Ayako Tani

Positive associations of polymorphisms in the metabotropic glutamate receptor type 3 gene (GRM3) with schizophrenia

Objectives Glutamatergic dysfunction is one of the major hypotheses of schizophrenia pathophysiology. We have been conducting systematic studies on the association between glutamate receptors and schizophrenia. We focused on the metabotropic glutamate receptor type 3 gene (GRM3) as a candidate for schizophrenia susceptibility. Methods We genotyped Japanese schizophrenics (n = 100) and controls (n = 100) for six single nucleotide polymorphisms (SNPs) located in the GRM3 region at intervals of approximately 50 kb. Statistical differences in genotype, allele and haplotype frequencies between cases and controls were evaluated by the &KHgr;2 test and Fishers exact probability test at a significance level of 0.05. Haplotype frequencies were estimated by the EM algorithm. Results A case‐control association study identified a significant difference in allele frequency distribution of a SNP, rs1468412, between schizophrenics and controls (P=0.011). We also observed significant differences in haplotype frequencies estimated from SNP frequencies between schizophrenics and controls. The haplotype constructed from three SNPs, including rs1468412, showed a significant association with schizophrenia (P=8.30 × 10−4). Conclusions Our data indicate that at least one susceptibility locus for schizophrenia is situated within or very close to the GRM3 region in the Japanese patients.

Positive association of the AMPA receptor subunit GluR4 gene (GRIA4) haplotype with schizophrenia: linkage disequilibrium mapping using SNPs evenly distributed across the gene region.

The glutamatergic dysfunction hypothesis suggests that genes involved in the glutamate neurotransmitter system are candidates for schizophrenia‐susceptibility genes. We have been conducting systematic studies of the association between glutamate receptors and schizophrenia. We report on a positive association of some haplotypes of the AMPA receptor subunit GluR4 gene (GRIA4) with schizophrenia. We genotyped 100 Japanese schizophrenics and 100 controls for six single nucleotide polymorphism (SNP) markers distributed at intervals of about 50 kb in the GRIA4 region, and estimated the degree of linkage disequilibrium (LD) between the SNPs. We constructed haplotypes of the SNPs in LD using the EM algorithm to test their association with schizophrenia. Significant associations were detected for the combination of SNP4‐5 (χ2 = 12.54, df = 3, P = 0.0057, P = 0.029 with Bonferroni correction) and for the combination of SNP3‐4‐5 (χ2 = 18.9, df = 7, P = 0.0085, P = 0.043 with Bonferroni correction). These results suggest that at least one susceptibility locus for schizophrenia is located within or very close to the GRIA4 region in Japanese.

Association study of polymorphisms in the group III metabotropic glutamate receptor genes, GRM4 and GRM7, with schizophrenia.

Based on the hypothesis that a glutamatergic dysfunction is involved in the pathophysiology of schizophrenia, we have been conducting systematic studies on the association between glutamate receptor genes and schizophrenia. Here we report association studies of schizophrenia with polymorphisms in group III metabotropic glutamate receptor genes, GRM4 and GRM7. We selected 8 and 43 common SNPs distributed in the entire gene regions of GRM4 (>111 kb) and GRM7 (>900 kb), respectively. We scanned significant associations with schizophrenia using 100 case-control pairs of Japanese. We identified two neighboring SNPs (rs12491620 and rs1450099) in GRM7 showing highly significant haplotype association with schizophrenia surviving the FDR correction. We then performed additional typing of the two SNPs using the expanded sample set (404 cases and 420 controls) and confirmed the significant association with the disease. We conclude that at least one susceptibility locus for schizophrenia is located within or nearby GRM7, whereas GRM4 is unlikely to be a major susceptibility gene for schizophrenia in the Japanese population.

Association study of polymorphisms in the Glur5 kainate receptor gene ( grik1 ) with schizophrenia

The glutamatergic dysfunction hypothesis suggests genes involved in glutamatergic transmission as candidates for schizophrenia susceptibility genes. We screened single nucleotide polymorphisms (SNPs) in the entire coding sequence of the GluR5 kainate receptor gene, GRIK1, by polymerase chain reaction-single strand conformation polymorphism and direct sequencing. We identified six SNPs including three known ones, 522A/C (174T, synonymous), 1173C/T (391D, synonymous), and 2705C/T (902L/S), as well as three novel ones, 995C/T (332A/V), 2400C/T (800L, synonymous), and 2585A/G (862R/Q). We genotyped Japanese samples of schizophrenia (n  = 193–203) and healthy controls (n  = 199–215) for three SNPs those were commonly observed in our samples, 522A/C, 1173C/T, and 2705C/T. We observed no significant associations of the SNPs and their haplotypes with schizophrenia. Therefore, we conclude that GRIK1 does not play a major role in schizophrenia pathogenesis in the Japanese population.

Lys-49-phospholipases A2 as active enzyme for β-arachidonoyl phospholipid bilayer membranes

Phospholipases A2 containing Lys‐49 have been reported to be extremely weak or inactive as enzyme. We have recently shown that basic proteins I and II (BP‐I and BP‐II), Lys‐49‐PLA2s isolated from the venom of Trimeresurus flavoviridis (Habu snake), are potent to hydrolyze the arachidonate of 2‐arachidonoyl‐1‐stearoyl‐L‐3‐phosphatidylcholine (ASPC) in bilayer vesicles. In order to ensure such enzymatic activity of Lys‐49‐PLA2s, two other Lys‐49‐PLA2s from different snake venoms, myotoxin II (from Bothrops asper) and App‐K49 (form Agkistrodon piscivorus piscivorus), were examined. Myotoxin II was found to be very active, even more potent than BP‐II, liberating about 80% of arachidonic acid from liposomes. App‐K49 was also active (about 50%) for ASPC liposomes. They were very weak or almost inactive for ASPC micelles and monomers. All these Lys‐49‐PLA2s were inactive for ASPC liposomes in the absence of Ca2+. These results clearly demonstrated that Lys‐49‐PLA2s are the enzymes to hydrolyze the C2‐ester bond of ASPC in bilayer membranes.

Polymorphism analysis of the upstream region of the human N-methyl-D-aspartate receptor subunit NR1 gene (GRIN1): Implications for schizophrenia

Dysfunction of the gene for the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor (GRIN1) has been implicated in the pathogenesis of schizophrenia. In support of this hypothesis are behavioral abnormalities reminiscent of schizophrenia in mice with an attenuated expression of the NR1 subunit receptor and the reduced level of NR1 mRNA in postmortem brains of patients with schizophrenia. We screened single nucleotide polymorphisms (SNPs) in the upstream region between +51 and -941 from the translation initiation codon of GRIN1 and identified 17 SNPs, 10 of which were located within the region containing the Sp1 motif and the GSG motifs. As genotyping of 191-196 Japanese patients with schizophrenia and 202-216 controls revealed no significant association between schizophrenia and the SNPs in the upstream region of GRIN1, these SNPs apparently do not play a critical role in the pathogenesis of schizophrenia in the Japanese population.

DISCRIMINATORY RECOGNITION OF MEMBRANE PHOSPHOLIPIDS BY LYSINE-49-PHOSPHOLIPASES A2 FROM TRIMERESURUS FLAVOVIRIDIS VENOM

Basic proteins I and II (BP‐I and BP‐II) isolated from Trimeresurus flavoviridis venom, which are classified into a group of lysine‐49‐phospholipases A2 (Lys‐49‐PLA2), exhibited only limited lipolytic activity for the mixed micelles of various phospholipids. Based on the finding that BP‐II elicits a strong contraction of guinea pig ileum due to the release of arachidonic acid, BP‐II together with BP‐I has been tested for their interaction with artificial phospholipid bilayer membranes. The dye leakage experiments indicated that BP‐II interacts strongly with liposomes of β‐arachidonoyl‐γ‐stearoyl‐Lα‐phosphatidylcholine. The perturbation of liposomes was observed only in the Ca2+‐containing buffer, and as demonstrated by HPLC analyses, accompanied by the release of arachidonic acid. The concentration of Ca2+ which gave a half maximal activity of BP‐II was 3.0 × 10−4 M, suggesting that the affinity of BP‐II for Ca2+ is more than 10 times stronger than that of BP‐II without liposomes. These observations clearly show that Lys‐49‐PLA2 of BP‐II is the enzyme responsible for the hydrolysis of membrane phospholipids and that Ca2+ is essential for such enzymatic activity. The interaction of BP‐I with liposomes was much weaker than BP‐II. BP‐I and BP‐II share a common sequence except for Asp‐67 (BP‐I) and Asn‐67 (BP‐II) in the aligned sequences. This implies that the amino acid at position 67 of Lys‐49‐PLA2s is the residue required for discriminatory recognition of phospholipid membranes.