An-a Kazuno

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.

Association of Mitochondrial Complex I Subunit Gene NDUFV2 at 18p11 with Bipolar Disorder in Japanese and the National Institute of Mental Health Pedigrees

BACKGROUND Linkage with 18p11 is one of the replicated findings in molecular genetics of bipolar disorder. Because mitochondrial dysfunction has been suggested in bipolar disorder, NDUFV2 at 18p11, encoding a subunit of the complex I, reduced nicotinamide adenine dinucleotide (NADH)ubiquinone oxidoreductase, is a candidate gene for this disorder. We previously reported that a polymorphism in the upstream region of NDUFV2, -602G> A, was associated with bipolar disorder in Japanese subjects; however, functional significance of -602G> A was not known. METHODS We screened the further upstream region of NDUFV2. We performed a case-control study in Japanese patients with bipolar disorder and control subjects and a transmission disequilibrium test in 104 parent and proband trios of the National Institute of Mental Health (NIMH) Genetics Initiative pedigrees. We also performed the promoter assay to examine functional consequence of the -602G> A polymorphism. RESULTS The -602G> A polymorphism was found to alter the promoter activity. We found that the other haplotype block surrounding -3542G> A was associated with bipolar disorder. The association of the haplotypes consisting of -602G> A and -3542G> A polymorphisms with bipolar disorder was seen both in Japanese case-control samples and NIMH trios. CONCLUSION Together these findings indicate that the polymorphisms in the promoter region of NDUFV2 are a genetic risk factor for bipolar disorder by affecting promoter activity.

Exome sequencing for bipolar disorder points to roles of de novo loss-of-function and protein-altering mutations

Although numerous genetic studies have been conducted for bipolar disorder (BD), its genetic architecture remains elusive. Here we perform, to the best of our knowledge, the first trio-based exome sequencing study for BD to investigate potential roles of de novo mutations in the disease etiology. We identified 71 de novo point mutations and one de novo copy-number mutation in 79 BD probands. Among the genes hit by de novo loss-of-function (LOF; nonsense, splice site or frameshift) or protein-altering (LOF, missense and inframe indel) mutations, we found significant enrichment of genes highly intolerant (first percentile of intolerant genes assessed by Residual Variation Intolerance Score) to protein-altering variants in general population, an observation that is also reported in autism and schizophrenia. When we performed a joint analysis using the data of schizoaffective disorder in published studies, we found global enrichment of de novo LOF and protein-altering mutations in the combined group of bipolar I and schizoaffective disorders. Considering relationship between de novo mutations and clinical phenotypes, we observed significantly earlier disease onset among the BD probands with de novo protein-altering mutations when compared with non-carriers. Gene ontology enrichment analysis of genes hit by de novo protein-altering mutations in bipolar I and schizoaffective disorders did not identify any significant enrichment. These results of exploratory analyses collectively point to the roles of de novo LOF and protein-altering mutations in the etiology of bipolar disorder and warrant further large-scale studies.

Exome sequencing in the knockin mice generated using the CRISPR/Cas system.

Knockin (KI) mouse carrying a point mutation has been an invaluable tool for disease modeling and analysis. Genome editing technologies using the CRISPR/Cas system has emerged as an alternative way to create KI mice. However, if the mice carry nucleotide insertions and/or deletions (InDels) in other genes, which could have unintentionally occurred during the establishment of the KI mouse line and potentially have larger impact than a point mutation, it would confound phenotyping of the KI mice. In this study, we performed whole exome sequencing of multiple lines of F1 heterozygous Ntrk1 KI mice generated using the CRISPR/Cas system in comparison to that of a wild-type mouse used as a control. We found three InDels in four KI mice but not in a control mouse. In vitro digestion assay suggested that each InDel occurred as a de novo mutation, was carried-over from the parental mice, or was incorporated through the Cas9 nuclease mediated off-target cleavage. These results suggest that frequency of InDels found in KI mice generated by the CRISPR/Cas technology is not high, but cannot be neglected and careful assessment of these mutations is warranted.

Mitochondrial DNA-dependent effects of valproate on mitochondrial calcium levels in transmitochondrial cybrids

Calcium plays important roles in various cellular processes. Using transmitochondrial hybrid cells (cybrids) carrying fluorescent calcium indicators, we previously found two mitochondrial DNA (mtDNA) polymorphism sites, 8701 and 10398, that alter intracellular calcium signalling and mitochondrial pH. The 10398A polymorphism is reportedly associated with bipolar disorder, Parkinsons disease, Alzheimers disease, and cancer, whereas 10398G is associated with longevity. In bipolar disorder, elevation of intracellular calcium levels in the platelets and lymphocytes is a well-replicated finding. Thus, we examined whether two mood stabilizers, lithium and valproate, affect the intracellular calcium signalling in cybrids with these mtDNA polymorphisms. After cybrids with 8701A/10398A and 8701G/10398G (three cell lines for each) derived from healthy controls were pretreated with lithium (0.75 mm or 1.5 mm) or valproate (0.6 mm or 1.2 mm) for 7 d, they were stimulated by 10 mum histamine. Valproate decreased mitochondrial calcium levels, compared with untreated cybrids, only in cybrids with 8701A/10398A. Moreover, valproate decreased cytosolic calcium levels at plateau after stimulation in cybrids with 8701A/10398A. These finding suggest that valproate may stabilize intracellular calcium only in cells with high mitochondrial calcium levels.

Proteomic Analysis of Lymphoblastoid Cells Derived from Monozygotic Twins Discordant for Bipolar Disorder: A Preliminary Study

Bipolar disorder is a severe mental illness characterized by recurrent manic and depressive episodes. In bipolar disorder, family and twin studies suggest contributions from genetic and environmental factors; however, the detailed molecular pathogenesis is yet unknown. Thus, identification of biomarkers may contribute to the clinical diagnosis of bipolar disorder. Monozygotic twins discordant for bipolar disorder are relatively rare but have been reported. Here we performed a comparative proteomic analysis of whole cell lysate derived from lymphoblastoid cells of monozygotic twins discordant for bipolar disorder by using two-dimensional differential in-gel electrophoresis (2D-DIGE). We found approximately 200 protein spots to be significantly differentially expressed between the patient and the co-twin (t test, p<0.05). Some of the proteins were subsequently identified by liquid chromatography tandem mass spectrometry and included proteins involved in cell death and glycolysis. To examine whether these proteins could serve as biomarkers of bipolar disorder, we performed Western blot analysis using case–control samples. Expression of phosphoglycerate mutase 1 (PGAM1), which is involved in glycolysis, was significantly up-regulated in patients with bipolar disorder (t test, p<0.05). Although PGAM1 cannot be regarded as a qualified biomarker of bipolar disorder from this preliminary finding, it could be one of the candidates for further study to identify biomarkers of bipolar disorder.

Mitochondrial DNA haplogroup analysis in patients with bipolar disorder.

Several lines of evidence support mitochondrial dysfunction in bipolar disorder. Elevated calcium level in platelets is reported in this disease. To verify mitochondrial DNA (mtDNA) haplogroups characteristic to bipolar disorder, we sequenced mtDNA of seven regions and performed haplogroup analysis in 195 patients with bipolar disorder and 255 controls. They belonged to 16 major mtDNA haplogroups, A, B4, B5, C, D4, D5, F, G, M7, M8, M9, M10‐12, N9a, N9b, Y, and Z. The logistic regression analysis revealed that the haplogroup N9a was over‐represented in bipolar disorder. We also performed a case–control study for two functional mtDNA polymorphisms, mtDNA5460G > A and 12358A > G, that altered intracellular calcium dynamics. While the mtDNA5460G > A polymorphism was not associated with bipolar disorder, the mtDNA12358A > G polymorphism was associated with bipolar disorder in 199 patients with bipolar disorder and 260 controls. However, this association was not replicated in an independent sample set. Possible significances of these findings are discussed.

Relationships between mitochondrial DNA subhaplogroups and intracellular calcium dynamics.

Although an association between mitochondrial DNA (mtDNA) subhaplogroups and complex traits has been suggested, few functional analyses have been reported. To identify the mtDNA subhaplogroups that alter intracellular calcium dynamics, we analysed data on intracellular calcium dynamics in 35 transmitochondrial hybrid cells (cybrids). One cybrid showing decreased calcium levels had mtDNA subhaplogroup G3 or G4, characterised by 1413T>C, 2109A>T, 3434A>G, 5460G>A, 7521G>A, 9011C>T, 9670A>G and 15940T>C. The cybrid having higher calcium levels was subhaplogroup D4a, characterised by a non-synonymous polymorphism, 13651A>G. These mtDNA subhaplogroups might have functional effects.

Loss of function mutations in ATP2A2 and psychoses: A case report and literature survey

Though genetic factors play a major role in the pathophysiology of psychoses including bipolar disorder (BD) and schizophrenia, lack of well‐established causative genetic mutations hampers their neurobiological studies. Dariers disease, an autosomal dominant skin disorder caused by mutations of ATP2A2 on chromosome 12q23‐24.1, encoding sarco/endoplasmic reticulum calcium transporting ATPase 2 (SERCA2), reportedly cosegregates with BD. A recent genome‐wide association study showed an association of schizophrenia with ATP2A2.

A 3-bp deletion of mitochondrial DNA tRNALys observed in lymphoblastoid cells.

During the course of our studies to sequence mitochondrial DNA (mtDNA) in patients with bipolar disorder and in healthy control subjects,1, 2 we found a 3-bp deletion, m.8348–8350, in a lymphoblastoid cell line (LCL) of a healthy Japanese man (49-years old). This deletion occurred in the region of mitochondrial tRNALys (Figure 1a). The deletion was not, however, detected in the lymphocyte DNA obtained from this subject (Figure 1a). Allele-specific polymerase chain reaction, which can detect a 0.1% mutation of total mtDNA, did not reveal the 3-bp deletion in the lymphocytes (Figure 1b). This finding suggests that the acquisition of this deletion occurred during formation of the LCL, although the possibility of a bottleneck effect cannot be ruled out.