that 102C is expressed but at lower levels than 102T in brains. In contrast to the biallelic expression observed in brains, monoallelic expression of HTR2A was common in PBLs. However, a family study revealed that imprinting was not responsible for the monoallelic expression in PBLs. CONCLUSIONS The present study revealed a tissue-specific modification of HTR2A expression, which makes allelic and epiallelic analyses necessary for genetic epidemiologic and pharmacogenomic studies of HTR2A.

Support for association of the PPP3CC gene with schizophrenia

Calcineurin is a calcium-dependent protein phosphatase that plays an important role in cellular responses and calcium signal transduction. Several studies have suggested that calcineurin is one of the key molecules in signal transduction in the brain and that dysfunction of calcineurin signaling could be linked to schizophrenia. Calcineurin is a heteromeric protein complex consisting of a catalytic subunit (calcineurin A) and a regulatory subunit (calcineurin B). PPP3CC encodes the calcineurin g-catalytic subunit and is located on chromosome 8p21.3 within a few cM of markers reported to be linked to schizophrenia. Gerber et al. reported genetic associations of the PPP3CC gene with schizophrenia in populations from the United States and South Africa. However, only one replication study has been published, and these associations were not confirmed in 457 Japanese schizophrenic patients and 429 control subjects. HapMap data indicated that a haplotype block spans almost the entire PPP3CC region in Japanese and European populations. The single nucleotide polymorphism (SNP) haplotype reported to be associated with schizophrenia by Gerber et al. is located in the haplotype block. Therefore, we examined the associations in a large case–control study of 1645 schizophrenic patients and 1673 control subjects. This sample size has a power > 0.98 to replicate the haplotypic association with the same magnitude as that reported by Gerber et al., assuming an a value = 0.05, two-tailed, a haplotype relative risk of 1.23, and haplotype frequency of 0.26 or effect size of 0.1. The haplotype frequency in the Japanese population was reported by Kinoshita et al. All subjects were of Japanese descent and were recruited from the main island of Japan. The study included 1645 unrelated patients with schizophrenia (mean age7s.d., 48.2714.5 years; 899 men and 745 women) diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). Control subjects were 1673 mentally healthy unrelated subjects (age, 47.9714.3, 886 men and 787 women) without self-reported family histories of mental illness within second-degree relatives. The subjects studied by Kinoshita et al. were not included in the present study. The present study was approved by the Ethics Committees of the University of Tsukuba, Niigata University, Fujita Health University, Nagoya University, Okayama University and Teikyo University and all participants provided written informed consent. To rule out population stratification between patients and controls in the present study, 35 SNPs that are not in linkage disequilibrium (LD) with each other were genotyped in all samples and analyzed with the STRUCTURE program 2.0. No stratification was observed. We genotyped five SNPs. SNP1 (rs10108011, CC21 in Gerber et al.) and SNP2 (rs2461491, CCS3 in Gerber et al.) were selected because Gerber et al. reported nominally significant allelic association of these SNPs with schizophrenia. SNP4 (rs2449340), SNP3 (rs2461490) and SNP5 (rs1116085) were genotyped to distinguish common haplotypes with frequencies X5% in the haplotype block based on HapMap data of the Japanese population. SNPs were genotyped with the TaqMan SNP Genotyping Assay (Applied Biosystems, Foster City, CA, USA) and ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). Deviation from predicted Hardy–Weinberg frequency was examined by w-test. Individual allelic and genotypic associations were examined by Fisher’s exact test. LD between polymorphisms and haplotypic associations were evaluated with Haploview software version 3.32. To deal with multiple testing, allelic associations were evaluated by permutation tests implemented in Haploview. The genotype distributions were evaluated by the Cochran–Armitage test without correction for multiple testing. The genotype and allele distributions of the five SNPs in the patient group and control group are shown in Table 1. Distributions of these SNPs did not differ significantly from Hardy–Weinberg equilibrium. All five SNPs showed nominally significant allelic associations with schizophrenia and permutation tests revealed significant allelic associations of SNP1 (P = 0.012), SNP3 (P = 0.005) and SNP4 (P = 0.013) with schizophrenia. The genotype distributions suggest that the minor allele of each SNP is likely to have an additive effect in the susceptibility to schizophrenia. These five SNPs are in LD; however, the LD is not complete. Therefore, these associations were not caused by a single SNP in the present study. As shown in Table 2, there were only two common haplotypes constructed from these SNPs. The most common haplotype in the control group was observed less frequently in the patient group (P = 0.034) and the second most common Molecular Psychiatry (2007) 12, 891–903 & 2007 Nature Publishing Group All rights reserved 1359-4184/07

Haplotype analysis of a 100 kb region spanning TNF-LTA identifies a polymorphism in the LTA promoter region that is associated with atopic asthma susceptibility in Japan.

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