Chihiro Kakiuchi

Molecular characterization of bipolar disorder by comparing gene expression profiles of postmortem brains of major mental disorders

We performed the oligonucleotide microarray analysis in bipolar disorder, major depression, schizophrenia, and control subjects using postmortem prefrontal cortices provided by the Stanley Foundation Brain Collection. By comparing the gene expression profiles of similar but distinctive mental disorders, we explored the uniqueness of bipolar disorder and its similarity to other mental disorders at the molecular level. Notably, most of the altered gene expressions in each disease were not shared by one another, suggesting the molecular distinctiveness of these mental disorders. We found a tendency of downregulation of the genes encoding receptor, channels or transporters, and upregulation of the genes encoding stress response proteins or molecular chaperons in bipolar disorder. Altered expressions in bipolar disorder shared by other mental disorders mainly consisted of upregulation of the genes encoding proteins for transcription or translation. The genes identified in this study would be useful for the understanding of the pathophysiology of bipolar disorder, as well as the common pathophysiological background in major mental disorders at the molecular level. In addition, we found the altered expression of LIM and HSPF1 both in the brains and lymphoblastoid cells in bipolar disorder. These genes may have pathophysiological importance and would be novel candidate genes for bipolar disorder.

Genetic or epigenetic difference causing discordance between monozygotic twins as a clue to molecular basis of mental disorders.

Classical twin research focused on differentiating genetic factors from environmental factors by comparing the concordance rate between monozygotic (MZ) and dizygotic twins. On the other hand, recent twin research tries to identify genetic or epigenetic differences between MZ twins discordant for mental disorders. There are a number of reports of MZ twins discordant for genetic disorders caused by genetic or epigenetic differences of known pathogenic genes. In the case of mental disorder research, for which the causative gene has not been established yet, we are trying to identify the ‘pathogenic gene’ by comprehensive analysis of genetic or epigenetic difference between discordant MZ twins. To date, no compelling evidence suggesting such difference between MZ twins has been reported. However, if the genetic or epigenetic difference responsible for the discordant phenotype is found, it will have impact on the biology of mental disorder, in which few conclusive molecular genetic evidences have been obtained.

HHV-8 infection status of AIDS-unrelated and AIDS-associated multicentric Castleman's disease.

Multicentric Castlemans disease (MCD) is a clinicopathologically defined entity characterized by systemic lymphadenopathy with unique pathomorphology such as angiosclerosis, blood vessel proliferation in and around follicles, and plasmacytosis. While its pathogenesis has remained unclarified for many years, identification of the human herpesvirus 8 (HHV‐8) in at least some MCD cases has opened new perspectives in this field. Because previous reports have described many inconsistencies regarding HHV‐8 positivity in MCD, we intended to clarify this issue by the introduction of more convincing methodologies. For this investigation, we introduced two antibodies produced in our laboratories that recognize a latent gene product ORF73 and a lytic gene product ORF59, together with two well‐recognized methods, in situ hybridization for the detection of lytic phase transcript T1.1/nut‐1, and genomic polymerase chain reaction (PCR). Eighty‐two cases of MCD were collected from Japan (n= 75) and France (n= 7). In three cases, the patients were suffering from acquired immunodeficiency syndrome (AIDS). Immunohistochemistry and in situ hybridization showed identical results: only three out of 82 cases were positively stained, and all the positive cases were found to be the patients with AIDS. Genomic PCR was done in 43 cases, and only one case produced positive results: the only AIDS case among the 43 cases studied by genomic PCR. Histopathologically, the HHV‐8‐positive cases showed the highest intensity of angiosclerosis and germinal center / perifollicular vascular proliferation, while plasmacytosis was not severe in the HHV‐8‐positive cases. Some of the HHV‐8‐negative MCD cases displayed similar histopathology, but at a far less intense level, except for the plasmacytosis. These results suggest that: (i) all three of the HHV‐8‐positive MCD patients in the present group are the patients with AIDS; and (ii) HHV‐8‐positive MCD patients develop typical but marked angiosclerosis and vascular proliferation that might be differentiated from HHV‐8‐negative MCD patients, who showed far less intense changes.

DNA methylation analysis of BDNF gene promoters in peripheral blood cells of schizophrenia patients.

Accumulating evidence suggests that epigenetic alterations in brain-derived neurotrophic factor (BDNF) promoters are associated with the pathophysiology of psychiatric disorders. Epigenetic changes in BDNF were reported not only in brain tissues but also in other tissues, including peripheral blood cells (PBC) and saliva. We examined DNA methylation levels of BDNF promoters I and IV using genomic DNA derived from PBC of healthy controls (n=100), and patients with schizophrenia (n=100), all from the Japanese population, by pyrosequencing. The examined CpG sites were chosen based on previous epigenetic studies that reported altered DNA methylation. We found a significantly higher level of methylation at BDNF promoter I in patients with schizophrenia compared to controls, although the difference was small. Subsequent analysis revealed that in controls, the methylation level of BDNF promoters was associated with sex, and the methylation difference observed in promoter I was more prominent in male patients with schizophrenia. Epigenetic alteration of BDNF in the PBC might reflect the pathophysiology of schizophrenia, and could be a potential biomarker.

The association between oxytocin receptor gene (OXTR) polymorphisms and affective temperaments, as measured by TEMPS-A.

BACKGROUNDnOxytocin is associated with social interaction, trust, and affectivity. Affective temperaments are traits based on Kraepelins typological definition of the fundamental states of manic-depressive illness. These states can be measured by the Temperament Evaluation of Memphis, Pisa, Paris and San Diego-Autoquestionnaire version (TEMPS-A). The objective of this study is to assess the association between oxytocin receptor gene (OXTR) polymorphisms and affective temperaments.nnnMETHODSnParticipants consisted of 493 genetically unrelated, non-clinical Japanese subjects (307 males and 186 females). The Mini-International Neuropsychiatric Interview (MINI) was used to screen and exclude those who had a lifetime diagnosis of schizophrenia or other psychotic disorders. Fifteen OXTR tag single nucleotide polymorphisms (SNPs) were genotyped using TaqMan® or direct sequencing. The Haploview 4.1. software determined the haplotype block structure. Haplotype-based quantitative trait association analysis with Bonferroni correction using PLINK 1.06 software was used to assess the association between haplotypes and the following affective temperaments: depressive, cyclothymic, hyperthymic, irritable, and anxious.nnnRESULTSnTwo haplotype blocks were identified on the OXTR. The depressive temperament was significantly associated with the most frequent haplotype GGGTGTC (rs11131149/rs2243370/rs2243369/rs13316193/rs2254298/rs2268493/rs2268491) (corrected P<0.05).nnnLIMITATIONSnThis study consisted of participants from a corporation and the effect sizes were small.nnnCONCLUSIONSnThe findings suggest that an OXTR haplotype is associated with a discrete depressive temperament. Clarification of the biological basis of this temperamental trait may help to elucidate the pathophysiology of depressive disorder.

Aspergillus tracheobronchitis after allogeneic bone marrow transplantation.

Recently Machidaet al presented in this journal a case of a patient with anAspergillus tracheobronchitis after allogeneic BMT together with a review of the literature of this serious complication after BMT. We would like to add our recent experience and comment on the role of galactomannan antigen testing in establishing the diagnosis of this Aspergillusinfection. A 56-year-old female patient underwent allogeneic BMT in our center after induction of first complete morphological and cytogenetic remission of acute myeloid leukemia. After conditioning with idarubicin, cyclophosphamide and busulfan, bone marrow-derived stem cells from her histocompatible sibling were infused. She developed grade II acute GVHD of the skin, for which she was treated with prednisone. Later on she experienced grade IV chronic GVHD of the gastro-intestinal tract and three episodes of CMVcolitis. Treatment with high-dose corticosteroids, cyclosporin, ganciclovir and CMV-hyperimmunoglobulins was given with limited success. On day 193 after BMT she was admitted to our hospital because of severe dyspnea, inspiratory wheezing and hoarseness. Temperature was not raised and persistent severe diarrhea was still present. Physical examination showed severe respiratory distress with use of accessory respiratory muscles and extreme inspiratory wheezing, and on auscultation her breath sounds were hardly audible. Chest X-ray and high-resolution CT scan were unremarkable. Lung function examination demonstrated extreme inspiratory and expiratory obstruction. Yellowish plaques, ulcerations and pseudomembranes were seen on bronchoscopy performed immediately and there were mucus plugs that almost completely obstructed the trachea and bronchi. Microscopic examination of the obstructing material showed hyphae and cultures yielded Aspergillus fumigatus . Biopsy was considered impossible because of the low platelet count. Daily bronchial flushing and treatment with high-dose amphotericin B (1 mg/kg/day, 45 mg daily) and nebulized amphotericin B was started with only temporary improvement. Liposomal amphotericin B 200 mg daily and itraconazole (200 mg, twice daily for 2 days, followed by 200 mg daily) as well as G-CSF (300 mg daily) were added without any improvement. Our patient became ventilator-dependent and could no longer cope with this regime. For this reason it was decided to abstain from further treatment and she died of respiratory failure the same day, 37 days after admission. Permission for autopsy was denied. Like Machidaet al we measured serum galactomannan antigen levels by ELISA (Platelia Aspergillus; Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France). In a series of six consecutive serum samples, the highest galactomannan serum ratio found was 0.6 which is below the recommended cut-off level of 1.0. This observation might have several explanations. Firstly, Clarke et al divided fungal tracheobronchitis into two different morphological types. The first type is characterized by intraluminal growth involving the entire circumference of the airway with only superficial invasion and ulceration; tenacious mucus or

Meta-analysis of genome-wide association studies for panic disorder in the Japanese population

Panic disorder (PD) is a moderately heritable anxiety disorder whose pathogenesis is not well understood. Due to the lack of power in previous association studies, genes that are truly associated with PD might not be detected. In this study, we conducted a genome-wide association study (GWAS) in two independent data sets using the Affymetrix Mapping 500K Array or Genome-Wide Human SNP Array 6.0. We obtained imputed genotypes for each GWAS and performed a meta-analysis of two GWAS data sets (718 cases and 1717 controls). For follow-up, 12 single-nucleotide polymorphisms (SNPs) were tested in 329 cases and 861 controls. Gene ontology enrichment and candidate gene analyses were conducted using the GWAS or meta-analysis results. We also applied the polygenic score analysis to our two GWAS samples to test the hypothesis of polygenic components contributing to PD. Although genome-wide significant SNPs were not detected in either of the GWAS nor the meta-analysis, suggestive associations were observed in several loci such as BDKRB2 (P=1.3 × 10−5, odds ratio=1.31). Among previous candidate genes, supportive evidence for association of NPY5R with PD was obtained (gene-wise corrected P=6.4 × 10−4). Polygenic scores calculated from weakly associated SNPs (P<0.3 and 0.4) in the discovery sample were significantly associated with PD status in the target sample in both directions (sample I to sample II and vice versa) (P<0.05). Our findings suggest that large sets of common variants of small effects collectively account for risk of PD.

Valproate, a mood stabilizer, induces WFS1 expression and modulates its interaction with ER stress protein GRP94

Background Valproate is a standard treatment for bipolar disorder and a first-line mood stabilizer. The molecular mechanisms underlying its actions in bipolar disorder are unclear. It has been suggested that the action of valproate is linked to changes in gene expression and induction of endoplasmic reticulum (ER) stress-response proteins. Principal Findings Here we show that valproate modulates the ER stress response through the regulation of WFS1, an important component for mitigating ER stress. Therapeutic concentrations of valproate induce expression of WFS1 mRNA and activate the WFS1 promoter. In addition, WFS1 forms a complex with GRP94, an ER stress-response protein, in which valproate dose-dependently enhances its dissociation from GRP94. Conclusions These results suggest that the therapeutic effects of valproate in bipolar disorder may be mediated by WFS1 expression and its dissociation from GRP94.

Effect of mood stabilizers on gene expression in lymphoblastoid cells

Lithium and valproate are widely used as effective mood stabilizers for the treatment of bipolar disorder. To elucidate the common molecular effect of these drugs on non-neuronal cells, we studied the gene expression changes induced by these drugs. Lymphoblastoid cell cultures derived from lymphocytes harvested from three healthy subjects were incubated in medium containing therapeutic concentrations of lithium (0.75xa0mM) or valproate (100xa0μgxa0ml−1) for 7xa0days. Gene expression profiling was performed using an Affymetrix HGU95Av2 array containing approximately 12,000 probe sets. We identified 44 and 416 genes that were regulated by lithium and valproate, respectively. Most of the genes were not commonly affected by the two drugs. Among the 18 genes commonly altered by both drugs, vascular endothelial growth factor A (VEGFA), which is one of the VEGF gene isoforms, showed the largest downregulation. Our findings indicate that these two structurally dissimilar mood stabilizers, lithium, and valproate, alter VEGFA expression. VEGFA might be a useful biomarker of their effects on peripheral tissue.

Genome-wide Association Study of Autism Spectrum Disorder in the East Asian Populations.

Autism spectrum disorder is a heterogeneous neurodevelopmental disorder with strong genetic basis. To identify common genetic variations conferring the risk of ASD, we performed a two‐stage genome‐wide association study using ASD family and healthy control samples obtained from East Asian populations. A total of 166 ASD families (nu2009=u2009500) and 642 healthy controls from the Japanese population were used as the discovery cohort. Approximately 900,000 single nucleotide polymorphisms (SNPs) were genotyped using Affymetrix Genome‐Wide Human SNP array 6.0 chips. In the replication stage, 205 Japanese ASD cases and 184 healthy controls, as well as 418 Chinese Han trios (nu2009=u20091,254), were genotyped by TaqMan platform. Case–control analysis, family based association test, and transmission/disequilibrium test (TDT) were then conducted to test the association. In the discovery stage, significant associations were suggested for 14 loci, including 5 known ASD candidate genes: GPC6, JARID2, YTHDC2, CNTN4, and CSMD1. In addition, significant associations were identified for several novel genes with intriguing functions, such as JPH3, PTPRD, CUX1, and RIT2. After a meta‐analysis combining the Japanese replication samples, the strongest signal was found at rs16976358 (Pu2009=u20096.04 × 10−7), which is located near the RIT2 gene. In summary, our results provide independent support to known ASD candidate genes and highlight a number of novel genes warranted to be further investigated in a larger sample set in an effort to improve our understanding of the genetic basis of ASD. Autism Res 2016, 9: 340–349.