Miki Bundo

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.

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.

Hypermethylation of serotonin transporter gene in bipolar disorder detected by epigenome analysis of discordant monozygotic twins

Bipolar disorder (BD) is a severe mental disorder characterized by recurrent episodes of mania and depression. Serotonin transporter (HTT) is a target of antidepressants and is one of the strongest candidate molecules of mood disorder, however, genetic study showed equivocal results. Here, we performed promoter-wide DNA methylation analysis of lymphoblastoid cell lines (LCLs) derived from two pairs of monozygotic twins discordant for BD. To rule out the possible discordance of copy number variation (CNV) between twins, we performed CNV analysis and found the copy number profiles were nearly identical between the twin pairs except for immunoglobulin-related regions. Among the three genes we obtained as candidate regions showing distinct difference of DNA methylation between one of the two pairs, hypermethylation of SLC6A4, encoding HTT, in the bipolar twin was only confirmed by bisulfite sequencing. Then, promoter hypermethylation of SLC6A4 in LCLs of BD patients was confirmed in a case–control analysis. DNA methylation of SLC6A4 was significantly correlated with its mRNA expression level in individuals with the S/S genotype of HTTLPR, and mRNA expression level was lower in BD patients carrying the S/S genotype. Finally, DNA methylation of the same site was also higher in the postmortem brains of BD patients. This is the first study to report the role of epigenetic modification of SLC6A4 in BD using an unbiased approach, which provides an insight for its pathophysiology.

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.

A family-based and case-control association study of SOX10 in schizophrenia.

Downregulation of oligodendrocyte‐related genes in postmortem brains of patients with schizophrenia has been reported by several DNA microarray studies. We recently reported that enhanced DNA methylation of SOX10, which encodes a transcription factor responsible for terminal differentiation of oligodendrocyte, correlated with lower expression of SOX10 and other oligodendrocyte‐related genes. Although we ruled out the possible role of SNPs of SOX10 in the altered expression and epigenetic status of oligodendrocyte genes by mutation screening of the SOX10 gene, it is not known whether its genetic polymorphisms contribute to susceptibility to schizophrenia. Here we performed a case‐control and family‐based association study of SOX10 in Japanese patients with schizophrenia using six SNPs and one microsatellite marker. None of these markers showed significant associations with schizophrenia by case‐control or family‐based association study. Haplotype analysis did not reveal significant associations between the two groups. We concluded that genetic variations in the SOX10 gene do not contribute to susceptibility to Japanese schizophrenia.

Epigenome-wide association study of DNA methylation in panic disorder

BackgroundPanic disorder (PD) is considered to be a multifactorial disorder emerging from interactions among multiple genetic and environmental factors. To date, although genetic studies reported several susceptibility genes with PD, few of them were replicated and the pathogenesis of PD remains to be clarified. Epigenetics is considered to play an important role in etiology of complex traits and diseases, and DNA methylation is one of the major forms of epigenetic modifications. In this study, we performed an epigenome-wide association study of PD using DNA methylation arrays so as to investigate the possibility that different levels of DNA methylation might be associated with PD.MethodsThe DNA methylation levels of CpG sites across the genome were examined with genomic DNA samples (PD, N = 48, control, N = 48) extracted from peripheral blood. Methylation arrays were used for the analysis. β values, which represent the levels of DNA methylation, were normalized via an appropriate pipeline. Then, β values were converted to M values via the logit transformation for epigenome-wide association study. The relationship between each DNA methylation site and PD was assessed by linear regression analysis with adjustments for the effects of leukocyte subsets.ResultsForty CpG sites showed significant association with PD at 5% FDR correction, though the differences of the DNA methylation levels were relatively small. Most of the significant CpG sites (37/40 CpG sites) were located in or around CpG islands. Many of the significant CpG sites (27/40 CpG sites) were located upstream of genes, and all such CpG sites with the exception of two were hypomethylated in PD subjects. A pathway analysis on the genes annotated to the significant CpG sites identified several pathways, including “positive regulation of lymphocyte activation.”ConclusionsAlthough future studies with larger number of samples are necessary to confirm the small DNA methylation abnormalities associated with PD, there is a possibility that several CpG sites might be associated, together as a group, with PD.

Effect of a functional single nucleotide polymorphism in the 2′,3′-cyclic nucleotide 3′-phosphodiesterase gene on the expression of oligodendrocyte-related genes in schizophrenia

Aims:  Although the expression of oligodendrocyte‐related genes in post‐mortem brains of patients with schizophrenia is consistently reported to be downregulated, the cause of the change remains unclear. The A‐allele of rs2070106 within the 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP), an oligodendrocyte‐related gene, was reported to show reduced expression compared with the G‐allele, and proposed to be associated with schizophrenia.

Evaluation of whole genome amplification methods using postmortem brain samples.

The importance of examining genomic DNA derived from human brain has been highlighted by recent findings such as the possible link between DNA methylation and behavior or mental disorders, as well as the possible genomic differences between neurons from the same individual caused by transposons and aneuploidy. Consequently, obtaining a sufficient amount of genomic DNA derived from human brain is a critical issue for further research. Whole genome amplification (WGA) methods, by which genomic DNA is typically amplified on the order of 10(4)-10(6), will be a valuable tool for providing a sufficient amount of DNA for various molecular genetic studies. Here we evaluated three methods, including both PCR-based and non-PCR based WGA, as well as DNA extraction methods using frozen postmortem brain tissue. We found that WGA products from postmortem brains can be used in molecular genetic analysis, if a particular protocol for DNA extraction is used, and the most appropriate method for WGA depends on the state of the genomic DNA to be amplified.