👤 Yusei Hayashi

Hypertrophic cardiomyopathy (HCM) and restrictive cardiomyopathy (RCM) present a high risk for sudden cardiac death in pediatric patients. The aim of this study was to identify disease-associated genetic variants in Japanese patients with pediatric HCM and RCM. We analyzed 67 cardiomyopathy-associated genes in 46 HCM and 7 RCM patients diagnosed before 16 years of age using a next-generation sequencing system. We found that 78% of HCM and 71% of RCM patients carried disease-associated genetic variants. Disease-associated genetic variants were identified in 80% of HCM patients with a family history and in 77% of HCM patients with no apparent family history (NFH). MYH7 and/or MYBPC3 variants comprised 76% of HCM-associated variants, whereas troponin complex-encoding genes comprised 75% of the RCM-associated variants. In addition, 91% of HCM patients with implantable cardioverter-defibrillators and infant cases had NFH, and the 88% of HCM patients carrying disease-associated genetic variants were males who carried MYH7 or MYBPC3 variants. Moreover, two disease-associated LAMP2, one DES and one FHOD3 variants, were identified in HCM patients. In this study, pediatric HCM and RCM patients were found to carry disease-associated genetic variants at a high rate. Most of the variants were in MYH7 or MYPBC3 for HCM and TNNT2 or TNNI3 for RCM. Show less

Excess carbohydrate intake causes obesity in humans. On the other hand, acute administration of fructose, glucose or sucrose in experimental animals has been shown to increase the plasma concentration of anti-obesity hormones such as glucagon-like peptide 1 (GLP-1) and Fibroblast growth factor 21 (FGF21), which contribute to reducing body weight. However, the secretion and action of GLP-1 and FGF21 in mice chronically fed a high-sucrose diet has not been investigated. To address the role of anti-obesity hormones in response to increased sucrose intake, we analyzed mice fed a high-sucrose diet, a high-starch diet or a normal diet for 15 weeks. Mice fed a high-sucrose diet showed resistance to body weight gain, in comparison with mice fed a high-starch diet or control diet, due to increased energy expenditure. Plasma FGF21 levels were highest among the three groups in mice fed a high-sucrose diet, whereas no significant difference in GLP-1 levels was observed. Expression levels of uncoupling protein 1 (UCP-1), FGF receptor 1c (FGFR1c) and β-klotho (KLB) mRNA in brown adipose tissue were significantly increased in high sucrose-fed mice, suggesting increases in FGF21 sensitivity and energy expenditure. Expression of carbohydrate responsive element binding protein (ChREBP) mRNA in liver and brown adipose tissue was also increased in high sucrose-fed mice. These results indicate that FGF21 production in liver and brown adipose tissue is increased in high-sucrose diet and participates in resistance to weight gain. Show less

The action of incretin hormones including glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) is potentiated in animal models defective in glucagon action. It has been reported that such animal models maintain normoglycaemia under streptozotocin (STZ)-induced beta cell damage. However, the role of GIP in regulation of glucose metabolism under a combination of glucagon deficiency and STZ-induced beta cell damage has not been fully explored. In this study, we investigated glucose metabolism in mice deficient in proglucagon-derived peptides (PGDPs)-namely glucagon gene knockout (GcgKO) mice-administered with STZ. Single high-dose STZ (200 mg/kg, hSTZ) or moderate-dose STZ for five consecutive days (50 mg/kg × 5, mSTZ) was administered to GcgKO mice. The contribution of GIP to glucose metabolism in GcgKO mice was also investigated by experiments employing dipeptidyl peptidase IV (DPP4) inhibitor (DPP4i) or Gcg-Gipr double knockout (DKO) mice. GcgKO mice developed severe diabetes by hSTZ administration despite the absence of glucagon. Administration of mSTZ decreased pancreatic insulin content to 18.8 ± 3.4 (%) in GcgKO mice, but ad libitum-fed blood glucose levels did not significantly increase. Glucose-induced insulin secretion was marginally impaired in mSTZ-treated GcgKO mice but was abolished in mSTZ-treated DKO mice. Although GcgKO mice lack GLP-1, treatment with DPP4i potentiated glucose-induced insulin secretion and ameliorated glucose intolerance in mSTZ-treated GcgKO mice, but did not increase beta cell area or significantly reduce apoptotic cells in islets. These results indicate that GIP has the potential to ameliorate glucose intolerance even under STZ-induced beta cell damage by increasing insulin secretion rather than by promoting beta cell survival. 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

Although many single nucleotide polymorphisms (SNPs) have been identified to be associated with metabolic syndrome (MetS), there was only a slight improvement in the ability to predict future MetS by the simply addition of SNPs to clinical risk markers. To improve the ability to predict future MetS, combinational effects, such as SNP-SNP interaction, SNP-environment interaction, and SNP-clinical parameter (SNP × CP) interaction should be also considered. We performed a case-control study to explore novel SNP × CP interactions as risk markers for MetS based on health check-up data of Japanese male employees. We selected 99 SNPs that were previously reported to be associated with MetS and components of MetS; subsequently, we genotyped these SNPs from 360 cases and 1983 control subjects. First, we performed logistic regression analyses to assess the association of each SNP with MetS. Of these SNPs, five SNPs were significantly associated with MetS (P < 0.05): LRP2 rs2544390, rs1800592 between UCP1 and TBC1D9, APOA5 rs662799, VWF rs7965413, and rs1411766 between MYO16 and IRS2. Furthermore, we performed multiple logistic regression analyses, including an SNP term, a CP term, and an SNP × CP interaction term for each CP and SNP that was significantly associated with MetS. We identified a novel SNP × CP interaction between rs7965413 and platelet count that was significantly associated with MetS [SNP term: odds ratio (OR) = 0.78, P = 0.004; SNP × CP interaction term: OR = 1.33, P = 0.001]. This association of the SNP × CP interaction with MetS remained nominally significant in multiple logistic regression analysis after adjustment for either the number of MetS components or MetS components excluding obesity. Our results reveal new insight into platelet count as a risk marker for MetS. Show less

Upon binding of a Wnt ligand to the frizzled (FZD)-low density lipoprotein receptor related protein 5/6 (LRP5/6) receptor complex, the β-catenin destruction complex, composed of Axin1, adenomatous polyposis coli (APC), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), is immediately inactivated, which causes β-catenin stabilization. However, the molecular mechanism of signal transduction from the receptor complex to the β-catenin destruction complex is controversial. Here we show that Wnt3a treatment promotes the dissociation of the Axin1-APC complex in glioblastoma cells cultured in serum-free medium. Experiments with the GSK3 inhibitor BIO suggest that Axin1-APC dissociation was controlled by phosphorylation. Introduction of a phosphomimetic mutation into Thr160 of Axin1, located in the APC-binding region RGS, abrogated the interaction of Axin1 with APC. Consistent with these observations, the Axin1 phosphomimetic mutant lost the ability to reduce β-catenin stability and to repress β-catenin/TCF-dependent transcription. Taken together, our results suggest a novel mechanism of Wnt signaling through the dissociation of the β-catenin destruction complex by Axin1 Thr160 modification. Show less

Basaloid squamous cell carcinoma (BSCC) is a rare and poorly differentiated variant of typical squamous cell carcinoma, and is characterised in part by activation of the Wnt signalling pathway. We previously demonstrated that constitutive activation of the Wnt signalling pathway by epigenetic silencing of secreted frizzled-related protein 4 (SFRP4) is observed in this tumour. Increasing evidence shows that the Wnt signalling pathway cross-talks with other developmental pathways, including the Hedgehog (HH) pathway. The HH pathway is stimulated by inactivating mutations of PTCH1, which have a well-described oncogenic role in basal cell carcinoma (BCC) of the skin. We employed polymerase chain reaction followed by direct sequencing to detect inactivating mutations of PTCH1 using archival tissue samples of 30 oesophageal BSCCs. The frequency of PTCH1 mutation was compared to that of Wnt component genes that we reported previously. We found PTCH1 mutations in 53.3% (16/30) of cases, revealing T1195S as a hotspot mutation. This frequency is quite high for cancers other than BCC of the skin, and PTCH1 mutations were almost mutually exclusive with mutations in APC, Axin1 and Axin2. Considering the fact that activation of Wnt signalling via down-regulation of APC and SFRP5 due to promoter methylation is observed in BCC of the skin, Wnt signalling activation in oesophageal BSCC might be a secondary effect of the PTCH1-inactivating mutations. These findings suggest that the HH and Wnt pathways coordinately contribute to tumourigenesis in oesophageal BSCC. Furthermore, this study provides a potential therapeutic application for HH pathway inhibitors in oesophageal BSCC with highly malignant potential. Show less

We propose Langerhans cell histiocytosis (LCH) is an inflammatory process that is prolonged by mutations. We hypothesize that Merkel cell polyomavirus (MCPyV) infection triggers an interleukin-1 (IL-1) activation loop that underlies the pathogenesis of LCH. Langerhans cells (LCs) are antigen presenting cells in the skin. When LCs encounter exogenous antigens, they migrate from the epidermis into draining lymphoid tissues to initiate T-cell activity. It has been proposed that LC migration-related factors, including E-cadherin, matrix metalloproteinase, and Notch ligand induce LCH activity. We found that the tyrosine phosphatase SHP-1, which binds IL-1 receptor-associated kinase 1, is expressed at a significantly higher level in LCH affecting multiple organ systems (MS-LCH) than in LCH affecting a single organ system (SS-LCH). IL-1 stimulates T helper 17 cells and their signature cytokine IL-17 had been a matter of controversy. We detected higher levels of IL-17A receptor expression in MS-LCH than in SS-LCH and proposed an IL-17 endocrine model that could settle the controversy. IL-1 is the first cytokine secreted in response to sensitizers and promotes LC migration from sentinel tissues. Myeloid differentiation primary response 88 (MyD88), downstream of the IL-1 receptor, has functions in both RAS signaling and inflammation, leading to human cell transformation. In 2010, an activating mutation in the B-rapidly accelerated fibrosarcoma gene (BRAF) V600E was found in LCH. This BRAF mutation induces phosphorylation of the extracellular signal-regulated kinase (ERK) that may play an important role with MyD88 in LCH pathogenesis. However, phosphorylated ERK (pERK) is rapidly dephosphorylated by dual specificity phosphatase 6 (DUSP6), and limited proliferation is predicted in BRAF mutant cells. MyD88 binds pERK via its D-domain, thereby preventing pERK-DUSP6 interaction and maintaining ERK in an active, phosphorylated state. We detected MCPyV-DNA in the peripheral blood cells of two out of three patients with LCH in high-risk organs but not in those of patients with LCH in non-high-risk organs (0/12; P = .029). MCPyV infection can trigger precursor LCH cells with BRAF mutation to produce IL-1; the IL-1 loop is amplified in all LCH subclasses. Our model indicates both BRAF mutation and IL-1 loop regulation as potential therapeutic targets. Show less

There is an overlap between the physiological cardiac remodeling associated with training in athletes, the so-called athlete's heart, and mild forms of hypertrophic cardiomyopathy (HCM), the most common hereditary cardiac disease. HCM is often accompanied by unfavorable outcomes including a sudden cardiac death in the adolescents. Because one of the initial signs of HCM is abnormality in electrocardiogram (ECG), athletes may need to monitor for ECG findings to prevent any unfavorable outcomes. HCM is caused by mutations in genes for sarcomere proteins, but there is no report on the systematic screening of gene mutations in athletes. One hundred and two genetically unrelated young Japanese athletes with abnormal ECG findings were the subjects for the analysis of four sarcomere genes, MYH7, MYBPC3, TNNT2 and TNNI3. We found that 5 out of 102 (4.9%) athletes carried mutations: a heterozygous MYH7 Glu935Lys mutation, a heterozygous MYBPC3 Arg160Trp mutation and another heterozygous MYBPC3 Thr1046Met mutation, all of which had been reported as HCM-associated mutations, in 1, 2 and 2 subjects, respectively. This is the first study of systematic screening of sarcomere gene mutations in a cohort of athletes with abnormal ECG, demonstrating the presence of sarcomere gene mutations in the athlete's heart. Show less

Occurrence of malignant ventricular tachyarrhythmias such as ventricular tachycardia and fibrillation (VT/VF) in hypertrophic cardiomyopathy (HCM) can be related to the extent of myocardial fibrosis. Although late gadolinium enhancement (LGE) on cardiovascular magnetic resonance (CMR) imaging has been used to detect myocardial fibrosis, few data exist regarding relationships between CMR-determined myocardial fibrosis and VT/VF in genotyped HCM populations. We retrospectively investigated whether the extent of LGE can be increased in HCM patients with VT/VF compared to those without VT/VF in the genotyped HCM population. We studied 35 HCM patients harboring sarcomere gene mutations (TNNI3=22, MYBPC3=12, MYH7=1) who underwent both CMR imaging and 24-h ambulatory electrocardiographic monitoring. VT/VF were identified in 6 patients (2 men, mean age 55.0 years). The extent of LGE was significantly increased in patients with VT/VF (n=6) compared with those without VT/VF (n=29) (18.6±14.4% vs. 8.3±11.4%, p=0.04), although the LGE extent was not an independent predictor for the occurrence of VT/VF. Applying a cut-off point ≥3.25%, episodes of VT/VF were identified with a sensitivity of 100%, specificity of 51.7%, positive predictive value of 30%, negative predictive value of 100%, and the area under the curve of 0.767 (95% confidence interval: 0.590-0.944). These results demonstrate that myocardial fibrosis determined by CMR imaging may be increased in genotyped HCM patients with episodes of VT/VF. A further prospective study will be needed to clarify the association between the LGE extent and arrhythmic events in HCM patients harboring sarcomere gene mutations. Show less

Gastric inhibitory polypeptide (GIP) is an incretin secreted from the gastrointestinal tract after an ingestion of nutrients, and stimulates an insulin secretion from the pancreatic islets. Additionally, GIP has important roles in extrapancreatic tissues: fat accumulation in adipose tissue, neuroprotective effects in the central nervous system and an inhibition of bone resorption. In the current study, we investigated the effects of GIP signaling on the peripheral nervous system (PNS). First, the presence of the GIP receptor (GIPR) in mouse dorsal root ganglion (DRG) was evaluated utilizing immunohistochemical analysis, western blotting and reverse transcription polymerase chain reaction. DRG neurons of male wild-type mice (WT) were cultured with or without GIP, and their neurite lengths were quantified. Functions of the PNS were evaluated in GIPR-deficient mice (gipr-/-) and WT by using current perception thresholds (CPTs), Thermal Plantar Test (TPT), and motor (MNCV) and sensory nerve conduction velocity (SNCV, respectively). Sciatic nerve blood flow (SNBF) and plantar skin blood flow (PSBF) were also evaluated. We confirmed the expression of GIPR in DRG neurons. The neurite outgrowths of DRG neurons were promoted by the GIP administrations. The gipr-/- showed impaired perception functions in the examination of CPTs and TPT. Both MNCV and SNCV were delayed in gipr-/- compared with these in WT. There was no difference in SNBF and PSBF between WT and gipr-/-. Our findings show that the GIP signal could exert direct physiological roles in the PNS, which might be directly exerted on the PNS. Show less

Basaloid squamous cell carcinoma (BSCC) of the esophagus is a rare variant of typical squamous cell carcinoma (SCC) associated with poor survival. A characteristic feature is nuclear accumulation of β-catenin, without a mutation of the gene. We studied the methylation status of Wnt antagonist genes, such as secreted frizzled-related protein (sFRP) gene family members, Wnt inhibitory factor-1 (WIF-1), Dickkopf-1 (Dkk-1), and human Dapper protein-1 (HDPR-1), and alterations of the APC, Axin1, and Axin2 genes in 30 cases of esophageal BSCC. β-catenin and sFRP (sFRP-1, sFRP-2, sFRP-4, sFRP-5) protein expression was examined by immunohistochemistry. APC, Axin1, and Axin2 gene mutations were detected in 3, 2, and 2 cases, respectively, and 6 cases (20 %) harbored at least 1 alteration in these genes. Methylation of the sFRP-2 promoter region was observed in all cases, and methylation was frequent in sFRP-1 and sFRP-5, but infrequent in Dkk-1, WIF-1, sFRP-4, and HDPR-1. sFRP-2 expression was almost completely absent in 25 cases (83 %), consistent with the methylation status. Nuclear accumulation of β-catenin was observed in all cases. sFRP-5 expression was associated with a low nuclear β-catenin labeling index. These results show that sFRP-2 is a target gene of hypermethylation in esophageal BSCC and suggest that sFRP-2 might contribute to BSCC tumorigenesis through the Wnt/β-catenin signaling pathway. 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

Although most founder mutation carriers of hypertrophic cardiomyopathy (HCM), such as the cardiac myosin-binding protein C gene (MYBPC3), arose from a common ancestor exhibit favorable clinical phenotypes, there still remain small fractions of these carriers associated with increased cardiovascular events. However, few data exist regarding the defining factors that modify phenotypes of these patients, particularly in terms of multiple gene mutations. Therefore, we assessed genotype-phenotype correlations and investigated factors that contribute to phenotypic diversities of mutation carriers from 488 unrelated HCM probands. A prevalent founder mutation (Val762Asp) in MYBPC3 was identified in 33 subjects from 19 families. Among them, 28 carriers harbored an isolated Val762Asp mutation and exhibited a late onset of overt HCM compared with other MYBPC3 mutation carriers (62.8 ± 3.0 vs 50.1 ± 2.6 yr, P < 0.05). In contrast, the remaining five carriers had additional sarcomere gene mutations (3 carriers in MYBPC3 and 2 carriers in the cardiac troponin T gene). Of these five carriers, two carriers showed early disease onset and one carrier exhibited end-stage HCM. These phenotypes were recapitulated in zebrafish models; injection of MYBPC3 Val762Asp alone did not alter ventricular size or function, but ventricular dimension was significantly increased when MYBPC3 Val762Asp mRNA was coinjected with MYBPC3 Arg820Gln mRNA. These results demonstrate that MYBPC3 Val762Asp may be associated with unfavorable HCM phenotypes in some cases when combined with another MYBPC3 mutation. Show less

We evaluated multiple effects of genetic variations of five candidate loci (LEP, LEPR, MC4R, PIK3C3 and VRTN) on four production traits (average daily weight gain (ADG); backfat thickness (BFT); loin eye muscle area (EMA); and intramuscular fat content (IMF)) in a closed nucleus herd of pure Duroc pigs. Polymorphisms in LEPR, MC4R and PIK3C3 had significant single gene effects on ADG and BFT. The additive genetic variance in ADG and BFT (16.99% and 22.51%, respectively) was explained by genetic effects of these three loci. No correlations were observed between the LEP genotype and production traits in this study. Although we detected marginally epistatic interactions between LEPR and PIK3C3 on the eye muscle area, there were no significant epistatic effects on any traits among all loci pairs. These results suggest that LEPR, MC4R, PIK3C3 and VRTN may independently influence growth rate and fat deposition. Furthermore, the statistical models for predicting the breeding values of each trait had the lowest Akaike's information criterion values when considering the effect of the MC4R, LEPR, PIK3C3 and VRTN genotype simultaneously. These results suggest that LEPR, MC4R, PIK3C3 and VRTN are useful markers for accurately predicting breeding values in Duroc pigs. Show less

Transient abnormal myelopoiesis (TAM) is a myeloid proliferation resembling acute megakaryoblastic leukemia (AMKL), mostly affecting perinatal infants with Down syndrome. Although self-limiting in a majority of cases, TAM may evolve as non-self-limiting AMKL after spontaneous remission (DS-AMKL). Pathogenesis of these Down syndrome-related myeloid disorders is poorly understood, except for GATA1 mutations found in most cases. Here we report genomic profiling of 41 TAM, 49 DS-AMKL and 19 non-DS-AMKL samples, including whole-genome and/or whole-exome sequencing of 15 TAM and 14 DS-AMKL samples. TAM appears to be caused by a single GATA1 mutation and constitutive trisomy 21. Subsequent AMKL evolves from a pre-existing TAM clone through the acquisition of additional mutations, with major mutational targets including multiple cohesin components (53%), CTCF (20%), and EZH2, KANSL1 and other epigenetic regulators (45%), as well as common signaling pathways, such as the JAK family kinases, MPL, SH2B3 (LNK) and multiple RAS pathway genes (47%). Show less

Hypertrophic cardiomyopathy (HCM) is a disease of the sarcomere, and approximately 5% of cases of HCM show systolic dysfunction with poor prognosis. Few data exist regarding the systolic dysfunction in a large population of genotyped HCM subjects. The aim of this study was to assess the systolic dysfunction and prognosis in sarcomere gene mutation carriers. The study included 157 sarcomere gene mutation carriers from 69 unrelated HCM families (87 males; mean age, 46.5 ± 20.5 years). After exclusions for systolic dysfunction at baseline, 107 subjects underwent serial echocardiograms. At a mean follow-up of 7.0 years, 12 subjects experienced systolic dysfunction. In multivariate Cox analysis, systolic dysfunction was related to age and ejection fraction at initial evaluation (P < 0.001 and P = 0.020, respectively), and was associated with the absence of mutations in the cardiac myosin-binding protein C gene (MYBPC3) (P = 0.042). When the subjects were divided into MYBPC3 and non-MYBPC3 mutation carriers, and time from birth to development of systolic dysfunction was compared, the rate of systolic dysfunction was higher in the non-MYBPC3 group than in MYBPC3 group (Kaplan-Meier, log-rank test, P = 0.010). After the onset of systolic dysfunction, 11 of 12 subjects died during a mean follow-up of 8.3 years. Non-MYBPC3 mutation carriers developed left ventricular systolic dysfunction more frequently than MYBPC3 mutation carriers, and the majority of sarcomere gene mutation carriers with systolic dysfunction had fatal outcomes during follow-up. This suggests that subjects with mutations in sarcomeric genes require careful management for systolic dysfunction. Show less

A C↔T single nucleotide polymorphism (SNP) on exon 24 of the porcine class 3 phosphoinositide-3-kinase (PIK3C3) gene is considered a possible genetic marker for selecting backfat (BF) thickness and carcass fat, although only one study has published results on its effects by performing experiments on a single resource family. We analyzed the association of this PIK3C3 polymorphism with production traits in 739 Duroc pigs. The C allele frequency was 67.9% in our study population. PIK3C3 polymorphism showed significant effects on average daily weight gain (ADG), BF thickness, intermuscular fat content (IMF), and the size of the loin eye muscle area (EMA). The C alleles increased ADG, BF and IMF, and decreased EMA. The predicted differences in traits between the homozygous pigs of the C and T alleles were 40 g/day for DG, 1.2 mm for BF, 0.44% for IMF, and 1.6 cm(2) for EMA. Furthermore, the statistical models for estimating the breeding values of each trait had lower Akaike's information criterion values when adding PIK3C3 genotype information. We therefore confirmed that the polymorphism in PIK3C3 (C2604T) has the potential to be a genetic marker for production traits in Duroc pigs. Show less

Vitreous samples collected in retinopathic surgeries have diverse properties, making proteomics analysis difficult. We report a cluster analysis to evade this difficulty. Vitreous and subretinal fluid samples were collected from 60 patients during surgical operation of non-proliferative diabetic retinopathy, proliferative diabetic retinopathy, proliferative vitreoretinopathy, and rhegmatogenous retinal detachment. For controls we collected vitreous fluid from patients of idiopathic macular hole, epiretinal, and from a healthy postmortem donor. Proteins from these samples were subjected to quantitative proteomics using two-dimensional gel electrophoresis. We selected 105 proteins robustly expressed among ca 400 protein spots and subjected them to permutation test. By using permutation test analysis we observed unique variations in the expression of some of these proteins in vitreoretinal diseases when compared to the control and to each other: 1) the levels of inflammation-associate proteins such as AAT, APOA4, ALB, and TF were significantly higher in all four types of vitreoretinal diseases, and 2) each vitreoretinal disease elevates a unique set of proteins which can be interpreted based on the pathology of retinopathy. Our protocol will be effective for the study of protein expression in other types of clinical samples of diverse property. Show less

Recent studies have shown that in the developing embryo, arterial and venous identity is established by genetic mechanisms before circulation begins. Vascular endothelial growth factor (VEGF) signaling and its downstream Notch pathway play critical roles in arterial cell fate determination. We have recently shown that Foxc1 and Foxc2, two closely related Fox transcription factors, are essential for arterial cell specification during development by directly inducing the transcription of Delta-like 4 (Dll4), a ligand for Notch receptors. However, the basic mechanisms whereby the VEGF and Notch signaling pathways control transcriptional regulation of arterial-specific genes have yet to be elucidated. In the current study, we examined whether and how Foxc transcription factors are involved in VEGF and Notch signaling in induction of Dll4 as well as the Notch target gene Hey2 in endothelial cells. We found that Foxc1 and Foxc2 directly activate the Hey2 promoter via Foxc binding elements. Significantly, Foxc2 physically and functionally interacts with a Notch transcriptional activation complex containing Su(H) and Notch intracellular domain to induce Hey2 promoter activity. Moreover, activation of the Dll4 and Hey2 promoters is induced by VEGF in conjunction with either Foxc1 or Foxc2 more than by either component alone. VEGF-activated PI3K and ERK intracellular pathways modulate the transcriptional activity of Foxc proteins in Dll4 and Hey2 induction. Our new findings demonstrate that Foxc transcriptional factors interact with VEGF and Notch signaling to regulate arterial gene expression in multiple steps of the VEGF-Dll4-Notch-Hey2 signaling pathway. Show less

In cochlear development, the Notch signaling pathway is required for both the early prosensory phase and a later lateral inhibition phase. While it is known that Hes genes are important downstream mediators of Notch function in lateral inhibition, it is not known what genes function as mediators of the early prosensory function of Notch. We report that two members of the Hes-related gene family, Hesr1 and Hesr2, are expressed in the developing cochlea at a time and place that makes them excellent candidates as downstream mediators of Notch during prosensory specification. We also show that treatment of cochlear explant cultures at the time of prosensory specification with a small-molecule inhibitor of the Notch pathway mimics the results of conditional Jag1 deletion. This treatment also reduces Hesr1 and Hesr2 expression by as much as 80%. These results support the hypothesis that Hesr1 and Hesr2 are the downstream mediators of the prosensory function of Notch in early cochlear development. Show less

Apolipoprotein (apo) A-V has been the focus of significant attention as a potential modulator of plasma triglyceride (TG) in spite of its very low plasma concentration. TG levels are frequently elevated in patients with end-stage renal disease (ESRD), which is associated with a high prevalence of cardiovascular disease among them. We measured plasma apo A-V levels in 20 control subjects and 70 patients with diabetic and nondiabetic ESRD to investigate whether low apo A-V levels could be involved in the pathogenesis of the hyper-TG in ESRD. The plasma TG levels were significantly elevated in diabetic patients with ESRD, whereas those in nondiabetic ESRD patients remained similar to those in the controls. High-density lipoprotein cholesterol levels were significantly lower in the patients with ESRD than in the controls, irrespective of the presence of diabetes. Apo A-V levels measured by an enzyme-linked immunosorbent assay were markedly reduced to 40% to 44% of the control levels in both diabetic and nondiabetic patients with ESRD. The apo A-V levels were not correlated with TG in the overall study population, but they were positively correlated with high-density lipoprotein cholesterol. These results suggest that reduced apo A-V levels do not necessarily lead to hyper-TG in ESRD, but we are unable to exclude the possibility that low apo A-V plays a role in raising the TG level in diabetic ESRD. Show less

The juvenile form of neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene, and is characterized by progressive loss of vision and development of motor deficits. A few patients exhibit a more protracted clinical course and are diagnosed with protracted JNCL (PJNCL). Here, we report the autopsy in a case of PJNCL in a 55-year-old male and immunohistochemical examination of the involvement of oxidative stress and glutamate excitotoxicity in neurodegeneration. The patient was born to consanguineous parents (I assume this means that the parents were related. If not, then the sentence will need to be changed again.) and had brothers with similar neurological disease. He showed mental retardation and visual impairment in the first decade which gradually developed along with motor dysfunction for over 40 years. At autopsy, the cerebral pyramidal neurons revealed deposition of lipopigments, which demonstrated 'finger print' and curvilinear profiles on electron microscopy. He also exhibited cerebellar cortical atrophy, fibrillary gliosis in the white matter, and rarefication in the globus pallidus. Immunohistochemically, the number of neurons immunoreactive for advanced glycation end product was elevated in the cerebellar cortex and midbrain. Immunoreactivity for excitatory amino acid transporter 1 was reduced in the cerebellar dentate and inferior olivary nuclei. These findings suggest that oxidative damage to proteins and disturbed glutamate transport can be involved in PJNCL. Show less

Embryonic stem (ES) cells are highlighted as promising cell sources for regenerative medicine. Here, we focused on providing the platform that forced ES cells to reproduce the vascular organization process, leading to efficiency and safety evaluation as preclinical testing of biological agents. Murine ES cell-derived embryoid bodies on matrigel, but not collagen or gelatin, could be differentiated into sprouting blood vessels without the addition of growth factors. The expression of endothelial cell marker CD31 and smooth muscle marker alpha-smooth muscle actin was partially colocalized and started to increase 7 days after culture on matrigel, accompanied by the induction of a number of growth factors, such as vascular endothelial growth factor, fibroblast growth factor-2, hepatocyte growth factor, transforming growth factor-beta, and angiopoietin-1. Moreover, notch-related genes, such as Del1 or Del4 (delta-like 1/4) and hey1 or hey2 (hairy/enhancer of split related TRPW motif 1/2), were upregulated in a similar time course. The treatment of neutralizing antibodies against these growth factors failed to inhibit the differentiation into the sprouting blood vessels, whereas arginine-glycine-aspartic peptide, a selective inhibitor for the alphavbeta3-integrins, did inhibit differentiation. An anticancer drug to inhibit angiogenesis, TNP-470, also blocked the vascular formation in this model. ES cells could reproduce the vascular organization process on the biosynthetic scaffolds, such as matrigel, without the addition of growth factors. In the future, a human ES-based tissue model would be an optional tool for the screening of pharmaceutical drugs for vascular disease. Show less

Tamoxifen is a potent antagonist of estrogen, and hepatic steatosis is a frequent complication in adjuvant tamoxifen for breast cancer. Impaired hepatic FA beta-oxidation in peroxisomes, microsomes, and mitochondria results in progression of massive hepatic steatosis in estrogen deficiency. This impairment, although latent, is potentially serious: About 3% of the general population in the United States is now suffering from nonalcoholic steatohepatitis associated with obesity and hyperlipidemia. Therefore, in the present study we tried to restore impaired hepatic FA beta-oxidation by administering a novel statin, pitavastatin, to aromatase-deficient (Ar-/-) mice defective in intrinsic estrogen synthesis. Northern blot analysis of Ar-/- mice liver revealed a significant restoration of mRNA expression of essential enzymes involved in FA beta-oxidation such as very long fatty acyl-CoA synthetase in peroxisome, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase. Severe hepatic steatosis observed in Ar-/- mice substantially regressed. Consistent findings were obtained in the in vitro assays of FA beta-oxidation activity. These findings demonstrate that pitavastatin is capable of restoring impaired FA beta-oxidation in vivo via the peroxisome proliferator-activated receptor-alpha-mediated signaling pathway and is potent enough to ameliorate severe hepatic steatosis in mice deficient in intrinsic estrogen. Show less

The recurrent translocation t(10;11) is associated with acute myeloid leukemia (AML). The AF10 gene on chromosome 10 at band p12 and MLL at 11q23 fuse in the t(10;11)(p12;q23). Recently, we have identified ABI1 as a new partner gene for MLL in an AML patient with a t(10;11)(p11.2;q23). The ABI1 is a human homologue of the mouse Abl-interactor 1 (Abi1), encoding an Abl-binding protein. The ABI1 protein exhibits sequence similarity to homeotic genes, and contains several polyproline stretches and a src homology 3 (SH3) domain. To clarify the clinical features of t(10;11)-leukemias, we investigated 6 samples from acute leukemia patients with t(10;11) and MLL rearrangement and detected MLL-AF10 chimeric transcripts in 5 samples and MLL-ABI1 in one. The patient with MLL-ABI1 chimeric transcript is the second case described, thus confirming that the fusion of the MLL and ABI1 genes is a recurring abnormality. Both of the patients with MLL-ABI1 chimeric transcript are surviving, suggesting that these patients have a better prognosis than the patients with MLL-AF10. To investigate the roles of AF10 and ABI1 further, we examined the expression of these genes in various cell lines and fresh tumor samples using the reverse transcriptase-polymerase chain reaction method. Although AF10 was expressed in almost all cell lines similarly, the expression patterns of ABI1 were different between leukemia and solid tumor cell lines, suggesting the distinctive role of each isoform of ABI1 in these cell lines. We also determined the complete mouse Abi1 sequence and found that the sequence matched with human ABI1 better than the originally reported Abi1 sequence. Further functional analysis of the MLL-AF10 and MLL-ABI1 fusion proteins will provide new insights into the leukemogenesis of t(10;11)-AML. Show less

Lymphatic lipid transport in the intestine of adult and ageing rats was compared. Adult (8-10 months old) and old (24-26 months old) male Wistar rats were cannulated into the mesenteric lymph under ethrane anesthesia. On the following day, lipid emulsion containing 35.4 mg/h of olive oil was infused intraduodenally for 7 h and lymph collected hourly was assayed for triglyceride and apolipoprotein A-IV (apo A-IV). The results showed there was no difference in lymphatic lipid and apo A-IV transport between adult and old rats. Since apo A-IV synthesis in the enterocytes is linked to the intracellular assembly of lipoprotein, it is likely that in addition to lymphatic transport, production of chylomicrons is not impaired in ageing rats. Show less

DUSP6 (alias PYST1), one of the dual-specificity tyrosine phosphatases, is localized on 12q21, one of the regions of frequent allelic loss in pancreatic cancer. This gene is composed of three exons, and two forms of alternatively spliced transcripts are ubiquitously expressed. Although no mutations were observed in 26 pancreatic cancer cell lines, reduced expressions of the full-length transcripts were observed in some cell lines, which may suggest some role for DUSP6 in pancreatic carcinogenesis. Show less

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder that is characterized by the appearance of multiple outgrowths of the long bones (exostoses) at their epiphyses. Genetical heterogeneities have segregated at least on chromosome 8, 11, and 19 and been designated EXT1, EXT2, and EXT3, respectively. Recently, the responsible genes for EXT1 and EXT2 have been isolated and appeared to define a structurally related gene family. In the present study, we have identified novel genes which share significant sequence homologies with the EXT genes. The predicted protein products of the novel EXT-related genes, EXTR and EXTR2 (for EXT-related genes 1 and 2), consist of 919 and 330 amino acid residues, respectively. These genes were transcribed ubiquitously in various tissues. Based on PCR-assisted analyses of both a human/rodent mono-chromosomal hybrid cell panel and a radiation hybrid mapping panel, EXTR1 was localized to the chromosome 8p21 region, where loss of heterozygosity has been frequently observed in various tumors, and EXTR2 was assigned to the chromosome 1p21 region, where osteopetrosis, a dominant hereditary disease of bone, has been mapped by genetic linkage analysis, implying that the protein products of these two EXT-related genes, as well as of the EXT genes, have potential tumor suppressor activity. Show less