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A predictive marker for the development of synchronous/metachronous gastric cancer (GC) would be highly desirable in order to establish an effective strategy for endoscopic surveillance. Herein, we examine the significance of gastric xanthelasma (GX) and molecular abnormalities for the prediction of synchronous/metachronous GC. Patients (n = 115) were followed up (range, 12-122; median, 55 months) in whom the presence of GX and molecular alterations, including microsatellite instability (MSI) and methylation of human mutL homolog 1 (hMLH1), cyclin-dependent kinase inhibitor 2A (CDKN2A) and adenomatous polyposis coli (APC) genes, had been confirmed in non-neoplastic gastric mucosa when undergoing endoscopic submucosal dissection (ESD) for early GC. At the start of surveillance, the numbers of positive subjects were as follows: GX, 59 (51.3%); MSI, 48 (41.7%); hMLH1, 37 (32.2%); CDKN2A, 7 (6.1%); APC, 18 (15.7%). After ESD treatment, synchronous/metachronous GCs occurred in patients with the following positive factors: GX, 16 (27.1%); MSI, 7 (14.6%); hMLH1, 6 (16.2%); CDKN2A, 3 (42.9%); APC, 3 (16.7%). The presence of GX had no significant relationship to positivity for MSI or methylation of hMLH1, CDKN2A or APC. GX was significantly (p = 0.0059) and independently (hazard ratio, 3.275; 95% confidence interval, 1.134-9.346) predictive for the development of synchronous/metachronous GC, whereas those genetic alterations were not predictive. GX is a simple and powerful marker for predicting the development of synchronous or metachronous GC.
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INTRODUCTION: Impaired intestinal epithelial barrier function is a hallmark of a variety of pathological conditions such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). IBD patients with IBS-like symptoms show higher interleukin-13 (IL-13) serum levels and poor psychological well-being. Supplementary glutamine reduced the daily bowel movement frequency, improved the stool form, and normalized intestinal hyperpermeability. This study was aimed at assessing the effects of IL-13 and supplementary glutamine on human intestinal epithelial function in vitro. METHODS: Caco-2 cells were grown on TranswellTM inserts. -IL-13 was added to the basolateral compartment, and transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) labeled-dextran permeability measured. Effects of glutamine or the phosphatidylinositol-3-kinase inhibitor LY294002 were assessed. Involvement of tight junction proteins was assessed using Western blotting and immunofluorescence staining. RESULTS: IL-13 significantly decreased TEER and increased FITC labeled-dextran epithelial permeability. IL-13 stimulation decreased the claudin-1 expression and increased the claudin-2 expression. Glutamine alleviated IL-13-induced decrease of TEER and increase of FITC labeled-dextran permeability. Further, the phosphatidylinositol-3-kinase inhibitor showed this alleviating effect while the signal transducer and activator of transcription 6 inhibitor did not. CONCLUSIONS: IL-13 induced barrier integrity impairment by decreasing claudin-1 and increasing claudin-2. Glutamine alleviated IL-13-induced barrier dysfunction by increasing claudin-1 expression, via disruption of the phosphatidylinositol-3-kinase/Akt signaling pathway.
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Glutamina , Interleucina-13 , Células CACO-2 , Claudina-1 , Células Epiteliales , Glutamina/farmacología , Humanos , Mucosa IntestinalRESUMEN
BACKGROUND AND AIM: Irritable bowel syndrome (IBS) is one of the most common functional gastrointestinal disorders, and bile acids are thought to be associated with the pathogenesis of IBS. Bile acid receptors are expressed on intestinal epithelial cells. However, no study has assessed bile acid receptor proteins in IBS. Therefore, we examined the intestinal mucosal expression of bile acid receptors in patients with IBS. METHODS: Intestinal biopsies were performed in patients with IBS and controls. Mast cells, vitamin D receptor (VDR), and somatostatin were stained with specific antibodies. Levels of VDR, farnesoid X receptor (FXR), takeda-G-protein-receptor-5 (TGR5), claudins, and transient-receptor-potential-cation-channel-subfamily-V-member 6 (TRPV6) were assessed by western blotting. RESULTS: 3Mast cell counts in the second part of the duodenum were significantly higher in patients with IBS than in controls. VDR protein levels were significantly elevated in the duodenum and terminal ileum of patients with IBS compared with controls, although this difference was not seen in the cecum or rectum. FXR and TGR5 protein levels did not differ in any part of the intestine. VDR-positive cryptal epithelia in IBS were distributed not only at basal crypt but also along the upper part of the basal crypt epithelial cells. In contrast, the pattern of gut somatostatin-positive cells, claudins, and TRPV6 levels did not differ. CONCLUSIONS: The number of mast cells in the duodenum was significantly increased, and the protein expression levels of VDR, but not those of FXR or TGR5, were elevated in the duodenal epithelial crypt in patients with IBS.
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Duodeno/metabolismo , Expresión Génica , Síndrome del Colon Irritable/genética , Síndrome del Colon Irritable/metabolismo , Receptores de Calcitriol/genética , Receptores de Calcitriol/metabolismo , Duodeno/citología , Femenino , Humanos , Masculino , Mastocitos/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
The polyglutamine (polyQ) diseases are a group of inherited neurodegenerative diseases that include Huntington's disease, various spinocerebellar ataxias, spinal and bulbar muscular atrophy, and dentatorubral pallidoluysian atrophy. They are caused by the abnormal expansion of a CAG repeat coding for the polyQ stretch in the causative gene of each disease. The expanded polyQ stretches trigger abnormal ß-sheet conformational transition and oligomerization followed by aggregation of the polyQ proteins in the affected neurons, leading to neuronal toxicity and neurodegeneration. Disease-modifying therapies that attenuate both symptoms and molecular pathogenesis of polyQ diseases remain an unmet clinical need. Here we identified arginine, a chemical chaperone that facilitates proper protein folding, as a novel compound that targets the upstream processes of polyQ protein aggregation by stabilizing the polyQ protein conformation. We first screened representative chemical chaperones using an in vitro polyQ aggregation assay, and identified arginine as a potent polyQ aggregation inhibitor. Our in vitro and cellular assays revealed that arginine exerts its anti-aggregation property by inhibiting the toxic ß-sheet conformational transition and oligomerization of polyQ proteins before the formation of insoluble aggregates. Arginine exhibited therapeutic effects on neurological symptoms and protein aggregation pathology in Caenorhabditis elegans, Drosophila, and two different mouse models of polyQ diseases. Arginine was also effective in a polyQ mouse model when administered after symptom onset. As arginine has been safely used for urea cycle defects and for mitochondrial myopathy, encephalopathy, lactic acid and stroke syndrome patients, and efficiently crosses the blood-brain barrier, a drug-repositioning approach for arginine would enable prompt clinical application as a promising disease-modifier drug for the polyQ diseases.
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Arginina/metabolismo , Arginina/farmacología , Péptidos/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Modelos Animales de Enfermedad , Drosophila/metabolismo , Femenino , Trastornos Heredodegenerativos del Sistema Nervioso/genética , Enfermedad de Huntington/genética , Masculino , Ratones , Ratones Endogámicos , Chaperonas Moleculares/genética , Péptidos/genética , Agregación Patológica de Proteínas , Conformación Proteica/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Ataxias Espinocerebelosas/genéticaRESUMEN
The risk of gastric cancer (GC) declines after Helicobacter pylori (H. pylori) eradication and long-term aspirin use. We evaluated the effects of H. pylori eradication (Cohort 1) and aspirin use (Cohort 2) on the methylation of microRNAs (miRNAs), such as miR-34c, miR-124a-3, miR-129-2, and miR-137, in the gastric mucosa with and without GC, i.e., in atrophic mucosal glands without intestinal metaplasia (non-IM) and intestinal metaplastic glands (IM). DNA was isolated from non-IM and IM separately using laser caption microdissection. In Cohort 1, H. pylori eradication was associated with a significant reduction of miR-124a-3 methylation only in non-IM, but not in IM. miR-129-2 methylation in non-IM may be a surrogate marker of GC in H. pylori-infected patients. In Cohort 2, aspirin did not reverse miRNA methylation in either non-IM or IM, irrespective of H. pylori infection. miR-129-2 methylation in non-IM was an independent predictive marker of GC in H. pylori-infected but not -eradicated patients. These results indicate that H. pylori eradication and aspirin use were less effective for improving methylation in IM than in non-IM; thus, these interventions are recommended at an early stage prior to the development of IM to prevent GC development. In addition, the effects of the interventions were not uniform for each miRNA gene.
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Aspirina/farmacología , Metilación de ADN/genética , Mucosa Gástrica/metabolismo , Helicobacter pylori/efectos de los fármacos , MicroARNs/genética , Lesiones Precancerosas/genética , Neoplasias Gástricas/genética , Anciano , Estudios de Cohortes , Femenino , Silenciador del Gen , Helicobacter pylori/fisiología , Humanos , Masculino , Persona de Mediana Edad , Lesiones Precancerosas/microbiología , Neoplasias Gástricas/microbiología , Factores de TiempoRESUMEN
Non-ampullary duodenal adenocarcinoma (NADC) is extremely rare. Little is known about its clinicopathological and molecular features or its management. Herein we retrospectively analyzed the cases of 32 NADC patients, focusing on microsatellite instability (MSI), genetic mutations, CpG island methylator phenotype (CIMP), and immunostaining including mucin phenotype and PD-L1 expression. The incidence of MSI, KRAS/BRAF/GNAS mutations and CIMP was 51.6%, 34.4%/3.1%/6.5% and 28.1%, respectively. PD-L1 expression was seen in 34.4% of patients. No significant associations between clinicopathological features and KRAS/BRAF/GNAS genetic mutations or CIMP were found. Histologically non-well-differentiated-type NADCs and those in the 1st portion of the duodenum were significantly associated with later stages (stages III-IV) (P = 0.006 and P = 0.003, respectively). Gastric-phenotype NADCs were frequently observed in the 1st portion and in late-stage patients; their cancer cells more frequently expressed PD-L1. Histologically, the non-well-differentiated type was an independent predictor of PD-L1 expression in cancer cells (OR 25.05, P = 0.04) and immune cells (OR 44.14, P = 0.02). Only late-stage disease (HR 12.23, P = 0.01) was a prognostic factor for worse overall survival in a Cox proportional hazards regression model. Our observation of high proportions of MSI and PD-L1 expression may prompt the consideration of immune checkpoint inhibitors as a new treatment option for NADCs.
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Adenocarcinoma/metabolismo , Antígeno B7-H1/biosíntesis , Neoplasias Duodenales/metabolismo , Proteínas de Neoplasias/biosíntesis , Adenocarcinoma/genética , Adenocarcinoma/mortalidad , Adenocarcinoma/patología , Anciano , Antígeno B7-H1/genética , Cromograninas/genética , Islas de CpG , Metilación de ADN , ADN de Neoplasias/química , Neoplasias Duodenales/genética , Neoplasias Duodenales/mortalidad , Neoplasias Duodenales/patología , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Genes Relacionados con las Neoplasias , Genes ras , Humanos , Estimación de Kaplan-Meier , Masculino , Inestabilidad de Microsatélites , Persona de Mediana Edad , Mutación , Proteínas de Neoplasias/genética , Fenotipo , Pronóstico , Modelos de Riesgos Proporcionales , Proteínas Proto-Oncogénicas B-raf/genética , Estudios RetrospectivosRESUMEN
Background Gastric cancers (GC) after H. pylori eradication are difficult to diagnose even by magnifying narrow-band imaging (NBI) or blue laser imaging (BLI) endoscopy. Little is known with regard to non-magnifying (NM)-NBI/BLI for early GC so we examined the efficacy of NM-NBI/BLI for early GC diagnosis. Methods We retrospectively analyzed the images of 29 small (≤â1âcm) intramucosal GC that had been treated with endoscopic submucosal dissection and 137 benign depressed lesions (BDLs). The brightness and shape of the GCs and BDLs by NM-NBI/BLI were assessed with ImageJ software. Results The NBI/BLI-index, which indicates the brightness of NBI/BLI for visualization, was significantly higher in GC than BDLs in both the H. pylori -infected ( P â=â0.009) and -eradicated group ( P â<â0.0001), indicating that GC exhibited brighter colors than the normal surrounding mucosa. The C-index, which refers to the circularity of the lesion, was also significantly higher in GC than BDLs in both H. pylori -infected ( P â=â0.006) and -eradicated cases ( P â=â0.004). Based on receiver-operating characteristic curve analysis, cutoff values for the NBI/BLI- and C-indices for GC were 1.04 and 0.58 in the H. pylori -infected cases, and 0.98 and 0.64 in the H. pylori -eradicated cases. With the reference value of the NBI/BLI-index set atâ≥â0.69 with the C-index at ≥â0.21 in the H. pylori -infected and the NBI/BLI-index at ≥â0.80 with the C-index at ≥â0.32 in the H. pylori -eradicated cases, both the sensitivity and negative predictive value for early GC were 100â%. A high NBI/BLI-index tended to be associated with a wide length of the intervening part histologically in the H. pylori -eradicated cases ( P â=â0.09). Conclusions The small depressed-type early GC had brighter color and rounder shape compared to BDLs in both H. pylori -infected and -eradicated cases. The NBI/BLI- and C-indices calculated by the image analysis may facilitate identification of small depressed-type GC.
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The risk of gastric cancer (GC) remains in precancerous conditions, including atrophic mucosa and intestinal mucosa (IM), even after H. pylori treatment. To define the molecular changes following H. pylori eradication, molecular alterations in the gastric mucosa with and without GC were evaluated in a long-term follow-up study. A total of 232 biopsy specimens from 78 consecutive patients, including atrophic gastritis patients with follow-up ≥3 y after successful H. pylori eradication (AG group), patients who developed early GC after successful eradication (≥3 y) (GC group), and patients with H. pylori-positive atrophic gastritis (Hp group), were analyzed. H. pylori eradication was associated with significant reductions of methylation of several genes/loci in atrophic mucosa (non-IM), but not in IM. In contrast, the incidence of CpG island methylator phenotype (CIMP) in IM was significantly higher in the GC group than in the AG group. miR-124a-3 methylation and miR-34c methylation were more frequently identified in IM, with very few in non-IM mucosa among the three groups. H. pylori eradication can reverse methylation only in non-IM mucosa. CIMP in IM may have potential as a surrogate maker of GC development, and methylation of miR-124a-3 and miR-34c is a molecular event in IM that may not be associated with GC development.
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Epigénesis Genética , Helicobacter pylori/fisiología , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiología , Anciano , Antibacterianos/farmacología , Cadherinas/metabolismo , Epigénesis Genética/efectos de los fármacos , Femenino , Helicobacter pylori/efectos de los fármacos , Humanos , Masculino , Neoplasias Gástricas/metabolismo , Factores de TiempoRESUMEN
Congenital muscular dystrophies (CMDs) are characterized by progressive weakness and degeneration of skeletal muscle. In several forms of CMD, abnormal glycosylation of α-dystroglycan (α-DG) results in conditions collectively known as dystroglycanopathies, which are associated with central nervous system involvement. We recently demonstrated that fukutin, the gene responsible for Fukuyama congenital muscular dystrophy, encodes the ribitol-phosphate transferase essential for dystroglycan function. Brain pathology in patients with dystroglycanopathy typically includes cobblestone lissencephaly, mental retardation, and refractory epilepsy; however, some patients exhibit average intelligence, with few or almost no structural defects. Currently, there is no effective treatment for dystroglycanopathy, and the mechanisms underlying the generation of this broad clinical spectrum remain unknown. Here, we analysed four distinct mouse models of dystroglycanopathy: two brain-selective fukutin conditional knockout strains (neuronal stem cell-selective Nestin-fukutin-cKO and forebrain-selective Emx1-fukutin-cKO), a FukutinHp strain with the founder retrotransposal insertion in the fukutin gene, and a spontaneous Large-mutant Largemyd strain. These models exhibit variations in the severity of brain pathology, replicating the clinical heterogeneity of dystroglycanopathy. Immunofluorescence analysis of the developing cortex suggested that residual glycosylation of α-DG at embryonic day 13.5 (E13.5), when cortical dysplasia is not yet apparent, may contribute to subsequent phenotypic heterogeneity. Surprisingly, delivery of fukutin or Large into the brains of mice at E12.5 prevented severe brain malformation in Emx1-fukutin-cKO and Largemyd/myd mice, respectively. These findings indicate that spatiotemporal persistence of functionally glycosylated α-DG may be crucial for brain development and modulation of glycosylation during the fetal stage could be a potential therapeutic strategy for dystroglycanopathy.
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Encéfalo/embriología , Distroglicanos/metabolismo , Feto/embriología , Técnicas de Transferencia de Gen , Terapia Genética , Malformaciones del Desarrollo Cortical/terapia , Animales , Encéfalo/patología , Distroglicanos/genética , Femenino , Feto/patología , Glicosilación , Masculino , Malformaciones del Desarrollo Cortical/embriología , Malformaciones del Desarrollo Cortical/genética , Malformaciones del Desarrollo Cortical/patología , Ratones , Ratones TransgénicosRESUMEN
The risk of gastric cancer (GC) remains even after H. pylori eradication; thus, other combination treatments, such as chemopreventive drugs, are needed. We evaluated the effects of aspirin on genetic/epigenetic alterations in precancerous conditions, i.e., atrophic mucosa (AM) and intestinal metaplasia (IM), in patients with chronic gastritis who had taken aspirin for more than 3 years. A total of 221 biopsy specimens from 74 patients, including atrophic gastritis (AG) cases without aspirin use (control), AG cases with aspirin use (AG group), and GC cases with aspirin use (GC group), were analyzed. Aspirin use was associated with a significant reduction of CDH1 methylation in AM (OR: 0.15, 95% CI: 0.06-0.41, p = 0.0002), but was less effective in reversing the methylation that occurred in IM. Frequent hypermethylation including that of CDH1 in AM increased in the GC group compared to the AG group, and CDH1 methylation was an independent predictive marker of GC (OR: 8.50, 95% CI: 2.64-25.33, p = 0.0003). In patients with long-term aspirin use, the changes of molecular events in AM but not IM may be an important factor in the reduction of cancer incidence. In addition, methylation of the CDH1 gene in AM may be a surrogate of GC.
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Aspirina/efectos adversos , Mucosa Gástrica/efectos de los fármacos , Mucosa Gástrica/patología , Lesiones Precancerosas/etiología , Lesiones Precancerosas/patología , Neoplasias Gástricas/etiología , Neoplasias Gástricas/patología , Anciano , Anciano de 80 o más Años , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/efectos adversos , Aspirina/administración & dosificación , Estudios de Casos y Controles , Islas de CpG , Estudios Transversales , Metilación de ADN , Epigénesis Genética , Femenino , Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/complicaciones , Humanos , Masculino , Inestabilidad de Microsatélites/efectos de los fármacos , Lesiones Precancerosas/epidemiología , Neoplasias Gástricas/epidemiología , Neoplasias Gástricas/metabolismo , Factores de TiempoRESUMEN
It is recommended that small (6-10 mm) lesions be treated with endoscopic resection (ER), whereas diminutive (≤5 mm) lesions are not currently an indication for ER according to the Japanese guidelines. The aim of this study was to evaluate the molecular alterations, and therefore treatment indications, in diminutive versus small tubular adenoma (TA). We prospectively analyzed genetic instability, including microsatellite instability and loss of heterozygosity, methylation status, KRAS/BRAF mutations, and Ki-67 staining in 96 TAs without a villous component. Although no microsatellite instability was identified in either diminutive or small TAs, genetic instability was seen in small TAs (9.1%) but not diminutive TAs (P = .04). In addition, the low-level CpG island methylator phenotype (CIMP-L) was more frequently observed in small TAs (31.8%) than in diminutive TAs (P = .01). Thus, genetic instability and CIMP-L were associated with small TAs, and only CIMP-L was an independent predictive marker for small TAs (odds ratio, 3.29; P = .03). Intriguingly, the Ki-67 proliferative index tended to be higher in small TAs than in diminutive TAs (P = .06) and higher in TAs with CIMP-L than in those without CIMP (P = .08). KRAS mutations were seen in codon 12 in 5.2% of TAs, but no BRAF gene mutations were found. As the molecular events and proliferative activity for the progression may increase from diminutive to small TAs, small TAs should be treated with ER, whereas a "predict, resect, and discard" strategy may be acceptable in most diminutive lesions except flat and depressed-type lesions, in keeping with the current strategy in the West.
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Adenoma/genética , Pólipos Adenomatosos/genética , Biomarcadores de Tumor/genética , Proliferación Celular , Pólipos del Colon/genética , Neoplasias Colorrectales/genética , Islas de CpG , Metilación de ADN , Inestabilidad Genómica , Adenoma/química , Adenoma/patología , Adenoma/cirugía , Pólipos Adenomatosos/química , Pólipos Adenomatosos/patología , Pólipos Adenomatosos/cirugía , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Biopsia , Pólipos del Colon/química , Pólipos del Colon/patología , Pólipos del Colon/cirugía , Colonoscopía , Neoplasias Colorrectales/química , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/cirugía , Análisis Mutacional de ADN , Progresión de la Enfermedad , Predisposición Genética a la Enfermedad , Humanos , Inmunohistoquímica , Antígeno Ki-67/análisis , Pérdida de Heterocigocidad , Masculino , Inestabilidad de Microsatélites , Persona de Mediana Edad , Mutación , Fenotipo , Estudios Prospectivos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Carga TumoralRESUMEN
α-Dystroglycanopathy (α-DGP) is a group of muscular dystrophy characterized by abnormal glycosylation of α-dystroglycan (α-DG), including Fukuyama congenital muscular dystrophy (FCMD), muscle-eye-brain disease, Walker-Warburg syndrome, and congenital muscular dystrophy type 1D (MDC1D), etc. LARGE, the causative gene for MDC1D, encodes a glycosyltransferase to form [-3Xyl-α1,3GlcAß1-] polymer in the terminal end of the post-phosphoryl moiety, which is essential for α-DG function. It has been proposed that LARGE possesses the great potential to rescue glycosylation defects in α-DGPs regardless of causative genes. However, the in vivo therapeutic benefit of using LARGE activity is controversial. To explore the conditions needed for successful LARGE gene therapy, here we used Large-deficient and fukutin-deficient mouse models for MDC1D and FCMD, respectively. Myofibre-selective LARGE expression via systemic adeno-associated viral gene transfer ameliorated dystrophic pathology of Large-deficient mice even when intervention occurred after disease manifestation. However, the same strategy failed to ameliorate the dystrophic phenotype of fukutin-conditional knockout mice. Furthermore, forced expression of Large in fukutin-deficient embryonic stem cells also failed to recover α-DG glycosylation, however coexpression with fukutin strongly enhanced α-DG glycosylation. Together, our data demonstrated that fukutin is required for LARGE-dependent rescue of α-DG glycosylation, and thus suggesting new directions for LARGE-utilizing therapy targeted to myofibres.
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Expresión Génica , Fibras Musculares Esqueléticas/metabolismo , N-Acetilglucosaminiltransferasas/genética , Proteínas/genética , Síndrome de Walker-Warburg/genética , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Distroglicanos/metabolismo , Técnicas de Transferencia de Gen , Terapia Genética , Vectores Genéticos/genética , Glicosilación , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/patología , Proteínas/metabolismo , Transducción Genética , Transferasas , Síndrome de Walker-Warburg/metabolismo , Síndrome de Walker-Warburg/terapiaRESUMEN
Defects in dystroglycan glycosylation are associated with a group of muscular dystrophies, termed dystroglycanopathies, that include Fukuyama congenital muscular dystrophy (FCMD). It is widely believed that abnormal glycosylation of dystroglycan leads to disease-causing membrane fragility. We previously generated knock-in mice carrying a founder retrotransposal insertion in fukutin, the gene responsible for FCMD, but these mice did not develop muscular dystrophy, which hindered exploring therapeutic strategies. We hypothesized that dysferlin functions may contribute to muscle cell viability in the knock-in mice; however, pathological interactions between glycosylation abnormalities and dysferlin defects remain unexplored. To investigate contributions of dysferlin deficiency to the pathology of dystroglycanopathy, we have crossed dysferlin-deficient dysferlin(sjl/sjl) mice to the fukutin-knock-in fukutin(Hp/-) and Large-deficient Largemyd/myd mice, which are phenotypically distinct models of dystroglycanopathy. The fukutin(Hp/-) mice do not show a dystrophic phenotype; however, (dysferlin(sjl/sjl): fukutin(Hp/-)) mice showed a deteriorated phenotype compared with (dysferlinsjl/sjl: fukutin(Hp/+)) mice. These data indicate that the absence of functional dysferlin in the asymptomatic fukutin(Hp/-) mice triggers disease manifestation and aggravates the dystrophic phenotype. A series of pathological analyses using double mutant mice for Large and dysferlin indicate that the protective effects of dysferlin appear diminished when the dystrophic pathology is severe and also may depend on the amount of dysferlin proteins. Together, our results show that dysferlin exerts protective effects on the fukutin(Hp/-) FCMD mouse model, and the (dysferlin(sjl/sjl): fukutin(Hp/-)) mice will be useful as a novel model for a recently proposed antisense oligonucleotide therapy for FCMD.
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Distroglicanos/metabolismo , Proteínas de la Membrana/genética , Modelos Biológicos , Proteínas Musculares/genética , Músculo Esquelético/patología , Síndrome de Walker-Warburg/genética , Animales , Disferlina , Humanos , Ratones , Ratones Transgénicos , Síndrome de Walker-Warburg/metabolismoRESUMEN
α-synuclein (SNCA) is an established susceptibility gene for Parkinson's disease (PD), one of the most common human neurodegenerative disorders. Increased SNCA is considered to lead to PD and dementia with Lewy bodies. Four single-nucleotide polymorphisms (SNPs) in SNCA 3' region were prominently associated with PD among different ethnic groups. To examine how these SNPs influence disease susceptibility, we analyzed their potential effects on SNCA gene expression. We found that rs356219 showed allele-specific features. Gel shift assay using nuclear extracts from SH-SY5Y cells showed binding of one or more proteins to the protective allele, rs356219-A. We purified the rs356219-A-protein complex with DNA affinity beads and identified a bound protein using mass spectrometry. This protein, YY1 (Yin Yang 1), is an ubiquitous transcription factor with multiple functions. We next investigated SNCA expression change in SH-SY5Y cells by YY1 transfection. We also analyzed the expression of antisense noncoding RNA (ncRNA) RP11-115D19.1 in SNCA 3'-flanking region, because rs356219 is located in intron of RP11-115D19.1. Little change was observed in SNCA expression levels; however, RP11-115D19.1 expression was prominently stimulated by YY1. In autopsied cortices, positive correlation was observed among RP11-115D19.1, SNCA and YY1 expression levels, suggesting their functional interactions in vivo. Knockdown of RP11-115D19.1 increased SNCA expression significantly in SH-SY5Y cells, suggesting its repressive effect on SNCA expression. Our findings of the protective allele-specific YY1 and antisense ncRNA raised a novel possible mechanism to regulate SNCA expression.
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Regiones no Traducidas 3' , Proteínas Nucleares/metabolismo , ARN sin Sentido/biosíntesis , ARN no Traducido/biosíntesis , Factores de Transcripción/metabolismo , alfa-Sinucleína/metabolismo , Adulto , Anciano , Pueblo Asiatico , Proteínas de Ciclo Celular , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas Nucleares/química , Polimorfismo de Nucleótido Simple , Unión Proteica , ARN sin Sentido/genética , ARN no Traducido/genética , Factores de Transcripción/química , Población Blanca , alfa-Sinucleína/genéticaRESUMEN
A group of muscular dystrophies, dystroglycanopathy is caused by abnormalities in post-translational modifications of dystroglycan (DG). To understand better the pathophysiological roles of DG modification and to establish effective clinical treatment for dystroglycanopathy, we here generated two distinct conditional knock-out (cKO) mice for fukutin, the first dystroglycanopathy gene identified for Fukuyama congenital muscular dystrophy. The first dystroglycanopathy model-myofiber-selective fukutin-cKO [muscle creatine kinase (MCK)-fukutin-cKO] mice-showed mild muscular dystrophy. Forced exercise experiments in presymptomatic MCK-fukutin-cKO mice revealed that myofiber membrane fragility triggered disease manifestation. The second dystroglycanopathy model-muscle precursor cell (MPC)-selective cKO (Myf5-fukutin-cKO) mice-exhibited more severe phenotypes of muscular dystrophy. Using an isolated MPC culture system, we demonstrated, for the first time, that defects in the fukutin-dependent modification of DG lead to impairment of MPC proliferation, differentiation and muscle regeneration. These results suggest that impaired MPC viability contributes to the pathology of dystroglycanopathy. Since our data suggested that frequent cycles of myofiber degeneration/regeneration accelerate substantial and/or functional loss of MPC, we expected that protection from disease-triggering myofiber degeneration provides therapeutic effects even in mouse models with MPC defects; therefore, we restored fukutin expression in myofibers. Adeno-associated virus (AAV)-mediated rescue of fukutin expression that was limited in myofibers successfully ameliorated the severe pathology even after disease progression. In addition, compared with other gene therapy studies, considerably low AAV titers were associated with therapeutic effects. Together, our findings indicated that fukutin-deficient dystroglycanopathy is a regeneration-defective disorder, and gene therapy is a feasible treatment for the wide range of dystroglycanopathy even after disease progression.
Asunto(s)
Distroglicanos/metabolismo , Expresión Génica , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Mioblastos/metabolismo , Fenotipo , Animales , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Glicosilación , Ratones , Ratones Noqueados , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Factor 5 Regulador Miogénico/genética , Proteínas/genética , Proteínas/metabolismo , TransferasasRESUMEN
The polyglutamine (polyQ) diseases such as Huntington's disease (HD), are neurodegenerative diseases caused by proteins with an expanded polyQ stretch, which misfold and aggregate, and eventually accumulate as inclusion bodies within neurons. Molecules that inhibit polyQ protein misfolding/aggregation, such as Polyglutamine Binding Peptide 1 (QBP1) and molecular chaperones, have been shown to exert therapeutic effects in vivo by crossing of transgenic animals. Towards developing a therapy using these aggregation inhibitors, we here investigated the effect of viral vector-mediated gene therapy using QBP1 and molecular chaperones on polyQ disease model mice. We found that injection of adeno-associated virus type 5 (AAV5) expressing QBP1 or Hsp40 into the striatum both dramatically suppresses inclusion body formation in the HD mouse R6/2. AAV5-Hsp40 injection also ameliorated the motor impairment and extended the lifespan of R6/2 mice. Unexpectedly, we found even in virus non-infected cells that AAV5-Hsp40 appreciably suppresses inclusion body formation, suggesting a non-cell autonomous therapeutic effect. We further show that Hsp40 inhibits secretion of the polyQ protein from cultured cells, implying that it inhibits the recently suggested cell-cell transmission of the polyQ protein. Our results demonstrate for the first time the therapeutic effect of Hsp40 gene therapy on the neurological phenotypes of polyQ disease mice.
Asunto(s)
Terapia Genética , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/uso terapéutico , Enfermedad de Huntington/genética , Enfermedad de Huntington/terapia , Péptidos/metabolismo , Animales , Encéfalo/patología , Encéfalo/fisiopatología , Células Cultivadas , Dependovirus/metabolismo , Modelos Animales de Enfermedad , Fuerza de la Mano/fisiología , Enfermedad de Huntington/patología , Enfermedad de Huntington/fisiopatología , Cuerpos de Inclusión/metabolismo , Longevidad , Ratones , Actividad Motora/fisiología , Neuronas/metabolismo , Neuronas/patología , Neuronas/virología , Oligopéptidos/genética , Oligopéptidos/uso terapéutico , Fenotipo , Pérdida de PesoRESUMEN
To identify susceptibility variants for Parkinson's disease (PD), we performed a genome-wide association study (GWAS) and two replication studies in a total of 2,011 cases and 18,381 controls from Japan. We identified a new susceptibility locus on 1q32 (P = 1.52 x 10(-12)) and designated this as PARK16, and we also identified BST1 on 4p15 as a second new risk locus (P = 3.94 x 10(-9)). We also detected strong associations at SNCA on 4q22 (P = 7.35 x 10(-17)) and LRRK2 on 12q12 (P = 2.72 x 10(-8)), both of which are implicated in autosomal dominant forms of parkinsonism. By comparing results of a GWAS performed on individuals of European ancestry, we identified PARK16, SNCA and LRRK2 as shared risk loci for PD and BST1 and MAPT as loci showing population differences. Our results identify two new PD susceptibility loci, show involvement of autosomal dominant parkinsonism loci in typical PD and suggest that population differences contribute to genetic heterogeneity in PD.
Asunto(s)
Predisposición Genética a la Enfermedad , Variación Genética , Enfermedad de Parkinson/genética , Estudios de Casos y Controles , Frecuencia de los Genes , Heterogeneidad Genética , Genoma Humano , Estudio de Asociación del Genoma Completo , Humanos , Japón , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Enfermedad de Parkinson/epidemiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Riesgo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismoRESUMEN
The genetic factors underlying mental retardation (MR) are very heterogeneous. Recent studies have identified a number of genes involved in MR, several of which lie on the X-chromosome, but the current understanding of the monogenic causes of MR is far from complete. Investigation of chromosomal rearrangements in patients with MR has proven particularly informative in the search for novel genes. Using array-based comparative genomic hybridization analysis, we identified a small copy number gain at Xq25, which was undetectable by conventional G-band analysis, in a boy with unexplained MR. Further characterization revealed a partial tandem duplication of GRIA3, an alteration also present on one allele in his mother. RT-PCR analysis of lymphoblastoid cell RNA revealed remarkably reduced GRIA3 transcript levels in the patient. The mother, whose cognitive level is normal, also demonstrated remarkably reduced GRIA3 transcript levels in lymphoblastoid cells, and X-chromosome inactivation (XCI) was completely skewed in her peripheral lymphocytes. It is possible that XCI in the brain is not completely skewed and that GRIA3 expression from the normal allele may account for the mother's normal cognitive function. Taken together with previous findings of GRIA3 disruptions in the patients with MR, our study strengthens the idea that GRIA3 is a candidate gene for X-linked MR and that severely reduced GRIA3 expression results in MR.
Asunto(s)
Duplicación de Gen , Discapacidad Intelectual Ligada al Cromosoma X/genética , Receptores AMPA/genética , Secuencia de Bases , Femenino , Humanos , Masculino , Discapacidad Intelectual Ligada al Cromosoma X/diagnóstico , Datos de Secuencia Molecular , Mutación , Transcripción Genética , Inactivación del Cromosoma XRESUMEN
A genetic association between the fibroblast growth factor 20 (FGF20) gene and Parkinson's disease has been found by the pedigree disequilibrium test. This association, however, was not replicated by a case-control association study. In order to clarify the association between the FGF20 gene and Parkinson's disease, we attempted to replicate this association by a case-control association study using a large number of Japanese samples (1388 patients and 1891 controls). rs1721100 exhibited a significant difference in allele C versus G (P=0.0089), and in genotype CC+CG versus GG (P=0.0053). Haplotype association analysis showed that haplotype 2 was the protective haplotype for Parkinson's disease (permutation-P=0.0075). These results suggest that the FGF20 gene is a susceptibility gene for Parkinson's disease in the Japanese population.
Asunto(s)
Factores de Crecimiento de Fibroblastos/genética , Enfermedad de Parkinson/genética , Anciano , Alelos , Estudios de Casos y Controles , Femenino , Genotipo , Haplotipos , Humanos , Japón/epidemiología , Desequilibrio de Ligamiento/genética , Masculino , Persona de Mediana Edad , Enfermedad de Parkinson/epidemiología , Polimorfismo de Nucleótido Simple , RiesgoRESUMEN
Parkinson's disease (PD), one of the most common human neurodegenerative diseases, is characterized by the loss of dopaminergic neurons in the substantia nigra of the midbrain. PD is a complex disorder with multiple genetic and environmental factors influencing disease risk. To identify susceptible genes for sporadic PD, we performed case-control association studies of 268 single nucleotide polymorphisms (SNPs) in 121 candidate genes. In two independent case-control populations, we found that a SNP in alpha-synuclein (SNCA), rs7684318, showed the strongest association with PD (P=5.0 x 10(-10)). Linkage disequilibrium (LD) analysis using 29 SNPs in a region around rs7684318 revealed that the entire SNCA gene lies within a single LD block (D'>0.9) spanning approximately 120 kb. A tight LD group (r2>0.85) of six SNPs, including rs7684318, associated most strongly with PD (P=2.0 x 10(-9)-1.7 x 10(-11)). Haplotype association analysis did not show lower P-values than any single SNP within this group. SNCA is a major component of Lewy bodies, the pathological hallmark of PD. Aggregation of SNCA is thought to play a crucial role in PD. SNCA expression levels tended to be positively correlated with the number of the associated allele in autopsied frontal cortices. These findings establish SNCA as a definite susceptibility gene for sporadic PD.