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Psychiatric disorders such as schizophrenia and bipolar disorder are caused by complex gene–environment interactions. While recent advances in genomic technologies have enabled the identification of several risk variants for psychiatric conditions, including single-nucleotide variants and copy-number variations, these factors can explain only a portion of the liability to these disorders. Although non-inherited factors had previously been attributed to environmental causes, recent genomic analyses have demonstrated that de novo mutations are among the main non-inherited risk factors for several psychiatric conditions. Somatic mutations in the brain may also explain how stochastic developmental events and environmental insults confer risk for a psychiatric disorder following fertilization. Here, we review evidence regarding somatic mutations in the brains of individuals with and without neuropsychiatric diseases. We further discuss the potential biological mechanisms underlying somatic mutations in the brain as well as the technical issues associated with the detection of somatic mutations in psychiatric research.

Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor, which is important for neuronal survival, development and synaptic plasticity. Accumulating evidence suggests that epigenetic modifications of BDNF are associated with the pathophysiology of psychiatric disorders, such as schizophrenia and mood disorders. Patients with psychiatric disorders generally show decreased neural BDNF levels, which are often associated with increased DNA methylation at the specific BDNF promoters. Importantly, observed DNA methylation changes are consistent across tissues including brain and peripheral blood, which suggests potential usefulness of these findings as a biomarker of psychiatric disorders. Here we review DNA methylation characteristics of BDNF promoters of cellular, animal and clinical samples and discuss future perspectives.

Bipolar disorder (BD) is a severe mental disorder characterized by repeated mood swings. Although genetic factors are collectively associated with the etiology of BD, the underlying molecular mechanisms, particularly how environmental factors affect the brain, remain largely unknown. We performed promoter-wide DNA methylation analysis of neuronal and nonneuronal nuclei in the prefrontal cortex of patients with BD (N = 34) and controls (N = 35). We found decreased DNA methylation at promoters in both cell types in the BD patients. Gene Ontology (GO) analysis of differentially methylated region (DMR)-associated genes revealed enrichment of molecular motor-related genes in neurons, chemokines in both cell types, and ion channel- and transporter-related genes in nonneurons. Detailed GO analysis further revealed that growth cone- and dendrite-related genes, including NTRK2 and GRIN1, were hypermethylated in neurons of BD patients. To assess the effect of medication, neuroblastoma cells were cultured under therapeutic concentrations of three mood stabilizers. We observed that up to 37.9% of DMRs detected in BD overlapped with mood stabilizer-induced DMRs. Interestingly, mood stabilizer-induced DMRs showed the opposite direction of changes in DMRs, suggesting the therapeutic effects of mood stabilizers. Among the DMRs, 12 overlapped with loci identified in a genome-wide association study (GWAS) of BD. We also found significant enrichment of neuronal DMRs in the loci reported in another GWAS of BD. Finally, we performed qPCR of DNA methylation-related genes and found that DNMT3B was overexpressed in BD. The cell-type-specific DMRs identified in this study will be useful for understanding the pathophysiology of BD.

The long interspersed nuclear element‐1 (LINE‐1) retrotransposons are a major family of mobile genetic elements, comprising approximately 17% of the human genome. The methylation state of LINE‐1 is often used as an indicator of global DNA methylation levels and it regulates the retrotransposition and somatic insertion of the genetic element. We have previously reported the significant relationship between LINE‐1 hypomethylation and poor prognosis in upper gastrointestinal (GI) cancers. However, the causal relationships between LINE‐1 hypomethylation, retrotransposition, and tumor‐specific insertion in upper GI cancers remain unknown. We used bisulfite‐pyrosequencing and quantitative real‐time PCR to verify LINE‐1 methylation and copy number in tissue samples of 101 patients with esophageal and 103 patients with gastric cancer. Furthermore, we analyzed the LINE‐1 retrotransposition profile with an originally developed L1Hs‐seq. In tumor samples, LINE‐1 methylation levels were significantly lower than non‐tumor controls, while LINE‐1 copy numbers were markedly increased. As such, there was a significant inverse correlation between the LINE‐1 methylation level and copy number in tumor tissues, with lower LINE‐1 methylation levels corresponding to higher LINE‐1 copy numbers. Of particular importance is that somatic LINE‐1 insertions were more numerous in tumor than normal tissues. Furthermore, we observed that LINE‐1 was inserted evenly across all chromosomes, and most often within genomic regions associated with tumor‐suppressive genes. LINE‐1 hypomethylation in upper GI cancers is related to increased LINE‐1 retrotransposition and tumor‐specific insertion events, which may collectively contribute to the acquisition of aggressive tumor features through the inactivation of tumor‐suppressive genes. Methylation of long interspersed nuclear element‐1 (LINE‐1) is indicative of global DNA methylation level and regulates LINE‐1 retrotransposition and somatic insertion. We have previously reported the significant relationship between LINE‐1 hypomethylation and poor prognosis in upper gastrointestinal (GI) cancers. LINE‐1 hypomethylation in upper GI cancers was related to increased LINE‐1 retrotransposition and tumor‐specific insertion events, which might collectively contribute to the acquisition of aggressive tumor features through the inactivation of tumor‐suppressive genes.

Epidemiological studies have revealed that schizophrenia is highly heritable. However, genetic studies have not fully elucidated its etiology. Accumulating evidence suggests that epigenetic alterations may provide an additional explanation of its pathophysiology. We investigated the methylation profiles of DNA in peripheral blood cells from 18 patients with first-episode schizophrenia (FESZ) and from 15 normal controls. Schizophrenia patients were confined to those at the stage of first-episode psychosis. We analyzed the DNA methylation status of 27,578 CpG sites by means of the Illumina Infinium HumanMethylation27 BeadChip array. Differentially methylated CpG sites, which were particularly abundant within CpG islands, were enriched in genes related to the nuclear lumen, to transcription factor binding, and to nucleotide binding. We also observed differential methylation of the promoters of HTR1E and COMTD1, which are functionally related to genes found to be differentially methylated in schizophrenia patients in previous studies. Our results indicate the site-specific epigenetic alterations in patients with FESZ.

Serotonin-transporter-linked polymorphic region (5-HTTLPR), a variable number of tandem repeats in the promoter region of serotonin transporter gene, is classified into short (S) and long (L) alleles. Initial case-control association studies claiming the risks of the S allele in depression and anxiety were not completely supported by recent studies. However, most studies, especially those on East Asian populations, have overlooked the complexity of 5-HTTLPR, which involves multiple different alleles with distinct functional properties. To address this issue, distinguishing multiple 5-HTTLPR alleles is essential. Here, using the 5-HTTLPR genotypes previously determined by exhaustive Sanger sequencing of approximately 1,500 Japanese subjects and their comprehensive SNP data, we constructed a method for 5-HTTLPR genotype imputation. We identified 28 tag SNPs for the imputation of four major 5-HTTLPR alleles, which collectively account for 97.6% of 5-HTTLPR alleles in the Japanese population. Our imputation method, achieved an accuracy of 0.872 in cross-validation, will contribute to association analysis of 5-HTTLPR in the Japanese subjects.

Accumulating evidence suggests that the epigenetic alterations induced by antipsychotics contribute to the therapeutic efficacy. However, global and site-specific epigenetic changes by antipsychotics and those shared by different classes of antipsychotics remain poorly understood. We conducted a comprehensive DNA methylation analysis of human neuroblastoma cells cultured with antipsychotics. The cells were cultured with low and high concentrations of haloperidol or risperidone for 8 days. DNA methylation assay was performed with the Illumina HumanMethylation450 BeadChip. We found that both haloperidol and risperidone tended to cause hypermethylation changes and showed similar DNA methylation changes closely related to neuronal functions. A total of 294 differentially methylated probes (DMPs), including 197 hypermethylated and 97 hypomethylated DMPs, were identified with both haloperidol and risperidone treatment. Gene ontology analysis of the hypermethylated probe-associated genes showed enrichment of genes related to the regulation of neurotransmitter receptor activity and lipoprotein lipase activity. Pathway analysis identified that among the DMP-associated genes, SHANK1 and SHANK2 were the major genes in the neuropsychiatric disorder-related pathways. Our data would be valuable for understanding the mechanisms of action of antipsychotics from an epigenetic viewpoint.

Quantifying cytosine modifications in various brain regions provides important insights into the gene expression regulation and pathophysiology of neuropsychiatric disorders. In this study, we quantified 5‐methylcytosine (5‐mC), 5‐hydroxymethylation (5‐hmC), and 5‐formylcytosine (5‐fC) levels in five brain regions (the frontal lobe, cerebral cortical region without frontal lobe, hippocampus, basal ganglia, and the cerebellum) and the heart at three developmental periods (12, 48, and 101 weeks). We observed significant regional variations in cytosine modification. Notably, regional variations were generally maintained throughout development, suggesting that epigenetic regulation is unique to each brain region and remains relatively stable with age. The 5‐mC and 5‐hmC levels were positively correlated, although the extent of the correlations seemed to differ in different brain regions. On the contrary, 5‐fC levels did not correlate with 5‐mC or 5‐hmC levels. Additionally, we observed an age‐dependent decrease in 5‐fC levels in the basal ganglia, suggesting a unique epigenetic regulation mechanism. Further high‐resolution studies using animal models of neuropsychiatric disorders as well as postmortem brain evaluation are warranted.

Introduction Epigenetic information such as DNA methylation is a useful biomarker that reflects complex gene‐environmental interaction. Peripheral tissues such as blood and saliva are commonly collected as the source of genomic DNA in cohort studies. Epigenetic studies mainly use blood, while a few studies have addressed the epigenetic characteristics of saliva. Methods The effects of methods for DNA extraction and purification from saliva on DNA methylation were surveyed using Illumina Infinium HumanMethylation450 BeadChip. Using 386 661 probes, DNA methylation differences between blood and saliva from 22 healthy volunteers, and their functional and structural characteristics were examined. CpG sites with DNA methylation levels showing large interindividual variations in blood were evaluated using saliva DNA methylation profiles. Results Genomic DNA prepared by simplified protocol from saliva showed a similar quality DNA methylation profile to that derived from the manufacturer provided protocol. Consistent with previous studies, the DNA methylation profiles of blood and saliva showed high correlations. Blood showed 1,514 hypomethylated and 2099 hypermethylated probes, suggesting source‐dependent DNA methylation patterns. CpG sites with large methylation difference between the two sources were underrepresented in the promoter regions and enriched within gene bodies. CpG sites with large interindividual methylation variations in blood also showed considerable variations in saliva. Conclusion In addition to high correlation in DNA methylation profiles, CpG sites showing large interindividual DNA methylation differences were similar between blood and saliva, ensuring saliva could be a suitable alternative source for genomic DNA in cohort studies. Consideration of source‐dependent DNA methylation differences will, however, be necessary. We compared quality of saliva methylome data collected by several DNA purification protocols and examined the characteristics of saliva methylome. Optimized protocol and identified characteristics such as common informative CpG sites to blood and unique epigenetic changes in saliva will contribute to promote the use of saliva for epigenetic studies in clinical settings and epidemiological cohort studies.

SLC6A4, which encodes the serotonin transporter, has a functional polymorphism called the serotonin transporter-linked polymorphic region (5-HTTLPR). The 5-HTTLPR consists of short (S) and long (L) alleles, each of which has 14 or 16 tandem repeats. In addition, the extralong (XL) and other rare alleles have been reported in 5-HTTLPR. Although they are more frequent in Asian and African than in other populations, the extent of variations and allele frequencies (AFs) were not addressed in a large population. Here, we report the AFs of the rare alleles in a large number of Japanese subjects (N = 2894) consisting of two cohorts. The first cohort (case-control study set, CCSS) consisted of 1366 subjects, including 485 controls and 881 patients with psychosis (bipolar disorder or schizophrenia). The second cohort (the Arao cohort study set, ACSS) consisted of 1528 elderly subjects. During genotyping, we identified 11 novel 5-HTTLPR alleles, including 3 XL alleles. One novel allele had the longest subunit ever reported, consisting of 28 tandem repeats. We named this XL28-A. An in vitro luciferase assay revealed that XL28-A has no transcriptional activity. XL28-A was found in two unrelated patients with bipolar disorder in the CCSS and one healthy subject in the ACSS who did not show depressive symptoms or a decline in cognitive function. Therefore, it is unlikely that XL28-A is associated with psychiatric disorders, despite its apparent functional deficit. Our results suggest that unraveling the complex genetic variations of 5-HTTLPR will be important for further understanding its role in psychiatric disorders.