Tsuyuka Ohtsuki

Human cannabinoid receptor 1: 5′ exons, candidate regulatory regions, polymorphisms, haplotypes and association with polysubstance abuse

A number of lines of evidence make the gene that encodes the G-protein-coupled CB1/Cnr1 receptor a strong candidate to harbor variants that might contribute to individual differences in human addiction vulnerability. The CB1/Cnr1 receptor is the major brain site at which cannabinoid marijuana constituents are psychoactive as well as the principal brain receptor for endogenous anandamide ligands. It is densely expressed in brain circuits likely to be important for both the reward and mnemonic processes important for addiction. Altered drug effects in CB1/Cnr1 knockout mice and initial association studies also make variants at the CB1/Cnr1 locus candidates for roles in human vulnerabilities to addictions. However, many features of this genes structure, regulation and variation remain poorly defined. This poor definition has limited the ability of previous association studies to adequately sample variation at this locus. We now report improved definition of the human CB1/Cnr1 locus and its variants. Novel exons 1–3, splice variant and candidate promoter region sequences add to the richness of the CB1/Cnr1 locus. Candidate promoter region sequences confer reporter gene expression in cells that express CB1/Cnr1. Common polymorphisms reveal patterns of linkage disequilibrium in European- and in African-American individuals. A 5′ CB1/Cnr1 ‘TAG’ haplotype displays significant allelic frequency differences between substance abusers and controls in European-American, African-American and Japanese samples. Post-mortem brain samples of heterozygous individuals contain less mRNA transcribed from the TAG alleles than from other CB1/Cnr1 haplotypes. CB1/ Cnr1 genomic variation thus appears to play roles in human addiction vulnerability.

Genomewide High-Density SNP Linkage Analysis of 236 Japanese Families Supports the Existence of Schizophrenia Susceptibility Loci on Chromosomes 1p, 14q, and 20p

The Japanese Schizophrenia Sib-Pair Linkage Group (JSSLG) is a multisite collaborative study group that was organized to create a national resource for affected sib pair (ASP) studies of schizophrenia in Japan. We used a high-density single-nucleotide-polymorphism (SNP) genotyping assay, the Illumina BeadArray linkage mapping panel (version 4) comprising 5,861 SNPs, to perform a genomewide linkage analysis of JSSLG samples comprising 236 Japanese families with 268 nonindependent ASPs with schizophrenia. All subjects were Japanese. Among these families, 122 families comprised the same subjects analyzed with short tandem repeat markers. All the probands and their siblings, with the exception of seven siblings with schizoaffective disorder, had schizophrenia. After excluding SNPs with high linkage disequilibrium, we found significant evidence of linkage of schizophrenia to chromosome 1p21.2-1p13.2 (LOD=3.39) and suggestive evidence of linkage to 14q11.2 (LOD=2.87), 14q11.2-q13.2 (LOD=2.33), and 20p12.1-p11.2 (LOD=2.33). Although linkage to these regions has received little attention, these regions are included in or partially overlap the 10 regions reported by Lewis et al. that passed the two aggregate criteria of a meta-analysis. Results of the present study--which, to our knowledge, is the first genomewide analysis of schizophrenia in ASPs of a single Asian ethnicity that is comparable to the analyses done of ASPs of European descent--indicate the existence of schizophrenia susceptibility loci that are common to different ethnic groups but that likely have different ethnicity-specific effects.

Screening for 22q11 deletions in a schizophrenia population

Since the recognition that adults with velocardiofacial syndrome (VCFS), which is associated with hemizygous interstitial deletions of chromosome 22q11, frequently show psychotic symptoms, deletion of the 22q11.2 region has been proposed as a common genetic abnormality associated with schizophrenia. In studies of schizophrenia patients, such deletions have been detected in more than 1% of schizophrenics, indicating the likely presence of this deletion in a significant number of patients. In this study, we screened for 22q11.2 deletions by genotyping microsatellite markers in 300 schizophrenics and 300 normal controls. The 22q11.2 deletion was confirmed by fluorescent in situ hybridization (FISH). One patient with schizophrenia was found to have a 22q11.2 deletion. The patient was mildly retarded but did not have craniofacial, palatal, or cardiac malformations characteristic of VCFS. Our results indicate that 22q11.2 deletion does not contribute substantially to the development of schizophrenia in general. However, our findings establish the existence of physically near-normal individuals with 22q11.2 deletion among learning disabled or mildly retarded persons with schizophrenia.

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.

Association between serotonin 4 receptor gene polymorphisms and bipolar disorder in Japanese case-control samples and the NIMH Genetics Initiative Bipolar Pedigrees.

Possible irregularities in serotonergic neurotransmission have been suggested as causes of a variety of neuropsychiatric diseases. We performed mutation and association analyses of the HTR4 gene, on 5q32, encoding the serotonin 4 receptor in mood disorders and schizophrenia. Mutation analysis was performed on the HTR4 exons and exon/intron boundaries in 48 Japanese patients with mood disorders and 48 patients with schizophrenia. Eight polymorphisms and four rare variants were identified. Of these, four polymorphisms at or in close proximity to exon d, g.83097C/T (HTR4-SVR (splice variant region) SNP1), g.83159G/A (HTR4-SVRSNP2), g.83164 (T)9–10 (HTR4-SVRSNP3), and g.83198A/G (HTR4-SVRSNP4), showed significant association with bipolar disorder with odds ratios of 1.5 to 2. These polymorphisms were in linkage disequilibrium, and only three common haplotypes were observed. One of the haplotypes showed significant association with bipolar disorder (P = 0.002). The genotypic and haplotypic associations with bipolar disorder were confirmed by transmission disequilibrium test in the NIMH Genetics Initiative Bipolar Pedigrees with ratios of transmitted to not transmitted alleles of 1.5 to 2.0 (P = 0.01). The same haplotype that showed association with bipolar disorder was suggested to be associated with schizophrenia in the case-control analysis (P = 0.003) but was not confirmed when Japanese schizophrenia families were tested. The polymorphisms associated with mood disorder were located within the region that encodes the divergent C-terminal tails of the 5-HT4 receptor. These findings suggest that genomic variations in the HTR4 gene may confer susceptibility to mood disorder.

A genome-wide linkage analysis of orchard grass- sensitive childhood seasonal allergic rhinitis in Japanese families

Seasonal allergic rhinitis (SAR) is an inflammatory disease of the nose and eyes that follows sensitization to air-born pollens. We conducted a genome-wide linkage screening of 48 Japanese families (188 members) with orchard grass (OG)-sensitive SAR children (67 affected sib-pairs) in a farming community in central Japan where OG was planted for apple farming and OG pollen is a major cause of SAR. We used the GENEHUNTER program to performed nonparametric multipoint linkage analysis for OG-sensitive SAR as a qualitative trait and for log total serum IgE levels and OG-RAST IgE levels as quantitative traits. Genotyping data of 400 microsatellite markers suggested linkage of SAR to chromosomes 1p36.2, 4q13.3, and 9q34.3 (P < 0.001), linkage of serum total IgE levels to 3p24.1, 5q33.1, 12p13.1, and 12q24.2 (P < 0.001), and linkage of OG-RAST IgE levels to 4p16.1, 11q14.3, and 16p12.3 (P < 0.001). Weak evidence for linkage of SAR to 5q33.1 was also observed (P = 0.01). All these regions, with the exception of 9q34.3, have been previously reported to be linked to asthma and/or atopy. These data suggest that, although loci linked to SAR are likely to be common to asthma, a strong contribution by specific gene(s) to OG-sensitive SAR is unlikely.

Failure to confirm association between AKT1 haplotype and schizophrenia in a Japanese case-control population.

SIR—AKT1 is a serine/threonine kinase known as protein kinase B. Modulation of AKT1 activity appears to play a key role in the mechanism of glutamate excitotoxicity and lithium neuroprotection.1 Glycogen synthase kinase 3 (GSK3) is one of the substrates of AKT1 and AKT1/GSK3 signaling is known as a target of lithium. Lithium can exert its acute effect on dopamine-dependent behaviors by interfering with the action of dopamine on an Akt/GSK-3 signaling cascade in the brain of living mice.2 Transmission of haplotypes construct from seven single-nucleotide polymorphisms (SNPs) in a highly variable region of the AKT1 gene has been shown to have a weak evidence of association with bipolar disorder.3

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.

Possible association between a haplotype of the GABA-A receptor alpha 1 subunit gene (GABRA1) and mood disorders

BACKGROUND The gamma-aminobutyric acid (GABA) neurotransmitter system has been implicated in the pathogenesis of mood disorders. The GABRA1 gene encodes one of the subunits of GABA-A receptor and is located on human chromosome 5q34-q35, which is a region reportedly linked to mood disorders. We examined the GABRA1 gene as a candidate for mood disorders. METHODS We performed mutation screening of GABRA1 in 24 Japanese bipolar patients and evaluated associations in Japanese case-control subjects consisting of 125 patients with bipolar disorder, 147 patients with depressive disorders, and 191 healthy control subjects. Associations were confirmed in the National Institute of Mental Health (NIMH) Initiative Bipolar Pedigrees, which consists of 88 multiplex pedigrees with 480 informative persons. RESULTS We identified 13 polymorphisms in the GABRA1 gene. Nonsynonymous mutations were not found. Association of a specific haplotype with affective disorders was suggested in the Japanese case-control population (corrected p=.0008). This haplotype association was confirmed in the NIMH pedigrees (p=.007). CONCLUSIONS These results indicate that the GABRA1 gene may play a role in the etiology of bipolar disorders.

WFS1 gene mutation search in depressive patients: detection of five missense polymorphisms but no association with depression or bipolar affective disorder

BACKGROUND Wolfram syndrome (WFS) is an autosomal recessive neurodegenerative disorder. Recently, the WFS1 gene was isolated, and approximately 80% of the mutations responsible for WFS were found in exon 8 of WFS1. It has been noted that heterozygous carriers of the WFS gene are 26-fold more likely to be hospitalized for depression, and it has been estimated that approximately 25% of all people hospitalized for depression may carry the WFS gene(s). METHODS We searched for mutations in exon 8 of WFS1 in 30 depressive patients with a history of hospitalization and whose age at onset was under 40 years. We also examined 47 bipolar affective patients and 62 control subjects for an association. RESULTS A were detected. Four of the six were novel. No nonsense or frameshift mutation was detected. Genotypic and allelic distributions were similar between the depressive patients and the controls. No association with bipolar affective disorder was suggested. LIMITATIONS Because of the small sample size, the probability of finding at least one patient with WFS-responsible mutation(s) was 70% if depression is associated with WFS1 mutation(s) in 5% of patients. CONCLUSION It is not likely that WFS1 mutations are responsible for as much as 25% of depressive illness.