👤 Kaito Suzuki

Liver X receptors (LXR) α and β are a family of nuclear receptors that regulate lipogenesis by controlling the expression of the genes involved in the synthesis of fatty acids. MID1IP1, which encodes MIG12, is a target gene of LXR. MIG12 induces fatty acid synthesis by stimulating the polymerization-mediated activation of acetyl-CoA carboxylase (ACC). Here, we show that LXR's activation stimulates ACC polymerization in HepG2 cells by increasing the expression of MIG12. A knockdown of MID1IP1 abrogated the stimulation completely. The mutations of MIG12's leucine-zipper domain reduced the interaction between MIG12 and ACC, thus decreasing the MIG12's capacity to stimulate ACC polymerization. These results indicate that LXR's activation stimulates lipogenesis not only through the induction of the genes encoding lipogenic enzymes but also through MIG12's stimulation of ACC polymerization. Show less

We aimed to determine the mechanism of epithelial-mesenchymal transition (EMT)-induced stemness in cancer cells. Cancer relapse and metastasis are caused by rare stem-like cells within tumors. Studies of stem cell reprogramming have linked Show less

Monogenic diabetes is clinically heterogeneous and differs from common forms of diabetes (type 1 and 2). We aimed to investigate the clinical usefulness of a comprehensive genetic testing system, comprised of targeted next-generation sequencing (NGS) with phenotype-driven bioinformatics analysis in patients with monogenic diabetes, which uses patient genotypic and phenotypic data to prioritize potentially causal variants. We performed targeted NGS of 383 genes associated with monogenic diabetes or common forms of diabetes in 13 Japanese patients with suspected (n = 10) or previously diagnosed (n = 3) monogenic diabetes or severe insulin resistance. We performed in silico structural analysis and phenotype-driven bioinformatics analysis of candidate variants from NGS data. Among the patients suspected having monogenic diabetes or insulin resistance, we diagnosed 3 patients as subtypes of monogenic diabetes due to disease-associated variants of INSR, LMNA, and HNF1B. Additionally, in 3 other patients, we detected rare variants with potential phenotypic effects. Notably, we identified a novel missense variant in TBC1D4 and an MC4R variant, which together may cause a mixed phenotype of severe insulin resistance. This comprehensive approach could assist in the early diagnosis of patients with monogenic diabetes and facilitate the provision of tailored therapy. Show less

Nonalcoholic steatohepatitis (NASH) is a hepatic phenotype of the metabolic syndrome, and increases the risk of cirrhosis and hepatocellular carcinoma (HCC). Although increasing evidence points to the therapeutic implications of certain types of anti-diabetic agents in NASH, it remains to be elucidated whether their effects on NASH are independent of their effects on diabetes. Genetically obese melanocortin 4 receptor-deficient (MC4R-KO) mice fed Western diet are a murine model that sequentially develops hepatic steatosis, NASH, and HCC in the presence of obesity and insulin resistance. In this study, we investigated the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor anagliptin on NASH and HCC development in MC4R-KO mice. Anagliptin treatment effectively prevented inflammation, fibrosis, and carcinogenesis in the liver of MC4R-KO mice. Interestingly, anagliptin only marginally affected body weight, systemic glucose and lipid metabolism, and hepatic steatosis. Histological data and gene expression analysis suggest that anagliptin treatment targets macrophage activation in the liver during the progression from simple steatosis to NASH. As a molecular mechanism underlying anagliptin action, we showed that glucagon-like peptide-1 suppressed proinflammatory and profibrotic phenotypes of macrophages in vitro. This study highlights the glucose metabolism-independent effects of anagliptin on NASH and HCC development. Show less

The early-phase wound repair response of the intestinal epithelium is characterized by rapid and organized cell migration. This response is regulated by several humoral factors, including TGF-β. However, due to a lack of appropriate models, the precise response of untransformed intestinal epithelial cells (IECs) to those factors is unclear. In this study, we established an in vitro wound repair model of untransformed IECs, based on native type-I collagen. In our system, IECs formed a uniform monolayer in a two-chamber culture insert and displayed a stable wound repair response. Gene expression analysis revealed significant induction of Apoa1, Apoa4, and Wnt4 during the collagen-guided wound repair response. The wound repair response was enhanced significantly by the addition of TGF-β. Surprisingly, addition of TGF-β induced a set of genes, including Slc28a2, Tubb2a, and Cpe, that were expressed preferentially in fetal IECs. Moreover, TGF-β significantly increased the peak velocity of migrating IECs and, conversely, reduced the time required to reach the peak velocity, as confirmed by the motion vector prediction (MVP) method. Our current in vitro system could be employed to assess other humoral factors involved in IEC migration and could contribute to a deeper understanding of the wound repair potentials of untransformed IECs. Show less

Triglyceride (TG) and cholesterol accumulation are known to occur in the liver of rats fed a histidine-excess (5%) diet, but there are few studies reporting histochemical and molecular biological analyses of the rat liver. The aim of this study was to elucidate the molecular basis of this lipid-accumulation mechanism. Lipid accumulations, tissue section images, and gene expression levels were compared in the livers of rats fed a control or histidine-excess diet for 5 wk (n=8/group). Serum levels of TGs, free fatty acids, total cholesterol, high-density lipoprotein cholesterol, glucose, albumin, and the enzyme activities of aspartate aminotransferase and alanine aminotransferase were also analyzed. In the livers of rats fed a histidine-excess diet, histochemical analyses showed what appeared to be a preliminary stage of nonalcoholic fatty liver, characterized by lipid accumulation around the central vein area and minor fibrosis. However, there were no changes in serum TG or free fatty acid levels. Quantitative PCR analyses showed the up-regulation of FAT/CD36, which is related to the uptake of fatty acids into cells, and the downregulation of two apolipoprotein genes, ApoC3 and ApoE. The mRNA levels of PPARγ, LXRα, and AMPKα in the liver were also reduced by excess histidine intake. The results of this study suggest that steatosis caused by excess histidine intake may be the result of an imbalance between lipid transport from the liver and the uptake of free fatty acids into hepatocytes. Show less

Bardet-Biedl syndrome (BBS) is a rare autosomal recessive disorder characterized by obesity, mental impairment, rod-cone dystrophy, polydactyly, male hypogonadism, and renal abnormalities. This disorder is caused by mutations in BBS1-21. Alström syndrome (AS), caused solely by mutations in ALMS1, is another genetic obesity syndrome clinically similar to BBS. We previously conducted the first nationwide survey of BBS in Japan and found four patients with genetically definite BBS. In this study, exome analyses were performed on new patients whose symptoms fulfilled the diagnostic criteria for BBS. We identified one reported heterozygous mutation in BBS1 (p.R429*) in one patient, two novel mutations (p.L493R and p.H719Y) in BBS20 in a second patient, and one novel mutation (p.Q920*) and one reported mutation (p.R2928*) in ALMS1 in a third patient, who was subsequently diagnosed with AS. The first patient with BBS was previously considered to have digenic heterozygous mutations in BBS1 and BBS4. RT-PCR and long-range genomic PCR analyses identified a new heterozygous mutation in BBS1, the deletion of exons 10 and 11. Thus, this patient was compound heterozygous for mutations in BBS1. Many studies have described digenic heterozygous mutations in BBS. However, undetected mutations might have existed in either one of the mutated genes. Show less

The production of 11-ketotestosterone (11KT), an important steroid hormone in piscine spermatogenesis, is regulated by the pituitary gonadotropins [Gths: follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh)] and it is synthesized by catalytic reactions involving several steroidogenic enzymes. Among these enzymes, the role of 17β-hydroxysteroid dehydrogenases (Hsd17bs) that exhibited 17-ketosteroid reducing activity (17KSR activity) responsible for 11KT synthesis is still poorly understood. In the present study, for the deeper understanding of testicular 11KT biosynthesis, we first investigated the steroidogenic pathway to produce 11KT in Japanese eel testis. In vitro incubation of the testis with androstenedione (A4) and the subsequent analysis of the metabolites by thin-layer chromatography indicated that 11KT was synthesized from A4 via 11β-hydroxyandrostenedione (11OHA4) and 11-ketoandrostenedione (11KA4), which indicated that the steroidogenic enzyme exhibiting the 17KSR activity responsible for converting 11KA4 to 11KT is crucial for 11KT production. Subsequently, cDNAs encoding three candidate enzymes, Hsd17b type3 (Hsd17b3), Hsd17b type12a (Hsd17b12a), and 20β-hydroxysteroid dehydrogenase type2 (Hsd20b2), potentially with the 17KSR activity were isolated and characterized in the Japanese eel. The isolated hsd17b3, hsd17b12a, and hsd20b2 cDNAs putatively encoded 308, 314, and 327 amino acid residues with high homology to those of other vertebrate counterparts, respectively. The Hsd17b3, Hsd17b12a, and Hsd20b2 expressed either in HEK293T or in Hepa-E1 converted 11KA4 to 11KT. Tissue-distribution analysis by quantitative real time PCR revealed that hsd17b12a and hsd20b2 mRNAs were detected in the testis, while hsd17b3 mRNA was not detectable. Furthermore, we examined the effects of Gths on the 17KSR activity and the expression of the candidate genes in the immature testis. The 17KSR activity was upregulated by administration of Gths. Furthermore, only expression of hsd17b12a among three candidates was upregulated by Gths as well as the 17KSR activity. These findings strongly suggested that Hsd17b12a is one of the enzymes with 17KSR activity responsible for 11KT synthesis in the testis of Japanese eel. Show less

Carbohydrate response element binding protein (ChREBP), a glucose responsive transcription factor, mainly regulates expression of genes involved in glucose metabolism and lipogenesis. Recently, ChREBP is speculated to be involved in the onset and progression of diabetic nephropathy (DN). However, there exists no report regarding the localization and function of ChREBP in the kidney. Therefore, we analyzed the localization of Chrebp mRNA expression in the wild type (WT) mice kidney using laser microdissection method, and observed its dominant expression in the proximal tubules. In diabetic mice, mRNA expression of Chrebp target genes in the proximal tubules, including Chrebpβ and thioredoxin-interacting protein (Txnip), significantly increased comparing with that of WT mice. Co-overexpression of ChREBP and its partner Mlx, in the absence of glucose, also increased TXNIP mRNA expression as well as high glucose in human proximal tubular epithelial cell line HK-2. Since TXNIP is well known to be involved in the production of reactive oxygen species (ROS), we next examined the effect of ChREBP/Mlx co-overexpression, in the absence of glucose, on ROS production in HK-2 cells. Interestingly, ChREBP/Mlx co-overexpression also induced ROS production significantly as well as high glucose. Moreover, both high glucose-induced increase of TXNIP mRNA expression and ROS production were abrogated by ChREBP small interfering RNA transfection. Taken together, high glucose-activated ChREBP in the renal proximal tubules induce the expression of TXNIP mRNA, resulting in the production of ROS which may cause renal tubular damage. It is therefore speculated that ChREBP is involved in the onset and progression of DN. Show less

A 67-year-old female was referred to our hospital with a sternal fracture in March 2008. She received a diagnosis of multiple myeloma (MM) BJP-κ type (ISS stage III). G-banding karyotype revealed 46, XX, t(11;22)(q23.3;q11.2) (Hubacek, Gene 592:193-9, 2016), which was later confirmed to be congenital. After repeated rounds of chemotherapy with bortezomib and lenalidomide, she obtained a very good partial response in August 2014, and she was followed up with no treatment. However, she relapsed in February 2016. At that time, fluorescence in situ hybridization identified del(13q) and t(4;14)(p16;q32), which are associated with a poor prognosis. Furthermore, PCR analysis showed that the chromosome 11 breakpoint was at the APOA5/APOA4 locus at 11q23.3, which is associated with malignancy, and that the chromosome 22 breakpoint was at the SEPT5 intron 1 locus, which also plays a role in leukemogenesis through formation of a fusion gene with MLL. Although she was treated with three further lines of therapy, she died from disease progression in August 2017. Synergism between t(11;22) and t(4;14) may have induced the double-refractory phenotype to proteasome inhibitor and lenalidomide, at least during the chemorefractory phase. We present a biological analysis of this case and a review of the literature. Show less

Statins have been reported to increase the plasma concentration of arachidonic acid (AA), an omega-6 long chain polyunsaturated fatty acid (LCPUFA) in several clinical studies indicating that statins affect the endogenous synthesis of LCUFAs. In the present study, we investigated the roles of the intrinsic mevalonate cascade and Rho-dependent pathway in LCPUFA synthesis, especially focusing on fatty acid desaturases (Fads) 2, using the human hepatocellular carcinoma cell line HepG2. Cell number and the activity of caspase-3 and 7 (caspase-3/7) was measured using a commercial kit. Gene expression was analyzed by quantitative real-time PCR. Protein expression was detected by Western blot analysis. Atorvastatin decreased cell viability and increased caspase-3/7 activity in a dose-dependent manner. At lower concentrations, atorvastatin stimulated both mRNA and protein expression of Fads2, and increased mRNA expression of FADS1 and ELVOL5. Both mevalonate and geranylgeranyl-pyrophosphate (GGPP), but not cholesterol, fully reversed atorvastatin-induced upregulation of Fads2, and mevalonate-effected reversal was inhibited by treatment with the Rho-associated protein kinase inhibitor Y-27632. These data clearly demonstrated that in human HepG2 cells, statins affect the endogenous synthesis of LCPUFAs by regulation of not only Fads2, but also Fads1 and Elovl5, through the GGPP-dependent Rho kinase pathway. Show less

Incretin hormones exert pleiotropic metabolic actions beyond the pancreas. Although the heart expresses both incretin receptors, the cardiac biology of GIP receptor (GIPR) action remains incompletely understood. Here we show that GIPR agonism did not impair the response to cardiac ischemia. In contrast, genetic elimination of the Gipr reduced myocardial infarction (MI)-induced ventricular injury and enhanced survival associated with reduced hormone sensitive lipase (HSL) phosphorylation; it also increased myocardial triacylglycerol (TAG) stores. Conversely, direct GIPR agonism in the isolated heart reduced myocardial TAG stores and increased fatty acid oxidation. The cardioprotective phenotype in Gipr Show less

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic neurons (GnRH neurons) and stimulates anterior pituitary gonadotrophs to synthesize and secrete gonadotropins. In addition to gonadotrophs, GnRH neurons also express GnRH receptors, and the autocrine action of GnRH is reportedly involved in the regulation of functions of GnRH neurons. There is accumulating evidence that extracellular signal-regulated kinase (ERK), one of mitogen-activated protein kinases (MAPKs), is activated by GnRH and involved in various effects of GnRH in GnRH neurons. In the present study, we performed microarray analysis to examine the types of genes whose expression was regulated by GnRH in immortalized mouse GnRH neurons (GT1-7 cells). We found that 257 genes among 55,681 genes examined were up-regulated after 30-min treatment of GT1-7 cells with GnRH. These up-regulated genes included four dual-specificity MAPK phosphatases (DUSPs), DUSP1, DUSP2, DUSP5, and DUSP6. Reverse transcription-polymerase chain reaction analysis confirmed that the mRNA levels of DUSP5 and DUSP6 were robustly increased within 30 min. U0126, an inhibitor of ERK activation, completely inhibited the increases in the mRNA levels of DUSP5 and DUSP6. Immunoblotting analysis revealed that ERK activation peaked at 5 min and declined steeply at 60 min, whereas DUSP5 and DUSP6 proteins were increased from 60 min. It was notable that down-regulation of DUSP6 augmented GnRH-induced ERK activation approximately 1.7-fold at 60 min. These results suggested that the up-regulation of DUSP6 regulates the duration of ERK activation at least in part. Show less

Donor cell leukemia (DCL) occurs after allogeneic hematopoietic stem cell transplantation. Several mechanisms, including occult leukemic/preleukemic subclones in the donor graft and germline predisposition to leukemia, are proposed to be associated with DCL's molecular pathogenesis. We report a comprehensive genetic analysis of a patient with KMT2A-rearranged DCL after allogeneic bone marrow transplantation for refractory cytopenia of childhood. We performed a whole-exome sequencing of the recipient's peripheral blood before transplant and the donor's peripheral blood and the recipient's bone marrow at the time of DCL diagnosis. RNA sequencing was also performed to detect fusion genes in DCL blasts. There were no germline mutations that were associated with a predisposition to leukemia in the recipient and donor. Furthermore, there were no detectable somatic alterations except KMT2A-MLLT10 and other related gene fusions in DCL. KMT2A-MLLT10 was not detectable in the donor's bone marrow. We propose a novel pattern of the molecular pathogenesis of DCL solely involving a genetic mutation acquired after transplant with no identifiable genetic factor related to the donor and recipient. Show less

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) are genetically and phenotypically heterogeneous. Cardiac function is improved after treatment in some cardiomyopathy patients, but little is known about genetic predictors of long-term outcomes and myocardial recovery following medical treatment. To elucidate the genetic basis of cardiomyopathy in Japan and the genotypes involved in prognosis and left ventricular reverse remodeling (LVRR), we performed targeted sequencing on 120 DCM (70 sporadic and 50 familial) and 52 HCM (15 sporadic and 37 familial) patients and integrated their genotypes with clinical phenotypes. Among the 120 DCM patients, 20 (16.7%) had TTN truncating variants and 13 (10.8%) had LMNA variants. TTN truncating variants were the major cause of sporadic DCM (21.4% of sporadic cases) as with Caucasians, whereas LMNA variants, which include a novel recurrent LMNA E115M variant, were the most frequent in familial DCM (24.0% of familial cases) unlike Caucasians. Of the 52 HCM patients, MYH7 and MYBPC3 variants were the most common (12 (23.1%) had MYH7 variants and 11 (21.2%) had MYBPC3 variants) as with Caucasians. DCM patients harboring TTN truncating variants had better prognosis than those with LMNA variants. Most patients with TTN truncating variants achieved LVRR, unlike most patients with LMNA variants. Show less

Gastric inhibitory polypeptide receptor (GIPR) directly induces energy accumulation in adipose tissue in vitro. However, the importance of the direct effect of GIPR signaling on adipose tissue in vivo remains unclear. In the current study, we generated adipose tissue-specific GIPR knockout (GIPR Show less

Numerous clinical studies have reported that statins increase the plasma concentration of arachidonic acid, which is an ω-6 long-chain polyunsaturated fatty acid (LCPUFA), and decrease the concentrations of eicosapentaenoic acid and docosahexaenoic acid, which are ω‑3 LCPUFAs. These findings indicate that statins may affect the endogenous synthesis of LCPUFAs, which is regulated by fatty acid desaturases (FADSs) and elongation of very long‑chain fatty acids proteins (ELOVLs). The present study aimed to investigate the roles of the intrinsic mevalonate cascade and Rho‑dependent pathway in statin‑induced regulation of these desaturases and elongases, as well as cell viability using mouse 3T3‑L1 cells. mRNA expression was analyzed by quantitative polymerase chain reaction. Treatment with atorvastatin decreased cell viability and increased the mRNA expression levels of Fads1, Fads2 and ELOVL fatty acid elongase 5 (Elovl5) in a dose‑dependent manner. Mevalonate and geranylgeranyl pyrophosphate (GGPP), but not cholesterol, fully reversed the atorvastatin‑induced downregulation of cell viability and upregulation of gene expression; however, mevalonate itself did not affect cell viability and gene expression. The Rho‑associated protein kinase inhibitor Y‑27632 inhibited the mevalonate‑ and GGPP‑mediated reversal of atorvastatin‑induced upregulation of Fads1, Fads2 and Elovl5. These findings indicated that statins may affect the endogenous synthesis of LCPUFAs by regulating Fads1, Fads2 and Elovl5 gene expression via the GGPP‑dependent Rho kinase pathway in mouse 3T3-L1 cells. Show less

Epigenetic regulation is required to ensure the precise spatial and temporal pattern of gene expression that is necessary for embryonic development. Although the roles of some epigenetic modifications in embryonic development have been investigated in depth, the role of methylation at lysine 79 (H3K79me) is poorly understood. Dot1L, a unique methyltransferase for H3K79, forms complexes with distinct sets of co-factors. To further understand the role of H3K79me in embryogenesis, we generated a mouse knockout of Mllt10, the gene encoding Af10, one Dot1L complex co-factor. We find homozygous Mllt10 knockout mutants (Mllt10-KO) exhibit midline facial cleft. The midfacial defects of Mllt10-KO embryos correspond to hyperterolism and are associated with reduced proliferation of mesenchyme in developing nasal processes and adjacent tissue. We demonstrate that H3K79me level is significantly decreased in nasal processes of Mllt10-KO embryos. Importantly, we find that expression of AP2α, a gene critical for midfacial development, is directly regulated by Af10-dependent H3K79me, and expression AP2α is reduced specifically in nasal processes of Mllt10-KO embryos. Suppression of H3K79me completely mimicked the Mllt10-KO phenotype. Together these data are the first to demonstrate that Af10-dependent H3K79me is essential for development of nasal processes and adjacent tissues, and consequent midfacial formation. Show less

Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR-Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations. Show less

Neurexins (NRXs) and neuroligins (NLs) play important roles in synapse specification. The alternatively spliced segment 4 (AS4) of NRX genes (Nrxn) is a critical element in selective trans-synaptic interactions. However, the role of splicing of NRXs and NLs in synapse specification is not fully understood. To investigate the exact role of splice-dependent NRX-NL interaction in the specification of glutamatergic and gamma-aminobutyric acid (GABA)-ergic synapses in the cerebellum, we evaluated the synaptogenic receptor activity of NL1/2/3 isoforms in a neuron-fibroblast co-culture system, in which the Nrxn AS4 segments are manipulated using SLM2, a selective and dominant regulator of AS4 splicing. We show that ectopic SLM2 expression (SLM2 E/E) causes marked skipping of exon 20 of AS4 in cerebellar neuron culture. Whereas NLs can induce VAMP2 Show less

The transcription factor cyclic AMP-responsive element-binding protein H (CREBH, encoded by To investigate the influence of intestinal CREBH on cholesterol metabolism, we compared plasma, bile, fecal, and tissue cholesterol levels between wild-type (WT) mice and mice overexpressing active human CREBH mainly in the small intestine (CREBH Tg mice) under different dietary conditions. Plasma cholesterol, hepatic lipid, and cholesterol crystal formation in the gallbladder were lower in CREBH Tg mice fed a lithogenic diet (LD) than in LD-fed WTs, while fecal cholesterol output was higher in the former. These results suggest that intestinal CREBH overexpression suppresses cholesterol absorption, leading to reduced plasma cholesterol, limited hepatic supply, and greater excretion. The expression of Niemann-Pick C1-like 1 ( Intestinal CREBH regulates dietary cholesterol flow from the small intestine by controlling the expression of multiple intestinal transporters. We propose that intestinal CREBH could be a therapeutic target for hypercholesterolemia. Show less

Fabry disease is caused by mutations in the α-galactosidase A (GLA) gene, which is located in X-chromosome coding for the lysosomal enzyme of GLA. Among many gene mutations, E66Q mutation is under discussion for its pathogenicity because there is no clinical report showing pathological evidence of Fabry disease with E66Q mutation. A 65-year-old Japanese female was referred to our hospital for chest discomfort on effort. Transthoracic echocardiography showed severe left ventricular (LV) hypertrophy with LV outflow obstruction. Maximum LV outflow pressure gradient was 87 mmHg, and Valsalva maneuver increased the pressure gradient up to 98 mmHg. According to medical interview, one of her younger sister and a nephew died suddenly at age 42 and 36, respectively. Another younger sister also presented LV hypertrophy with outflow obstruction. Maximum LV outflow pressure gradient was 100 mmHg, and the E66Q mutation was detected similar to the case. Endomyocardial biopsy specimens presented vacuolation of cardiomyocytes, in which zebra bodies were detected by electron microscopic examination. Although the enzymatic activity of GLA was within normal range, the c. 196G>C nucleotide change, which lead to the E66Q mutation of GLA gene, was detected. We initially diagnosed her as cardiac Fabry disease based on the findings of zebra body. However, immunostaining showed few deposition of globotriaosylceramide in left ventricular myocardium, and gene mutations in the disease genes for hypertrophic cardiomyopathy (HCM), MYBPC3 and MYH6, were detected. Although the pathogenicity of the E66Q mutation cannot be ruled out, hypertrophic obstructive cardiomyopathy (HOCM) was more reasonable to explain the pathophysiology in the case. This is the confusable case of HOCM with Fabry disease with the GLA E66Q mutation. We have to take into consideration the possibility that some patients with the E66Q mutation may have similar histological findings of Fabry disease, and should be examed the possibility for harboring gene mutations associated with HCM. Show less

Association between gastric inhibitory polypeptide receptor polymorphism, rs10423928, and body mass index in olanzapine-treated schizophrenia was examined. Body mass index change for the A/T+A/A genotypes was significantly higher than that for the T/T genotype. rs10423928 may predict weight gain in schizophrenia. Show less

Mammals are composed of hundreds of different cell types with specialized functions. Each of these cellular phenotypes are controlled by different combinations of transcription factors. Using a human non islet cell insulinoma cell line (TC-YIK) which expresses insulin and the majority of known pancreatic beta cell specific genes as an example, we describe a general approach to identify key cell-type-specific transcription factors (TFs) and their direct and indirect targets. By ranking all human TFs by their level of enriched expression in TC-YIK relative to a broad collection of samples (FANTOM5), we confirmed known key regulators of pancreatic function and development. Systematic siRNA mediated perturbation of these TFs followed by qRT-PCR revealed their interconnections with NEUROD1 at the top of the regulation hierarchy and its depletion drastically reducing insulin levels. For 15 of the TF knock-downs (KD), we then used Cap Analysis of Gene Expression (CAGE) to identify thousands of their targets genome-wide (KD-CAGE). The data confirm NEUROD1 as a key positive regulator in the transcriptional regulatory network (TRN), and ISL1, and PROX1 as antagonists. As a complimentary approach we used ChIP-seq on four of these factors to identify NEUROD1, LMX1A, PAX6, and RFX6 binding sites in the human genome. Examining the overlap between genes perturbed in the KD-CAGE experiments and genes with a ChIP-seq peak within 50 kb of their promoter, we identified direct transcriptional targets of these TFs. Integration of KD-CAGE and ChIP-seq data shows that both NEUROD1 and LMX1A work as the main transcriptional activators. In the core TRN (i.e., TF-TF only), NEUROD1 directly transcriptionally activates the pancreatic TFs HSF4, INSM1, MLXIPL, MYT1, NKX6-3, ONECUT2, PAX4, PROX1, RFX6, ST18, DACH1, and SHOX2, while LMX1A directly transcriptionally activates DACH1, SHOX2, PAX6, and PDX1. Analysis of these complementary datasets suggests the need for caution in interpreting ChIP-seq datasets. (1) A large fraction of binding sites are at distal enhancer sites and cannot be directly associated to their targets, without chromatin conformation data. (2) Many peaks may be non-functional: even when there is a peak at a promoter, the expression of the gene may not be affected in the matching perturbation experiment. Show less

Although the expression of hepatic lipogenic genes is enhanced in insulin resistance, the underlying mechanism is unclear. To reveal the details, the aim of this study was to investigate whether the expression of hepatic lipogenic genes are mediated by epigenetic regulation and specific transcription factors in an insulin resistance model of rats. Using a rat model of insulin resistance (SHR/NDmc-cp), we investigated the relationship between hepatic expression of the lipogenic gene fatty-acid synthase (Fasn), binding of the transcription factor carbohydrate-responsive element-binding protein (ChREBP) to the Fasn gene, and histone modifications in the region of the Fasn gene by real-time reverse transcriptase polymerase chain reaction, immunoblotting, and chromatin immunoprecipitation assay. Compared with control rats, Fasn mRNA expression and protein levels were higher in the livers of SHR/NDmc-cp rats, as were protein expression levels and Fasn binding of ChREBP and RNA polymerase II. Moreover, compared with the livers of control rats, levels of mono-methylated histone H3 lysine (K) 4 and acetylated histone H4 were higher in the promoter/enhancer region of the Fasn gene in the livers of SHR/NDmc-cp rats. Levels of trimethylated histone H3K4 and acetylated histone H3 were higher in the transcribed region. The results of this study indicate that expression of the Fasn gene in the livers of insulin-resistant rats is associated with increased H3K4 methylation, increased histone H3 acetylation, and increased H4 acetylation, and also, binding levels of ChREBP to promoter/enhancer region of Fasn gene is involved in the Fasn gene expression caused by hyperglycemia. Show less

Chronic kidney disease (CKD) is known to be one of the causes of cardiovascular disease and end-stage renal disease. Among the several treatable risk factors of CKD, that of dyslipidemia is relatively controversial. To clarify the association of polymorphisms in genes involved in lipid metabolism with the risk of CKD in the Japanese population, we used cross-sectional data from the Japan Multi-Institutional Collaborative Cohort (J-MICC) Study. A total of 3,268 men and women, aged 35-69 years, were selected from J-MICC Study participants for inclusion in this study. Twenty-eight candidate single nucleotide polymorphisms (SNPs) were selected in 17 genes associated with the risk of lipid metabolism disorders, and genotyping of the subjects was conducted using the multiplex PCR-based invader assay. The prevalence of CKD was determined for stages 3-5 (defined as estimated glomerular filtration rate <60 ml/min/1.73 m2). Logistic regression analysis revealed that SNPs APOA5 T - 1131C (rs662799), APOA5 T1259C (rs2266788), TOMM40 A/G (rs157580), and CETP TaqIB (rs708272) were significantly associated with CKD risk in those individuals genotyped, with age- and sex-adjusted odds ratios (ORs) per minor allele (and 95% confidence intervals (CIs)) of OR 1.22 (95% CI: 1.06-1.39), 1.19 (1.03-1.37), 1.27 (1.12-1.45), and 0.81 (0.71-0.92), respectively. Analysis of the gene-environment interaction revealed that body mass index (BMI) was a significant effect modifier for APOA5 T - 1131C (rs662799) and a marginally significant effect modifier for APOA5 T/C (rs2266788), with the interaction between BMI ≥30 and individuals with at least one minor allele of each genotype of OR 10.43 (95% CI: 1.29-84.19) and 3.36 (0.87-13.01), respectively. Four polymorphisms in APOA5, TOMM40, and CETP were shown to be significantly associated with CKD risk, and a significant interaction between the two APOA5 SNPs and BMI on CKD risk was also demonstrated. This suggests the future possibility of personalized risk estimation for this life-limiting disease. Show less

Valproic acid, which is widely used to treat various types of epilepsy, may cause severe hyperammonemia. However, the mechanism responsible for this side effect is not readily apparent. Polymorphisms in the genes encoding carbamoyl-phosphate synthase 1 (CPS1) and N-acetylglutamate synthase (NAGS) were recently reported to be risk factors for the development of hyperammonemia during valproic acid-based therapy. This study aimed to examine the influence of patient characteristics, including polymorphisms in CPS1 4217C>A and NAGS -3064C>A, on the development of hyperammonemia in Japanese pediatric epilepsy patients. The study included 177 pediatric epilepsy patients. The presence of a 4217C>A polymorphism in CPS1 was determined using an allele-specific polymerase chain reaction (PCR)-based method, and the presence of a -3064C>A polymorphism in NAGS was determined using a PCR-based restriction fragment length polymorphism method. Hyperammonemia was defined as a plasma ammonia level exceeding 200 μg/dL. We observed a significant difference between the combination of valproic acid with phenytoin and the development of hyperammonemia in both univariate and multivariate analyses. With regard to the CPS1 4217C>A polymorphism, we did not observe a significant association with the development of hyperammonemia. In conclusion, CPS1 4217C>A polymorphism may not be associated with the development of hyperammonemia in Japanese population. Show less

Intracranial germ cell tumours (IGCTs) are a group of rare heterogeneous brain tumours that are clinically and histologically similar to the more common gonadal GCTs. IGCTs show great variation in their geographical and gender distribution, histological composition and treatment outcomes. The incidence of IGCTs is historically five- to eightfold greater in Japan and other East Asian countries than in Western countries, with peak incidence near the time of puberty. About half of the tumours are located in the pineal region. The male-to-female incidence ratio is approximately 3-4:1 overall, but is even higher for tumours located in the pineal region. Owing to the scarcity of tumour specimens available for research, little is currently known about this rare disease. Here we report the analysis of 62 cases by next-generation sequencing, single nucleotide polymorphism array and expression array. We find the KIT/RAS signalling pathway frequently mutated in more than 50% of IGCTs, including novel recurrent somatic mutations in KIT, its downstream mediators KRAS and NRAS, and its negative regulator CBL. Novel somatic alterations in the AKT/mTOR pathway included copy number gains of the AKT1 locus at 14q32.33 in 19% of patients, with corresponding upregulation of AKT1 expression. We identified loss-of-function mutations in BCORL1, a transcriptional co-repressor and tumour suppressor. We report significant enrichment of novel and rare germline variants in JMJD1C, which codes for a histone demethylase and is a coactivator of the androgen receptor, among Japanese IGCT patients. This study establishes a molecular foundation for understanding the biology of IGCTs and suggests potentially promising therapeutic strategies focusing on the inhibition of KIT/RAS activation and the AKT1/mTOR pathway. Show less