Kazuya Iwamoto

Aberrant DNA methylation associated with bipolar disorder identified from discordant monozygotic twins

To search DNA methylation difference between monozygotic twins discordant for bipolar disorder, we applied a comprehensive genome scan method, methylation-sensitive representational difference analysis (MS-RDA) to lymphoblastoid cells derived from the twins. MS-RDA isolated 10 DNA fragments derived from 5′ region of known genes/ESTs. Among these 10 regions, four regions showed DNA methylation differences between bipolar twin and control co-twin confirmed by bisulfite sequencing. We performed a case–control study of DNA methylation status of these four regions by pyrosequencing. Two regions, upstream regions of spermine synthase (SMS) and peptidylprolyl isomerase E-like (PPIEL) (CN265253), showed aberrant DNA methylation status in bipolar disorder. SMS, a gene on X chromosome, showed significantly higher DNA methylation level in female patients with bipolar disorder compared with control females. However, there was no difference of mRNA expression. In PPIEL, DNA methylation level was significantly lower in patients with bipolar II disorder than in controls. The expression level of PPIEL was significantly higher in bipolar II disorder than in controls. We found strong inverse correlation between gene expression and DNA methylation levels of PPIEL. These results suggest that altered DNA methylation statuses of PPIEL might have some significance in pathophysiology of bipolar disorder.

Methylation status of the reelin promoter region in the brain of schizophrenic patients.

BACKGROUND Hypermethylation of the reelin (RELN) promoter region and the reduced levels of its messenger RNA and protein have been implicated in the pathophysiology of schizophrenia. We intended a technical replication of recent studies that observed hypermethylation of CpG or CpNpG sites in the RELN promoter region in the brain of schizophrenic patients. METHODS The DNA methylation status of the promoter region of RELN was examined by using the pyrosequencing method in the prefrontal cortices of 14 patients with schizophrenia and 13 control subjects. RESULTS All of the CpG and two proposed CpNpG sites analyzed showed no detectable DNA methylation (< 5%) in both control subjects and patients with schizophrenia. No detectable DNA methylation was observed in both gray and white matter, excluding the possibility of cellular heterogeneity of start materials. CONCLUSIONS We did not confirm the hypermethylation of the RELN promoter region in the brains of schizophrenic patients, suggested in the previous studies.

Mitochondrial DNA 3243A>G Mutation and Increased Expression of LARS2 Gene in the Brains of Patients with Bipolar Disorder and Schizophrenia

BACKGROUND Accumulating evidence suggests mitochondrial dysfunction in bipolar disorder. Analyses of mitochondria-related genes using DNA microarray showed significantly increased LARS2 (mitochondrial leucyl-tRNA synthetase) in the postmortem prefrontal cortices of patients with bipolar disorder provided by the Stanley Foundation Brain Collection. LARS2 is a nuclear gene encoding the enzyme catalyzing the aminoacylation of mitochondrial tRNA(Leu). A well-studied mitochondrial DNA point mutation, 3243A>G, in the region of tRNA(Leu (UUR)), related with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), is known to decrease the efficiency of aminoacylation of tRNA(Leu (UUR)). METHODS The steady state level of LARS2 was examined in the transmitochondrial cybrids carrying 3243A>G. We examined the 3243A>G mutation in these brains using the peptide nucleic acid-clamped polymerase chain reaction restriction fragment length polymorphism method. RESULTS LARS2 was upregulated in the transmitochrondrial cybrids carrying 3243A>G. The 3243A>G was detected in the postmortem brains of two patients with bipolar disorder and one with schizophrenia. These patients also showed higher levels of the mutation in their livers and significantly higher gene expression of LARS2 compared with other subjects. CONCLUSIONS These results suggest that upregulation of LARS2 is a hallmark of 324A>G mutation. The accumulation of 3243A>G mutation in the brain may have a pathophysiologic role in bipolar disorder and schizophrenia.

Gene expression and association analyses of LIM (PDLIM5) in bipolar disorder and schizophrenia

We previously reported that expression level of LIM (ENH, PDLIM5) was significantly and commonly increased in the brains of patients with bipolar disorder, schizophrenia, and major depression. Expression of LIM was decreased in the lymphoblastoid cells derived from patients with bipolar disorders and schizophrenia. LIM protein reportedly plays an important role in linking protein kinase C with calcium channel. These findings suggested the role of LIM in the pathophysiology of bipolar disorder and schizophrenia. To further investigate the role of LIM in these mental disorders, we performed a replication study of gene expression analysis and performed genetic association studies. Upregulation of LIM was confirmed in the independent sample set obtained from Stanley Array Collection. No effect of sample pH or medication was observed. Genetic association study revealed the association of single nucleotide polymorphism (SNP)1 (rs10008257) with bipolar disorder. In an independent sample set, SNP2 (rs2433320) close to SNP1 was associated with bipolar disorder. In total samples, haplotype of these two SNPs was associated with bipolar disorder. No association was observed in case–control analysis and family-based association analysis in schizophrenia. These results suggest that SNPs in the upstream region of LIM may confer the genetic risk for bipolar disorder.

Genome-wide association study of schizophrenia in Japanese population.

Schizophrenia is a devastating neuropsychiatric disorder with genetically complex traits. Genetic variants should explain a considerable portion of the risk for schizophrenia, and genome-wide association study (GWAS) is a potentially powerful tool for identifying the risk variants that underlie the disease. Here, we report the results of a three-stage analysis of three independent cohorts consisting of a total of 2,535 samples from Japanese and Chinese populations for searching schizophrenia susceptibility genes using a GWAS approach. Firstly, we examined 115,770 single nucleotide polymorphisms (SNPs) in 120 patient-parents trio samples from Japanese schizophrenia pedigrees. In stage II, we evaluated 1,632 SNPs (1,159 SNPs of p<0.01 and 473 SNPs of p<0.05 that located in previously reported linkage regions). The second sample consisted of 1,012 case-control samples of Japanese origin. The most significant p value was obtained for the SNP in the ELAVL2 [(embryonic lethal, abnormal vision, Drosophila)-like 2] gene located on 9p21.3 (p = 0.00087). In stage III, we scrutinized the ELAVL2 gene by genotyping gene-centric tagSNPs in the third sample set of 293 family samples (1,163 individuals) of Chinese descent and the SNP in the gene showed a nominal association with schizophrenia in Chinese population (p = 0.026). The current data in Asian population would be helpful for deciphering ethnic diversity of schizophrenia etiology.

Behavioral and gene expression analyses of Wfs1 knockout mice as a possible animal model of mood disorder

Wolfram disease is a rare genetic disorder frequently accompanying depression and psychosis. Non-symptomatic mutation carriers also have higher rates of depression and suicide. Because WfS1, the causative gene of Wolfram disease, is located at 4p16, a linkage locus for bipolar disorder, mutations of WfS1 were suggested to be involved in the pathophysiology of bipolar disorder. In this study, we performed behavioral and gene expression analyses of Wfs1 knockout mice to assess the validity as an animal model of mood disorder. In addition, the distribution of Wfs1 protein was examined in mouse brain. Wfs1 knockout mice did not show abnormalities in circadian rhythm and periodic fluctuation of wheel-running activity. Behavioral analysis showed that Wfs1 knockout mice had retardation in emotionally triggered behavior, decreased social interaction, and altered behavioral despair depending on experimental conditions. Wfs1-like immunoreactivity in mouse brain showed a similar distribution pattern to that in rats, including several nuclei potentially relevant to the symptoms of mood disorders. Gene expression analysis showed down-regulation of Cdc42ep5 and Rnd1, both of which are related to Rho GTPase, which plays a role in dendrite development. These findings may be relevant to the mood disorder observed in patients with Wolfram disease.

Detection of Chromosomal Structural Alterations in Single Cells by SNP Arrays: A Systematic Survey of Amplification Bias and Optimized Workflow

Background In single-cell human genome analysis using whole-genome amplified product, a strong amplification bias involving allele dropout and preferential amplification hampers the quality of results. Using an oligonucleotide single nucleotide polymorphism (SNP) array, we systematically examined the nature of this amplification bias, including frequency, degree, and preference for genomic location, and we assessed the effects of this amplification bias on subsequent genotype and chromosomal copy number analyses. Methodology/Principal Findings We found a large variability in amplification bias among the amplified products obtained by multiple displacement amplification (MDA), and this bias had a severe effect on the genotype and chromosomal copy number analyses. We established optimal experimental conditions for pre-screening for high-quality amplified products, processing array data, and analyzing chromosomal structural alterations. Using this optimized protocol, we successfully detected previously unidentified chromosomal structural alterations in single cells from a lymphoblastoid cell line. These alterations were subsequently confirmed by karyotype analysis. In addition, we successfully obtained reproducible chromosomal copy number profiles of single cells from the cell line with a complex karyotype, indicating the applicability and potential of our optimized workflow. Conclusions/Significance Our results suggest that the quality of amplification products should be critically assessed before using them for genomic analyses. The method of MDA-based whole-genome amplification followed by SNP array analysis described here will be useful for exploring chromosomal alterations in single cells.

Comprehensive gene expression analysis in bipolar disorder.

Objective: To review recent findings by DNA microarray in bipolar disorder (BD). Method: A literature search was performed. Results: Comprehensive gene expression analysis in the brain, peripheral blood cells, and olfactory neuroepithelium would be a promising strategy for the research of BD. To date, alterations in glutamate receptors (GR), mitochondria-related genes, chaperone genes, oligodendrocyte genes, and markers of gamma amino butyric acidergic (GABAergic) neurons in postmortem brains are replicated by several different strategies. However, alterations in mitochondria-related genes are associated with agonal factors, sample pH, and effects of drugs. Analysis of blood cells showed altered endoplasmic reticulum stress pathway and other molecular cascades. Analysis of olfactory epithelium showed altered expression of genes associated with apoptosis. Conclusions: These findings warrant that comprehensive gene expression analysis by DNA microarray will be useful to identify the molecular cascades responsible for BD.

Epigenetic Profiling in Schizophrenia and Major Mental Disorders

Epigenetic alterations such as DNA methylation and histone modifications are important in the etiology or pathophysiology of mental disorders. Here, we review recent studies on the relationship between DNA methylation and major mental disorders. We will focus on the role of DNA methylation in postmitotic neurons, intra- and interindividual variations in DNA methylation, the possible involvement of methionine metabolism pathways, and candidate and genome-wide DNA methylation as they relate to mental disorders.

Therapeutic implications of down-regulation of cyclophilin D in bipolar disorder.

We previously reported that neuron-specific mutant Polg1 (mitochondrial DNA polymerase) transgenic (Tg) mice exhibited bipolar disorder (BD)-like phenotypes such as periodic activity change and altered circadian rhythm. In this study, we re-evaluated two datasets resulting from DNA microarray analysis to estimate a biological pathway associated with the disorder. The gene lists were derived from the comparison between post-mortem brains of BD patients and control subjects, and from the comparison between the brains of Tg and wild-type mice. Gene ontology analysis showed that 16 categories overlapped in the altered gene expression profiles of BD patients and the mouse model. In the brains of Tg mice, 33 genes showed similar changes in the frontal cortex and hippocampus compared to wild-type mice. Among the 33 genes, SFPQ and PPIF were differentially expressed in post-mortem brains of BD patients compared to control subjects. The only gene consistently down-regulated in both patients and the mouse model was PPIF, which encodes cyclophilin D (CypD), a component of the mitochondrial permeability transition pore. A blood-brain barrier-permeable CypD inhibitor significantly improved the abnormal behaviour of Tg mice at 40 mg/kg.d. These findings collectively suggest that CypD is a promising target for a new drug for BD.