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In preparation for a collaborative multidisciplinary study of the pathogenesis of esophageal cancer, the authors reviewed the published literature to identify similarities and differences between Japan and China in esophageal cancer epidemiology. Esophageal squamous cell carcinoma (ESCC) is the predominant histologic type, while the incidence of esophageal adenocarcinoma remains extremely low in both countries. Numerous epidemiologic studies in both countries show that alcohol consumption and cigarette smoking are contributing risk factors for ESCC. There are differences, however, in many aspects of esophageal cancer between Japan and China, including cancer burden, patterns of incidence and mortality, sex ratio of mortality, risk factor profiles, and genetic variants. Overall incidence and mortality rates are higher in China than in Japan, and variation in mortality and incidence patterns is greater in China than in Japan. During the study period (1987-2000), the decline in age-adjusted mortality rates was more apparent in China than in Japan. Risk factor profiles differed between high- and low-incidence areas within China, but not in Japan. The association of smoking and drinking with ESCC risk appears to be weaker in China than in Japan. Genome-wide association studies in China showed that variants in several chromosome regions conferred increased risk, but only genetic variants in alcohol-metabolizing genes were significantly associated with ESCC risk in Japan. A well-designed multidisciplinary epidemiologic study is needed to examine the role of diet and eating habits in ESCC risk. [PUBLICATION ABSTRACT]

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.

Bipolar disorder (BD) is a global medical issue, afflicting around 1% of the population with manic and depressive episodes. Despite various genetic studies, the genetic architecture and pathogenesis of BD have not been fully resolved. Besides germline variants, postzygotic mosaic variants are proposed as new candidate mechanisms contributing to BD. Here, we performed extensive deep exome sequencing (DES, ~300×) and validation experiments to investigate the roles of mosaic variants in BD with 235 BD cases (194 probands of trios and 41 single cases) and 39 controls. We found an enrichment of developmental disorder (DD) genes in the genes hit by deleterious mosaic variants in BD ( P  = 0.000552), including a ClinVar-registered pathogenic variant in ARID2 . An enrichment of deleterious mosaic variants was also observed for autism spectrum disorder (ASD) genes ( P  = 0.000428). The proteins coded by the DD/ASD genes with non-synonymous mosaic variants in BD form more protein-protein interaction than expected, suggesting molecular mechanisms shared with DD/ASD but restricted to a subset of cells in BD. We also found significant enrichment of mitochondrial heteroplasmic variants, another class of mosaic variants, in mitochondrial tRNA genes in BD ( P  = 0.0102). Among them, recurrent m.3243 A > G variants known as causal for mitochondrial diseases were found in two unrelated BD probands with allele fractions of 5–12%, lower than in mitochondrial diseases. Despite the limitation of using peripheral tissues, our DES investigation supports the possible contribution of deleterious mosaic variants in the nuclear genome responsible for severer phenotypes, such as DD/ASD, to the risk of BD and further demonstrates that the same paradigm can be applied to the mitochondrial genome. These results, as well as the enrichment of heteroplasmic mitochondrial tRNA variants in BD, add a new piece to the understanding of the genetic architecture of BD and provide general insights into the pathological roles of mosaic variants in human diseases.

Paenibacillus thermoaerophilus strain TC22-2b, a thermophilic bacterium with an optimum growth temperature of 50–55 °C isolated from compost (55 °C) in Japan, secreted a chitinase into culture medium in the presence of colloidal chitin. Adding glucose, lactose, mannose, or sucrose to culture medium decreased the amount of chitinase in culture supernatants. This chitinase was purified by ammonium sulfate precipitation, colloidal chitin adsorption, and ion exchange chromatography. The apparent molecular mass of this enzyme was approximately 48 kDa, and its N-terminal amino acid sequence was determined to be Ala–Val–Ser–Thr–Gly–Lys–Lys. The optimum temperature and pH for chitinase activity were 60 °C and pH 4, respectively. The chitinase retained 68 % of its initial activity after incubation at 50 °C for 2 h. Using p-nitrophenyl N,N′-diacetyl-β-D-chitobioside [pNP-(GlcNAc)₂] as a substrate, the Kₘ, Vₘₐₓ, and kcₐₜvalues for this enzyme were 1.4 mM, 0.058 mM min⁻¹, and 9.6 s⁻¹, respectively. Analysis of hydrolysis products showed that the chitinase digested N-acetyl-chitooligosaccharides in an endo manner. N-acetylglucosamine dimers were not degraded by the enzyme. When colloidal chitin was used as the substrate, N-acetylglucosamine dimers, -trimers, and -tetramers were detected as hydrolysis products. Thus, the thermophilic chitinase may prove useful for industrial applications in chitooligosaccharide production from chitin biomass.

Recent studies have consistently demonstrated that the regulation of chromatin and gene transcription plays a pivotal role in the pathogenesis of neurodevelopmental disorders. Among many genes involved in these pathways, KMT2C , encoding one of the six known histone H3 lysine 4 (H3K4) methyltransferases in humans and rodents, was identified as a gene whose heterozygous loss-of-function variants are causally associated with autism spectrum disorder (ASD) and the Kleefstra syndrome phenotypic spectrum. However, little is known about how KMT2C haploinsufficiency causes neurodevelopmental deficits and how these conditions can be treated. To address this, we developed and analyzed genetically engineered mice with a heterozygous frameshift mutation of Kmt2c ( Kmt2c +/fs mice) as a disease model with high etiological validity. In a series of behavioral analyses, the mutant mice exhibit autistic-like behaviors such as impairments in sociality, flexibility, and working memory, demonstrating their face validity as an ASD model. To investigate the molecular basis of the observed abnormalities, we performed a transcriptomic analysis of their bulk adult brains and found that ASD risk genes were specifically enriched in the upregulated differentially expressed genes (DEGs), whereas KMT2C peaks detected by ChIP-seq were significantly co-localized with the downregulated genes, suggesting an important role of putative indirect effects of Kmt2c haploinsufficiency. We further performed single-cell RNA sequencing of newborn mouse brains to obtain cell type-resolved insights at an earlier stage. By integrating findings from ASD exome sequencing, genome-wide association, and postmortem brain studies to characterize DEGs in each cell cluster, we found strong ASD-associated transcriptomic changes in radial glia and immature neurons with no obvious bias toward upregulated or downregulated DEGs. On the other hand, there was no significant gross change in the cellular composition. Lastly, we explored potential therapeutic agents and demonstrate that vafidemstat, a lysine-specific histone demethylase 1 (LSD1) inhibitor that was effective in other models of neuropsychiatric/neurodevelopmental disorders, ameliorates impairments in sociality but not working memory in adult Kmt2c +/fs mice. Intriguingly, the administration of vafidemstat was shown to alter the vast majority of DEGs in the direction to normalize the transcriptomic abnormalities in the mutant mice (94.3 and 82.5% of the significant upregulated and downregulated DEGs, respectively, P  < 2.2 × 10 −16 , binomial test), which could be the molecular mechanism underlying the behavioral rescuing. In summary, our study expands the repertoire of ASD models with high etiological and face validity, elucidates the cell-type resolved molecular alterations due to Kmt2c haploinsufficiency, and demonstrates the efficacy of an LSD1 inhibitor that might be generalizable to multiple categories of psychiatric disorders along with a better understanding of its presumed mechanisms of action.

Background: A number of noninvasive diagnostic tests are available to detect Helicobacter pylori infection. Data on serologic testing of children are lacking, however, and thus it remains unclear whether the serology cutoff points used for adults are appropriate for children. Methods: Serum and stool samples were obtained from 73 children who visited 5 hospitals in Japan between March 1993 and December 2009. Analysis of stool samples was carried out using an H pylori stool antigen enzyme-linked immunosorbent assay (HpSA ELISA), and serum antibodies to H pylori were examined using an antibody determination kit (E-Plate Eiken H pylori antibody). The validity of the serologic test was evaluated based on its sensitivity, specificity, and receiver operating characteristics curve. Results: Of the 73 children included in this study, 34 were HpSA-positive and 39 were negative. Among the 34 HpSA-positive patients, 32 were IgG-positive and 2 were IgG-negative. Of the 39 patients who were HpSA-negative, 38 were IgG-negative and 1 was IgG-positive. The sensitivity, specificity, and positive likelihood ratio for IgG antibody testing were 91.2%, 97.4%, and 35.6, respectively, based on the recommended adult cutoff point of 10 U/ml. Among children, use of cutoff points in the range of 7 to 9 U/ml yielded optimal values for sensitivity and specificity, as well as a positive likelihood ratio. Conclusions: The performance of the E-plate anti-H pylori IgG antibody test was comparable to that of the stool antigen test and is therefore suitable for epidemiologic studies of H pylori infection in large samples.

Unraveling the epigenetic status of neuronal cells in the brain is critical to our understanding of the pathophysiology of psychiatric disorders, which may reflect a complex interaction between genetic and environmental factors. Several epigenetic studies of mood disorders have been conducted with postmortem brains. However, proper interpretation of the results is hampered by our scant understanding of the effects of mood stabilizers on the epigenetic status of neuronal cells. We performed both comprehensive and gene-specific analyses to examine DNA methylation in human neuroblastoma SK-N-SH cells treated with three mood stabilizers: lithium, valproate and carbamazepine. Measurement of the level of DNA methylation of about 27 000 CpG sites revealed a profound epigenetic effect of lithium, compared with the two other mood stabilizers. In addition, we found that the mood stabilizers have common epigenetic targets and a propensity to increase DNA methylation. Gene-specific analysis involved detailed analysis of the methylation of promoter regions of SLC6A4 and BDNF, both of which have been reported to show altered DNA methylation in bipolar disorder patients or suicide victims, by extensive bisulfite sequencing. We did not observe significant changes in DNA methylation at BDNF promoter IV. However, we found that CpG sites of SLC6A4, which were hypermethylated in patients with bipolar disorder, were hypomethylated in the neuroblastoma cells treated with mood stabilizers. Our results will contribute to a better understanding of the epigenetic changes associated with mood disorders, and they also provide new insight into the mechanisms of action of mood stabilizers.

Background It is clear that genetic variations in the fat mass and obesity-associated (FTO) gene affect body mass index and the risk of obesity. Given the mounting evidence showing a positive association between obesity and pancreatic cancer, this study aimed to investigate the relation between variants in the FTO gene, obesity and pancreatic cancer risk. Methods We conducted a hospital-based case–control study in Japan to investigate whether genetic variations in the FTO gene were associated with pancreatic cancer risk. We genotyped rs9939609 in the FTO gene of 360 cases and 400 control subjects. An unconditional logistic model was used to estimate the odds ratio (OR) and 95% confidence interval (CI) for the association between rs9939609 and pancreatic cancer risk. Results The minor allele frequency of rs9939609 was 0.18 among control subjects. BMI was not associated with pancreatic cancer risk. Compared with individuals with the common homozygous TT genotype, those with the heterozygous TA genotype and the minor homozygous AA genotype had a 48% (OR=1.48; 95%CI: 1.07–2.04), and 66% increased risk (OR=1.66; 95%CI: 0.70–3.90), respectively, of pancreatic cancer after adjustment for sex, age, body mass index, cigarette smoking and history of diabetes. The per-allele OR was 1.41 (95%CI: 1.07–1.85). There were no significant interactions between TA/AA genotypes and body mass index. Conclusions Our findings indicate that rs9939609 in the FTO gene is associated with pancreatic cancer risk in Japanese subjects, possibly through a mechanism that is independent of obesity. Further investigation and replication of our results is required in other independent samples.

Bipolar disorder (BD) is a severe psychiatric disorder characterized by repeated conflicting manic and depressive states. In addition to genetic factors, complex gene–environment interactions, which alter the epigenetic status in the brain, contribute to the etiology and pathophysiology of BD. Here, we performed a promoter-wide DNA methylation analysis of neurons and nonneurons derived from the frontal cortices of mutant Polg1 transgenic (n = 6) and wild-type mice (n = 6). The mutant mice expressed a proofreading-deficient mitochondrial DNA (mtDNA) polymerase under the neuron-specific CamK2a promoter and showed BD-like behavioral abnormalities, such as activity changes and altered circadian rhythms. We identified a total of 469 differentially methylated regions (DMRs), consisting of 267 neuronal and 202 nonneuronal DMRs. Gene ontology analysis of DMR-associated genes showed that cell cycle-, cell division-, and inhibition of peptide activity-related genes were enriched in neurons, whereas synapse- and GABA-related genes were enriched in nonneurons. Among the DMR-associated genes, Trim2 and Lrpprc showed an inverse relationship between DNA methylation and gene expression status. In addition, we observed that mutant Polg1 transgenic mice shared several features of DNA methylation changes in postmortem brains of patients with BD, such as dominant hypomethylation changes in neurons, which include hypomethylation of the molecular motor gene and altered DNA methylation of synapse-related genes in nonneurons. Taken together, the DMRs identified in this study will contribute to understanding the pathophysiology of BD from an epigenetic perspective.