High-resolution copy number variation analysis of schizophrenia in Japan.

Recent schizophrenia (SCZ) studies have reported an increased burden of de novo copy number variants (CNVs) and identified specific high-risk CNVs, although with variable phenotype expressivity. However, the pathogenesis of SCZ has not been fully elucidated. Using array comparative genomic hybridization, we performed a high-resolution genome-wide CNV analysis on a mainly (92%) Japanese population (1699 SCZ cases and 824 controls) and identified 7066 rare CNVs, 70.0% of which were small (<100 kb). Clinically significant CNVs were significantly more frequent in cases than in controls (odds ratio=3.04, P=9.3 × 10-9, 9.0% of cases). We confirmed a significant association of X-chromosome aneuploidies with SCZ and identified 11 de novo CNVs (e.g., MBD5 deletion) in cases. In patients with clinically significant CNVs, 41.7% had a history of congenital/developmental phenotypes, and the rate of treatment resistance was significantly higher (odds ratio=2.79, P=0.0036). We found more severe clinical manifestations in patients with two clinically significant CNVs. Gene set analysis replicated previous findings (e.g., synapse, calcium signaling) and identified novel biological pathways including oxidative stress response, genomic integrity, kinase and small GTPase signaling. Furthermore, involvement of multiple SCZ candidate genes and biological pathways in the pathogenesis of SCZ was suggested in established SCZ-associated CNV loci. Our study shows the high genetic heterogeneity of SCZ and its clinical features and raises the possibility that genomic instability is involved in its pathogenesis, which may be related to the increased burden of de novo CNVs and variable expressivity of CNVs.

Micrometer-sized network structure of novel DNA-lipid conjugates induced by heat stimulation.

We have developed a novel lipid-bearing DNA that forms hairpin modules, including a single RNA monomer; this can be used to create micrometer-sized structures from nanometer-sized building blocks during breakage at the RNA site. In the presence of divalent metal ions and heat stimulation, we observed transition of the self-assembly, which results in the formation of a three-dimensional network structure. To our knowledge, this is also the first report of heat-induced micrometer-sized molecular self-assembly of molecules that carry biological information.

22q11.2 deletion carriers and schizophrenia-associated novel variants.

The penetrance of schizophrenia risk in carriers of the 22q11.2 deletion is high but incomplete, suggesting the possibility of additional genetic defects. We performed whole exome sequencing on two individuals with 22q11.2 deletion, one with schizophrenia and the other who was psychosis-free. The results revealed novel genetic variants related to neuronal function exclusively in the person with schizophrenia (frameshift: KAT8, APOH and SNX31; nonsense: EFCAB11 and CLVS2). This study paves the way towards a more complete understanding of variant dose and genetic architecture in schizophrenia.

Schizophrenia with the 22q11.2 deletion and additional genetic defects: case history.

The 22q11.2 deletion is the most prominent known genetic risk factor for schizophrenia, but its penetrance is at most approximately 50% suggesting that additional risk factors are required for disease progression. We examined a woman with schizophrenia with this deletion for such risk factors. She had high plasma pentosidine levels ('carbonyl stress') and a frameshift mutation in the responsible gene, GLO1. She also had a constant exotropia, so we examined the PHOX2B gene associated with both schizophrenia and strabismus, and detected a 5-alanine deletion. We propose that the combination of these genetic defects may have exceeded the threshold for the manifestation of schizophrenia.

Autoantibodies against CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) that impair glucose-induced insulin secretion in noninsulin- dependent diabetes patients.

Cyclic ADP-ribose (cADPR) has been shown to be a mediator for intracellular Ca2+ mobilization for insulin secretion by glucose in pancreatic beta cells, and CD38 shows both ADP-ribosyl cyclase to synthesize cADPR from NAD+ and cADPR hydrolase to hydrolyze cADPR to ADP-ribose. We show here that 13.8% of Japanese non-insulin-dependent diabetes (NIDDM) patients examined have autoantibodies against CD38 and that the sera containing anti-CD38 autoantibodies inhibit the ADP-ribosyl cyclase activity of CD38 (P

Fatty acid composition of the postmortem corpus callosum of patients with schizophrenia, bipolar disorder, or major depressive disorder.

BACKGROUND: Studies investigating the relationship between n-3 polyunsaturated fatty acid (PUFA) levels and psychiatric disorders have thus far focused mainly on analyzing gray matter, rather than white matter, in the postmortem brain. In this study, we investigated whether PUFA levels showed abnormalities in the corpus callosum, the largest area of white matter, in the postmortem brain tissue of patients with schizophrenia, bipolar disorder, or major depressive disorder. METHODS: Fatty acids in the phospholipids of the postmortem corpus callosum were evaluated by thin-layer chromatography and gas chromatography. Specimens were evaluated for patients with schizophrenia (n=15), bipolar disorder (n=15), or major depressive disorder (n=15) and compared with unaffected controls (n=15). RESULTS: In contrast to some previous studies, no significant differences were found in the levels of PUFAs or other fatty acids in the corpus callosum between patients and controls. A subanalysis by sex gave the same results. No significant differences were found in any PUFAs between suicide completers and non-suicide cases regardless of psychiatric disorder diagnosis. CONCLUSIONS: Patients with psychiatric disorders did not exhibit n-3 PUFAs deficits in the postmortem corpus callosum relative to the unaffected controls, and the corpus callosum might not be involved in abnormalities of PUFA metabolism. This area of research is still at an early stage and requires further investigation.

Analysis of induced pluripotent stem cells carrying 22q11.2 deletion.

Given the complexity and heterogeneity of the genomic architecture underlying schizophrenia, molecular analyses of these patients with defined and large effect-size genomic defects could provide valuable clues. We established human-induced pluripotent stem cells from two schizophrenia patients with the 22q11.2 deletion (two cell lines from each subject, total of four cell lines) and three controls (total of four cell lines). Neurosphere size, neural differentiation efficiency, neurite outgrowth, cellular migration and the neurogenic-to-gliogenic competence ratio were significantly reduced in patient-derived cells. As an underlying mechanism, we focused on the role of DGCR8, a key gene for microRNA (miRNA) processing and mapped in the deleted region. In mice, Dgcr8 hetero-knockout is known to show a similar phenotype of reduced neurosphere size (Ouchi et al., 2013). The miRNA profiling detected reduced expression levels of miRNAs belonging to miR-17/92 cluster and miR-106a/b in the patient-derived neurospheres. Those miRNAs are reported to target p38α, and conformingly the levels of p38α were upregulated in the patient-derived cells. p38α is known to drive gliogenic differentiation. The inhibition of p38 activity by SB203580 in patient-derived neurospheres partially restored neurogenic competence. Furthermore, we detected elevated expression of GFAP, a gliogenic (astrocyte) marker, in postmortem brains from schizophrenia patients without the 22q11.2 deletion, whereas inflammation markers (IL1B and IL6) remained unchanged. In contrast, a neuronal marker, MAP2 expressions were decreased in schizophrenia brains. These results suggest that a dysregulated balance of neurogenic-to-gliogenic competence may underlie neurodevelopmental disorders such as schizophrenia.

T lymphocytes increase the synthesis of esterified cholesterol in human monocyte-derived macrophages by activation of the scavenger pathway.

Immunohistochemical analyses have shown the presence of T lymphocytes (T-cells) in atherosclerotic places in addition to macrophages and smooth muscle cells. To elucidate the role of T-cells in the formation of atherosclerotic lesions, we studied whether T-cells can stimulate the scavenger pathway and promote esterified cholesterol (EC) synthesis by [14C]oleate incorporation in macrophages. Macrophages and T-cells were co-cultured in two ways. In one culture, macrophages were in direct contact with T-cells (direct contact form). In the other, macrophages and T-cells were separated by Transwell membrane, but shared the same culture medium via the membrane (indirect contact form). Based on the incorporation of [14C]oleate into EC, macrophages strikingly increased EC synthesis in both forms of co-culture. This increase was proportional to the number of T-cells present and was inhibited by cyclosporin A. When macrophages were co-cultured indirectly in contact with T-cells in the presence of AcLDL for 24 h, and the T-cells were subsequently removed, EC synthesis in macrophages increased. However, this increase was not observed in macrophages that were rinsed twice with PBS. When macrophages, previously incubated with AcLDL for 24 h, were co-cultured indirectly in contact with T-cells for 24 h, the medium were prepared as activated T-cell-conditioned medium (aTCM). EC synthesis in macrophages cultured with aTCM increased. The ability of aTCM to increase EC synthesis disappeared upon repeated freezing/thawing, boiling and trypsin treatment. T-cells (indirect contact form) and aTCM similarly increased AcLDL-binding and -degradation in macrophages. These results indicated that T-cells secreted an active substance(s), protein in nature, which could activate the scavenger pathway and increase EC synthesis in macrophages. These observations suggest that T-cells can promote the uptake of modified lipoproteins by macrophages to induce foam cell-formation.

Absence of angiogenesis-inhibitory activity in aqueous humor of diabetic rabbits.

We studied inhibitory activity of angiogenesis in the eye to explore the formation of new blood vessels in diabetic microvascular diseases. When we examined the effects of extracts from various ocular tissues of nondiabetic rabbits on the proliferation of bovine aortic endothelial cells, potent inhibitory activity was found in lens and aqueous humor. Aqueous humor inhibited both angiogenesis on chorioallantoic membrane in vivo and promotion of endothelial cell growth in vitro, which were induced by retinal extracts. However, in diabetic rabbits, aqueous humor lost the ability to inhibit the growth of endothelial cells. The loss of activity became evident 1 mo after the alloxan injection and lasted while the animals were hyperglycemic. In addition, diabetic aqueous humor failed to suppress retinal extract-induced angiogenesis promotion and endothelial cell growth. The absence of the inhibitory activity of angiogenesis in diabetic aqueous humor might be involved in promotion of neovascularization in diabetic ocular disease.

Streptococcal preparation (OK-432) inhibits development of type I diabetes in NOD mice.

OK-432 (a streptococcal preparation) has been widely used for cancer immunotherapy in Japan. It is the most potent immunomodulator in activating both macrophages and killer T cells and in increasing interleukin 2 production. Two K.E. (Klinische Einheit, clinical unit) of OK-432 were given intraperitoneally to each of 17 female nonobese diabetic (NOD) mice every week from 4-24 wk of age. NOD mice as well as BB rats spontaneously develop type I diabetes. During administration of OK-432, the development of diabetes was inhibited in 17 of 17 mice over the 24-wk observation period, whereas 14 of 17 female NOD mice given physiological saline had developed diabetes by 24 wk of age. At the onset of diabetes, nonfasting blood glucose was 511 +/- 82 mg/dl. Histologic examination showed that in the OK-432-treated NOD mice, 98% of total islets were intact or mildly infiltrated with mononuclear cells, whereas in saline-treated NOD mice, 79% of total islets exhibited severe insulitis. In OK-432-treated NOD mice, both the number of the mononuclear spleen cells and their natural killer cell activity was significantly increased.

Insulin stimulates hydrolysis of plasmanylinositol-glycan and phosphatidylinositol-glycan in rat adipocytes. Insulin-induced generation of inositol glycan, alkylacylglycerol, and diacylglycerol.

Insulin binding to its receptor has been known to induce hydrolysis of phosphatidylinositol-glycan and release inositol-glycan and diacylglycerol, two putative second messengers of insulin actions. We metabolically labeled and purified PIG in rat cultured adipocytes. The treatment of [3H]glycerol-labeled PIG with phosphatidylinositol-specific phospholipase C released [3H]glycerol-labeled DAG and [3H]glycerol-labeled 1-alkyl,2-acyl-glycerol, suggesting that PIG has not only PIG but also plasmanylinositol-glycan moiety. Insulin induced hydrolysis of PIG/PMIG and generation of IG, DAG, and AAG in a dose-dependent manner. This report shows the first demonstration of insulin-sensitive PMIG in rat adipocytes. These results provide evidence that insulin-induced generation of IG, DAG, and AAG might be early events in the insulin-signaling mechanism in rat adipocytes, and insulin-releasable AAG seems to mediate some actions of insulin.

Mutated insulin receptor Val996 reduces insulin-dependent generation of inositol glycan and diacylglycerol.

We evaluated whether insulin-receptor tyrosine kinase activity is required for activation of PDH, insulin-induced hydrolysis of PIG and generation of IG and 1,2-DAG. For the analysis, we used stable-transfected CHO cell lines expressing wild-type human insulin receptor (CHO-wt cells) or the mutant receptor (Val996) that lacks tyrosine kinase activity (CHO-mut cells) (1,2). Insulin stimulated PDH activity in three CHO cell lines in a dose-dependent manner. Half-maximal concentrations of insulin to activate PDH was 7 x 10(-11) M in the CHO-wt cells, 10(-9) M in the parental cells, and 8 x 10(-9) M in the CHO-mut cells. Insulin stimulated hydrolysis of PIG and generation of IG and DAG in three CHO cell lines in a dose-dependent manner. Half-maximal concentrations of insulin to induce generation of IG was 8 x 10(-11) M in the CHO-wt cells, 10(-9) M in the parental CHO cells, and 10(-8) M in the CHO-mut cells. ED50 for the stimulation of DAG generation was 7 x 10(-11) M in the CHO-wt cells, 10(-9) M in the parental cells, and 10(-8) M in the CHO-mut cells. It is concluded that insulin-dependent PDH activation, PIG hydrolysis, and IG and DAG generation are mediated by the wild-type but not by the mutated insulin receptor of Val996. This study suggests that tyrosine kinase activity of the insulin receptor might be a prerequisite for insulin-stimulated generation of IG and DAG.

Human arterial smooth muscle cell proliferation in diabetes.

In the present study, we focus on the proliferation of human arterial smooth muscle cells (SMCs) from NIDDM patients (DM-SMCs) to clarify the reactivity to the growth factor(s) in fetal calf serum (FCS) and the factor(s) secreted by T-cells. The proliferation of DM-SMCs was significantly greater than SMCs from nondiabetic patients (nonDM-SMC). DM-SMC conditioned medium (DM-condMed) increased the growth of nonDM-SMCs. These results suggest that the growth factor is secreted from DM-SMCs as an autocrine system, which increases the proliferation of nonDM-SMCs. T-cells increased DNA synthesis of SMCs, and DM-SMCs strikingly reacted to T-cells. The present results support a function of T-cells in stimulating SMC growth. In conclusion, human arterial SMC proliferation is increased in diabetes in the same fashion as in experimentally induced diabetes in animals through responses to growth factors and an increased autocrine system. These results provide a mechanism for the increase in atherosclerotic disease in diabetes.

Treatment with streptococcal preparation (OK-432) suppresses anti-islet autoimmunity and prevents diabetes in BB rats.

We have recently shown that a streptococcal preparation (OK-432) inhibits insulitis and prevents diabetes in nonobese diabetic (NOD) mice, an animal model of insulin-dependent diabetes mellitus (IDDM). We extended this study to another model of IDDM, namely BB rats. Male and female BB rats were injected weekly with 0.2 mg OK-432 i.p. starting from 5 to 6 wk and continuing through 20 or 30 wk of age. The cumulative incidence of IDDM over 20 wk in the OK-432-treated BB rats (4 of 54, 7.4%) was significantly (P less than .01) lower than that found in the nontreated BB rats (13 of 47, 27.7%). We examined some of these rats as follows. All of the OK-432-treated BB rats tested showed normal glucose levels before and after oral glucose administrations, as did the nontreated and nondiabetic BB rats. Histological examination of pancreatic sections revealed that the OK-432-treated rats retained a greater number of intact islets without infiltration of the mononuclear cells than did the nontreated BB rats. A preliminary in vitro study further demonstrated that the cytotoxic activities of spleen cells against a rat insulinoma cell line, RIN, were suppressed in the OK-432-treated rat. However, the treatment of BB rats with OK-432 showed no suppressive effects in the spleen cell number, the responsiveness of spleen cells to concanavalin A, the populations of OX19+, W3/25+, and OX8+ peripheral blood lymphocytes, or in the titers of cell surface antibody against RIN. These results suggest that a nonimmunosuppressive immunomodulator such as OK-432 may be useful as an agent for immunotherapy of IDDM.

Immunologic aspects of the nonobese diabetic (NOD) mouse. Abnormalities of cellular immunity.

Experiments were performed on 12-wk-old nonobese diabetic (NOD) mice to investigate the immunologic background of the condition, using ICR mice as controls. The results indicate the following: (1) absolute decreases in number of T lymphocytes, (2) depression of natural killer activity, (3) normal responsiveness in delayed type hypersensitivity and functional depression of killer T cells against allogeneic tumors, (4) diminished resistance to herpes virus infection, and (5) enhanced production of polyclonal antibodies to T cell-dependent antigens. These features are similar to those noted in other autoimmune diseases of man and in their experimental models in laboratory animals. Elucidation of the pathogenetic mechanism of autoimmune diabetes mellitus in NOD mice, therefore, may contribute to the diagnosis, treatment, and prevention of a wide variety of autoimmune diseases.

Comparison of 1,5-anhydroglucitol, HbA1c, and fructosamine for detection of diabetes mellitus.

To evaluate the use of serum 1,5-anhydroglucitol (AG) levels in screening for diabetes mellitus, we compared the sensitivity and specificity of HbA1c, fructosamine (FA), and AG in 1620 randomly selected subjects in 11 institutions throughout Japan. Most individuals were receiving diet and/or drug therapy for diabetes. Subjects were separated into four groups based on World Health Organization criteria: nondiabetic control subjects, subjects with impaired glucose tolerance (IGT), patients with diabetes, and patients with other disorders without IGT. The overlap of AG values between each group was less than that of HbA1c or FA values. AG levels were significantly correlated with fasting plasma glucose (r = -0.627), HbA1c (r = -0.629), and FA (r = -0.590) levels. If we took 14 micrograms/ml as the normal lower limit, AG level was highly specific (93.1%), and a decreased AG level indicated diabetes mellitus (84.2% sensitivity). According to the selectivity index (sensitivity value times specificity value), AG determinations were superior to both HbA1c and FA measurements for diabetes screening. When combinations of these tests were used, only AG and HbA1c together were slightly better than AG alone. Thus, together with other advantages of AG, e.g., its wide variance with relatively fair glycemic control and the negligible influence of the sampling conditions, AG level has more potential than HbA1c or FA level as a screening criterion for diabetes.

Prevention of autoimmune diabetes with lymphotoxin in NOD mice.

We have reported previously that chronic and systemic administration of a streptococcal preparation (OK-432), an inducer of TNF, or of recombinant hTNF prevented the development of IDDM in the two animal models of IDDM-NOD mice and BB rats. In this study, we examined the effect of LT, which is structurally and functionally related to TNF, on NOD mice with diabetes. The cumulative incidence of diabetes at 30 wk of age was 22 of 40 (55%) in nontreated female NOD mice and was 4 of 8 (50%; NS), 3 of 29 (10%; P < 0.001), and 0 of 8 (0%; P < 0.001) in female mice treated three times a week from 4 to 30 wk of age with 5, 50, or 500 U of recombinant hLT, respectively. Intensity of insulitis was slightly reduced in the long-term LT-treated mice. LT productivity by ConA-stimulated spleen cells was examined in vitro. Although no significant difference was found between NOD mice and the other mouse strains, female NOD mice were slightly but significantly (P < 0.01) lower producers of LT immunoreactivity than male NOD mice, the diabetes incidence of which is lower than that of females. The SMLR as a marker of normal immune response, which was reported to be impaired in autoimmune animals including NOD mice, was significantly lower in female than male NOD mice. However, the low SMLR in female NOD mice was significantly increased by the administration of LT, and the increase was mediated by the responder cells of the LT-treated mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Association study on the DUSP6 gene, an affective disorder candidate gene on 12q23, performed by using fluorescence resonance energy transfer-based melting curve analysis on the LightCycler.

We introduced a new genotyping method, fluorescence resonance energy transfer-based melting curve analysis on the LightCycler, for the analysis of the gene, DUSP6 (dual specificity MAP kinase phosphatase 6), in affective disorder patients. The DUSP6 gene is located on chromosome 12q22-23, which overlaps one of the reported bipolar disorder susceptibility loci. Because of its role in intracellular signalling pathways, the gene may be involved in the pathogenesis of affective disorders not only on the basis of its position but also of its function. We performed association analysis using a T>G polymorphism that gives rise to a missense mutation (Leu114Val). No evidence for a significant disease-causing effect was found in Japanese unipolars (n = 132) and bipolars (n = 122), when compared with controls (n = 299). More importantly, this study demonstrates that melting curve analysis on the LightCycler is an accurate, rapid and robust method for discriminating genotypes from biallelic markers. This strategy has the potential for use in high throughput scanning for and genotyping of single nucleotide polymorphisms (SNPs). Molecular Psychiatry (2000) 5, 489-494.

Clinical efficacy of fidarestat, a novel aldose reductase inhibitor, for diabetic peripheral neuropathy: a 52-week multicenter placebo-controlled double-blind parallel group study.

OBJECTIVE: The purpose of this study was to evaluate the efficacy of fidarestat, a novel aldose reductase (AR) inhibitor, in a double-blind placebo controlled study in patients with type 1 and type 2 diabetes and associated peripheral neuropathy. RESEARCH DESIGN AND METHODS: A total of 279 patients with diabetic neuropathy were treated with placebo or fidarestat at a daily dose of 1 mg for 52 weeks. The efficacy evaluation was based on change in electrophysiological measurements of median and tibial motor nerve conduction velocity, F-wave minimum latency, F-wave conduction velocity (FCV), and median sensory nerve conduction velocity (forearm and distal), as well as an assessment of subjective symptoms. RESULTS: Over the course of the study, five of the eight electrophysiological measures assessed showed significant improvement from baseline in the fidarestat-treated group, whereas no measure showed significant deterioration. In contrast, in the placebo group, no electrophysiological measure was improved, and one measure significantly deteriorated (i.e., median nerve FCV). At the study conclusion, the fidarestat-treated group was significantly improved compared with the placebo group in two electrophysiological measures (i.e., median nerve FCV and minimal latency). Subjective symptoms (including numbness, spontaneous pain, sensation of rigidity, paresthesia in the sole upon walking, heaviness in the foot, and hypesthesia) benefited from fidarestat treatment, and all were significantly improved in the treated versus placebo group at the study conclusion. At the dose used, fidarestat was well tolerated, with an adverse event profile that did not significantly differ from that seen in the placebo group. CONCLUSIONS: The effects of fidarestat-treatment on nerve conduction and the subjective symptoms of diabetic neuropathy provide evidence that this treatment alters the progression of diabetic neuropathy.