RESUMEN
The antigenic peptides processed by ß-cells and presented through surface HLA class I molecules are poorly characterized. Each HLA variant (e.g., the most common being HLA-A2 and HLA-A3) carries some peptide-binding specificity. Hence, features that, despite these specificities, remain shared across variants may reveal factors favoring ß-cell immunogenicity. Building on our previous description of the HLA-A2/A3 peptidome of ß-cells, we analyzed the HLA-A3-restricted peptides targeted by circulating CD8+ T cells. Several peptides were recognized by CD8+ T cells within a narrow frequency (1-50/106), which was similar in donors with and without type 1 diabetes and harbored variable effector/memory fractions. These epitopes could be classified as conventional peptides or neoepitopes, generated either via peptide cis-splicing or mRNA splicing (e.g., secretogranin-5 [SCG5]-009). As reported for HLA-A2-restricted peptides, several epitopes originated from ß-cell granule proteins (e.g., SCG3, SCG5, and urocortin-3). Similarly, H-2Kd-restricted CD8+ T cells recognizing the murine orthologs of SCG5, urocortin-3, and proconvertase-2 infiltrated the islets of NOD mice and transferred diabetes into NOD/scid recipients. The finding of granule proteins targeted in both humans and NOD mice supports their disease relevance and identifies the insulin granule as a rich source of epitopes, possibly reflecting its impaired processing in type 1 diabetes.
Asunto(s)
Cromograninas/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Adulto , Empalme Alternativo , Animales , Linfocitos T CD8-positivos , Estudios de Casos y Controles , Cromograninas/genética , Simulación por Computador , Minería de Datos , Diabetes Mellitus Tipo 1/genética , Epítopos , Femenino , Regulación de la Expresión Génica , Antígeno HLA-A3 , Humanos , Insulina , Masculino , Ratones , Ratones Endogámicos NOD , Proteína 7B2 Secretora Neuroendocrina/genética , Proteína 7B2 Secretora Neuroendocrina/metabolismo , Unión Proteica , ARN Mensajero/genética , Urocortinas/genética , Urocortinas/metabolismo , Adulto JovenRESUMEN
Because of variable inconvenient living conditions in some places around the world, it is difficult to collect reliable physiological data for ostriches. Therefore, this study aims to provide a comprehensive in silico insight for the nature of polymorphism of important genetic loci that are related to physiological and reproductive traits. Sixty-nine mature ostriches ranging over half of Iraq were screened. Six exonic genetic loci, including cytochrome c oxidase I (COX1), cytochrome b (CYTB), secretogranin V (SCG5), feather keratin 2-like (FK2), prolactin (PRL) and placenta growth factor (PGF) were genotyped by PCR-single stranded conformation polymorphism (SSCP). Thirty-six novel SNPs, including seventeen nonsynonymous (ns) SNPs, were observed. Several computational software programs were utilized to assess the extent of the nsSNPs on their corresponding proteins structure, function and stability. The results showed several deleterious functional and stability changes in almost all the proteins studied. The total severity of each missense mutation was evaluated and compared with other nsSNPs accumulatively. It is evident from the extensive cumulative in silico computation that both p.E34D and p.E60K in PGF have the highest deleterious effect. The cumulative predictions from the present study are an impressive guide for the genotypes of African ostriches, which bypassed the expensive protocols for wet laboratory screening, to identify the effects of variants. To the best of our knowledge, this is the first investigation of its kind on the analyses and prediction outcome of missense mutations in African ostrich populations. The highly deleterious nsSNPs in the placenta growth factor are possible adaptive mutations which might be associated with adaptation in extreme and new environments. The flow and protocol of the computational predictions can be extended for various wild animals to identify the molecular nature of adaptations.
Asunto(s)
Adaptación Fisiológica , Proteínas Aviares/genética , Ciclooxigenasa 1/genética , Citocromos b/genética , Queratina-2/genética , Mutación Missense , Proteína 7B2 Secretora Neuroendocrina/genética , Factor de Crecimiento Placentario/genética , Polimorfismo de Nucleótido Simple , Prolactina/genética , Struthioniformes/genética , África , Animales , Genética de PoblaciónRESUMEN
Impairment of neuronal proteostasis is a hallmark of Alzheimer's and other neurodegenerative diseases. However, the underlying molecular mechanisms leading to pathogenic protein aggregation, and the role of secretory chaperone proteins in this process, are poorly understood. We have previously shown that the neural-and endocrine-specific secretory chaperone 7B2 potently blocks in vitro fibrillation of Aß42. To determine whether 7B2 can function as a chaperone in vivo, we measured plaque formation and performed behavioral assays in 7B2-deficient mice in an hAPPswe/PS1dE9 Alzheimer's model mouse background. Surprisingly, immunocytochemical analysis of cortical levels of thioflavin S- and Aß-reactive plaques showed that APP mice with a partial or complete lack of 7B2 expression exhibited a significantly lower number and burden of thioflavin S-reactive, as well as Aß-immunoreactive, plaques. However, 7B2 knockout did not affect total brain levels of either soluble or insoluble Aß. While hAPP model mice performed poorly in the Morris water maze, their brain 7B2 levels did not impact performance. Since 7B2 loss reduced amyloid plaque burden, we conclude that brain 7B2 can impact Aß disposition in a manner that facilitates plaque formation. These results are reminiscent of prior findings in hAPP model mice lacking the ubiquitous secretory chaperone clusterin.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Proteína 7B2 Secretora Neuroendocrina/deficiencia , Placa Amiloide/metabolismo , Placa Amiloide/patología , Animales , Benzotiazoles/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/fisiopatología , Clusterina/metabolismo , Modelos Animales de Enfermedad , Femenino , Genotipo , Heterocigoto , Humanos , Memoria , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína 7B2 Secretora Neuroendocrina/genética , Proteína 7B2 Secretora Neuroendocrina/metabolismo , Placa Amiloide/inmunología , Placa Amiloide/fisiopatología , Solubilidad , TransgenesRESUMEN
Since first characterized in 1997, patients with hereditary mixed polyposis syndrome (HMPS) have been difficult to identify because of lack of well-established diagnostic criteria. Recently, HMPS was found to be caused by a duplication on chromosome 15 spanning the 3' end of the SCG5 gene and a region upstream of the GREM1 locus. Clinical testing for the duplication is available; however, the clinical characteristics of hereditary mixed polyposis to support testing are ill defined. The clinicopathological findings of 10 HMPS patients with confirmed germline SCG5-GREM1 duplication were reviewed. Mean age at presentation was 33.3 years. Fifty-one colonoscopies yielded 207 polyp specimens, all of which were reexamined. Adenomas (n = 80) and a fairly unique polyp composed of a mixture of hyperplastic polyp and inflammatory polyp-type changes (n = 74) were the most common findings; however, other polyps, including hyperplastic (n = 28), mixed inflammatory polyp/adenoma (n = 8), inflammatory polyp (n = 7), prolapse-type polyp (n = 6), and lymphoid aggregates (n = 4), were encountered. None of the patients developed colorectal malignancy during surveillance, demonstrated extracolonic manifestations, or underwent colectomy on follow-up (mean, 26.2 years). SCG5-GREM1 duplication-associated polyposis is characterized by a few polyps per endoscopy with a mixture of phenotypes, most commonly adenoma and nondysplastic mixed hyperplastic/inflammatory polyps. Nine of 10 patients had at least 1 mixed hyperplastic-inflammatory polyp, which is the characteristic lesion of SCG5-GREM1 duplication-associated HMPS.
Asunto(s)
Poliposis Adenomatosa del Colon/genética , Biomarcadores de Tumor/genética , Pólipos del Colon/genética , Duplicación de Gen , Fusión Génica , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína 7B2 Secretora Neuroendocrina/genética , Poliposis Adenomatosa del Colon/etnología , Poliposis Adenomatosa del Colon/patología , Poliposis Adenomatosa del Colon/cirugía , Adulto , Anciano , Biopsia , Niño , Pólipos del Colon/etnología , Pólipos del Colon/patología , Pólipos del Colon/cirugía , Colonoscopía , Bases de Datos Factuales , Predisposición Genética a la Enfermedad , Herencia , Humanos , Judíos/genética , Persona de Mediana Edad , Linaje , Fenotipo , Adulto JovenRESUMEN
FAM20C is a secretory kinase responsible for the phosphorylation of multiple secreted proteins in mammalian cells; it has been shown to phosphorylate serine residues within a variety of different bone proteins. In this work we demonstrate that FAM20C also phosphorylates threonines, specifically those within the N-terminal domain of the neuroendocrine chaperone 7B2. Analysis of the primary sequence of 7B2 revealed that three threonine residues in its N-terminal domain are located within FAM20C consensus motifs: Thr73, Thr99, and Thr111. The individual substitution of Thr73 and Thr111 residues by neutral alanines caused a marked decrease in the total phosphorylation of 7B2. Furthermore, the phosphomimetic substitution of Thr111 by Glu clearly diminished the ability of 7B2 to activate pro-prohormone convertase 2 (PC2) in 7B2-lacking SK-N-MC neuroblastoma cells, suggesting that the phosphorylation of this residue critically impacts the 7B2-proPC2 interaction. However, the phosphomimetic mutation did not alter 7B2's ability to function as an antiaggregant for human islet amyloid polypeptide. FAM20C-mediated phosphorylation of a common alternatively spliced variant of human 7B2 that lacks Ala100 (thus eliminating the Thr99 phosphorylation consensus site) was similar to the Ala-containing protein, but this variant did not activate proPC2 as efficiently as the Ala-containing protein. Although threonines within 7B2 were phosphorylated efficiently, FAM20C was incapable of performing the well-known regulatory threonine phosphorylation of the molecular chaperone binding immunoglobulin protein. Taken together, these results indicate that FAM20C plays a role in 7B2-mediated proPC2 activation by phosphorylating residue Thr111; and that 7B2 function is regulated by alternative splicing.
Asunto(s)
Proteína 7B2 Secretora Neuroendocrina/metabolismo , Proproteína Convertasa 2/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Quinasa de la Caseína I/metabolismo , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Activación Enzimática , Proteínas de la Matriz Extracelular/metabolismo , Células HEK293 , Proteínas de Choque Térmico/metabolismo , Humanos , Datos de Secuencia Molecular , Proteína 7B2 Secretora Neuroendocrina/genética , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
Reliable preoperative diagnosis of malignant thyroid tumors remains challenging because of the inconclusive cytological examination of fine-needle aspiration biopsies. Although numerous studies have successfully demonstrated the use of high-throughput molecular diagnostics in cancer prediction, the application of microarrays in routine clinical use remains limited. Our aim was, therefore, to identify a small subset of genes to develop a practical and inexpensive diagnostic tool for clinical use. We developed a two-step feature selection method composed of a linear models for microarray data (LIMMA) linear model and an iterative Bayesian model averaging model to identify a suitable gene set signature. Using one public dataset for training, we discovered a three-gene signature dipeptidyl-peptidase 4 (DPP4), secretogranin V (SCG5) and carbonic anhydrase XII (CA12). We then evaluated the robustness of our gene set using three other independent public datasets. The gene signature accuracy was 85.7, 78.8 and 85.7%, respectively. For experimental validation, we collected 70 thyroid samples from surgery and our three-gene signature method achieved an accuracy of 94.3% by quantitative polymerase chain reaction (QPCR) experiment. Furthermore, immunohistochemistry in 29 samples showed proteins expressed by these three genes are also differentially expressed in thyroid samples. Our protocol discovered a robust three-gene signature that can distinguish benign from malignant thyroid tumors, which will have daily clinical application.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Nódulo Tiroideo/genética , Nódulo Tiroideo/patología , Adulto , Anciano , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/metabolismo , Conjuntos de Datos como Asunto , Diagnóstico Diferencial , Dipeptidil Peptidasa 4/genética , Dipeptidil Peptidasa 4/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Proteína 7B2 Secretora Neuroendocrina/genética , Proteína 7B2 Secretora Neuroendocrina/metabolismo , Pronóstico , Reproducibilidad de los Resultados , Neoplasias de la Tiroides/diagnóstico , Nódulo Tiroideo/diagnóstico , Adulto JovenRESUMEN
PURPOSE: Several researchers have suggested that the rs4779584 (15q13.3) polymorphism is associated with an increased risk of developing colorectal cancer (CRC). However, past results remain inconclusive. We addressed this controversy by performing a meta-analysis of the relationship between rs4779584 of GREM1-SCG5 and colorectal cancer. METHODS: We selected 12 case-control studies involving 11,769 cases of CRC and 14,328 healthy controls. The association between the rs4779584 polymorphism and CRC was examined by the overall odds ratio (OR) with a 95% confidence interval (CI). We used different genetic model analyses, sensitivity analyses, and assessments of bias in our meta-analysis. RESULTS: GREM1-SCG5 rs4779584 polymorphisms were associated with CRC in all of the genetic models that were examined in this meta-analysis of 12 case-control studies. CONCLUSION: GREM1-SCG5 rs4779584 polymorphisms may increase the risk of developing colorectal cancer.
Asunto(s)
Cromosomas Humanos Par 15/genética , Neoplasias Colorrectales/genética , Predisposición Genética a la Enfermedad/genética , Polimorfismo de Nucleótido Simple/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Modelos Genéticos , Proteína 7B2 Secretora Neuroendocrina/genética , Oportunidad Relativa , Análisis de RegresiónRESUMEN
Inactivating mutations of the "phosphate regulating gene with homologies to endopeptidases on the X chromosome" (PHEX/Phex) underlie disease in patients with X-linked hypophosphatemia (XLH) and the hyp-mouse, a murine homologue of the human disorder. Although increased serum fibroblast growth factor 23 (FGF-23) underlies the HYP phenotype, the mechanism(s) by which PHEX mutations inhibit FGF-23 degradation and/or enhance production remains unknown. Here we show that treatment of wild-type mice with the proprotein convertase (PC) inhibitor, decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (Dec), increases serum FGF-23 and produces the HYP phenotype. Because PC2 is uniquely colocalized with PHEX in osteoblasts/bone, we examined if PC2 regulates PHEX-dependent FGF-23 cleavage and production. Transfection of murine osteoblasts with PC2 and its chaperone protein 7B2 cleaved FGF-23, whereas Signe1 (7B2) RNA interference (RNAi) transfection, which limited 7B2 protein production, decreased FGF-23 degradation and increased Fgf-23 mRNA and protein. The mechanism by which decreased 7B2â¢PC2 activity influences Fgf-23 mRNA was linked to reduced conversion of the precursor to bone morphogenetic protein 1 (proBMP1) to active BMP1, which resulted in limited cleavage of dentin matrix acidic phosphoprotein 1 (DMP1), and consequent increased Fgf-23 mRNA. The significance of decreased 7B2â¢PC2 activity in XLH was confirmed by studies of hyp-mouse bone, which revealed significantly decreased Sgne1 (7B2) mRNA and 7B2 protein, and limited cleavage of proPC2 to active PC2. The expected downstream effects of these changes included decreased FGF-23 cleavage and increased FGF-23 synthesis, secondary to decreased BMP1-mediated degradation of DMP1. Subsequent Hexa-D-Arginine treatment of hyp-mice enhanced bone 7B2â¢PC2 activity, normalized FGF-23 degradation and production, and rescued the HYP phenotype. These data suggest that decreased PHEX-dependent 7B2â¢PC2 activity is central to the pathogenesis of XLH.
Asunto(s)
Raquitismo Hipofosfatémico Familiar/tratamiento farmacológico , Raquitismo Hipofosfatémico Familiar/patología , Enfermedades Genéticas Ligadas al Cromosoma X , Proteína 7B2 Secretora Neuroendocrina/metabolismo , Oligopéptidos/uso terapéutico , Osteoblastos/patología , Proproteína Convertasa 2/metabolismo , Animales , Huesos/diagnóstico por imagen , Huesos/efectos de los fármacos , Huesos/metabolismo , Huesos/patología , Proteínas de la Matriz Extracelular/metabolismo , Raquitismo Hipofosfatémico Familiar/diagnóstico por imagen , Raquitismo Hipofosfatémico Familiar/metabolismo , Femenino , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Proteína 7B2 Secretora Neuroendocrina/genética , Oligopéptidos/farmacología , Osteoblastos/efectos de los fármacos , Osteoblastos/enzimología , Fenotipo , Proproteína Convertasa 2/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , RadiografíaRESUMEN
In a genome-wide screen using DMH (differential methylation hybridization) we have identified a CpG island within the 5' region and untranslated first exon of the secretory granule neuroendocrine protein 1 gene (SGNE1/7B2) that showed hypermethylation in low- and high-grade astrocytomas compared to normal brain tissue. Pyrosequencing was performed to confirm the methylation status of this CpG island in 89 astrocytic gliomas of different malignancy grades and six glioma cell lines. Hypermethylation of SGNE1/7B2 was significantly more frequent in diffuse low-grade astrocytomas as well as secondary glioblastomas and anaplastic astrocytomas as compared to primary glioblastomas. mRNA expression analysis by real-time RT-PCR indicates that SGNE1/7B2 expression is downregulated in astrocytic gliomas compared to white matter samples. Treatment of glioma cells with the demethylating agent 5-aza-2'-deoxycytidine restores the transcription of SGNE1/7B2. Overexpression of SGNE1/7B2 in T98G, A172 and U373MG glioblastoma cells significantly suppressed focus formation and led to a significant increase in apoptotic cells as determined by flow cytometric analysis in T98G cells. In summary, we have identified SGNE1/7B2 as a novel target silenced by DNA methylation in astrocytic gliomas. The high incidence of this alteration and the significant effects of SGNE1/7B2 on the growth and apoptosis of glioblastoma cells provide a first proof for a functional implication of SGNE1/7B2 inactivation in the molecular pathology of gliomas.
Asunto(s)
Regiones no Traducidas 5' , Astrocitoma/genética , Islas de CpG , Metilación de ADN , Proteína 7B2 Secretora Neuroendocrina/genética , Proteína 7B2 Secretora Neuroendocrina/metabolismo , Apoptosis , Astrocitoma/metabolismo , Astrocitoma/patología , Azacitidina/análogos & derivados , Azacitidina/farmacología , Secuencia de Bases , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Decitabina , Epigénesis Genética , Exones , Regulación Neoplásica de la Expresión Génica , Humanos , Proteína 7B2 Secretora Neuroendocrina/biosíntesis , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Transcripción Genética/efectos de los fármacosRESUMEN
M cells assist mucosal immune surveillance by transcytosis of particles to underlying lymphoid tissue, but the mechanisms of M cell differentiation are poorly understood. To develop a better defined cell culture model of M cell differentiation, we treated human (Caco-2BBe) and rat (IEC-6) intestinal epithelial cell lines with lymphotoxin beta receptor (LTbetaR) and TNF receptor (TNFR) agonists. Treated cells were studied for regulation of genes associated with M cell and follicle-associated epithelium (FAE). We found that LTbetaR and TNFR agonists induce transcription of FAE-specific genes (Ccl20 and Lamb3) in Caco2-BBe cells and IEC-6 cells as well as rodent M cell specific genes such as Sgne-1/Scg5, Cldn4, and Gp2. The cytokines have distinct but complementary effects; TNFR agonists mainly induced FAE-specific genes, while the LTbetaR agonist induced M cell specific genes. The combination of cytokines showed additive induction of the FAE-associated Ccl20, Lamb3 and a surprising induction of CD137/Tnfrsf9. On the other hand TNF agonists appeared to suppress expression of some LTbetaR-induced genes. Functionally, cytokine treatment led to the reorganization of microvilli and Claudin-4 redistribution. These studies suggest complex interactions between these cytokines in the context of either inflammation or tissue differentiation.
Asunto(s)
Células Epiteliales/metabolismo , Mucosa Intestinal/citología , Receptor beta de Linfotoxina/agonistas , Receptores del Factor de Necrosis Tumoral/agonistas , Regulación hacia Arriba/efectos de los fármacos , Animales , Anexina A5/genética , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Células CACO-2 , Moléculas de Adhesión Celular/genética , Línea Celular , Línea Celular Tumoral , Quimiocina CCL20/genética , Claudina-4 , Citocinas/genética , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/microbiología , Proteínas Ligadas a GPI , Humanos , Receptor beta de Linfotoxina/inmunología , Glicoproteínas de Membrana/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microvellosidades/efectos de los fármacos , Proteína 7B2 Secretora Neuroendocrina/genética , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Transporte de Proteínas/efectos de los fármacos , Ratas , Staphylococcus aureus/inmunología , Factor de Transcripción ReIB/genética , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Factor de Necrosis Tumoral alfa/farmacología , KalininaRESUMEN
Pax6 is important in the development of the pancreas and was previously shown to regulate pancreatic endocrine differentiation, as well as the insulin, glucagon, and somatostatin genes. Prohormone convertase 2 (PC2) is the main processing enzyme in pancreatic alpha cells, where it processes proglucagon to produce glucagon under the spatial and temporal control of 7B2, which functions as a molecular chaperone. To investigate the role of Pax6 in glucagon biosynthesis, we studied potential target genes in InR1G9 alpha cells transfected with Pax6 small interfering RNA and in InR1G9 clones expressing a dominant-negative form of Pax6. We now report that Pax6 controls the expression of the PC2 and 7B2 genes. By binding and transactivation studies, we found that Pax6 indirectly regulates PC2 gene transcription through cMaf and Beta2/NeuroD1 while it activates the 7B2 gene both directly and indirectly through the same transcription factors, cMaf and Beta2/NeuroD1. We conclude that Pax6 is critical for glucagon biosynthesis and processing by directly and indirectly activating the glucagon gene through cMaf and Beta2/NeuroD1, as well as the PC2 and 7B2 genes.
Asunto(s)
Proteínas del Ojo/fisiología , Regulación de la Expresión Génica/fisiología , Proteínas de Homeodominio/fisiología , Proteína 7B2 Secretora Neuroendocrina/genética , Factores de Transcripción Paired Box/fisiología , Proproteína Convertasa 2/genética , Proteínas Represoras/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular , Cricetinae , Glucagón/biosíntesis , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Proteínas del Tejido Nervioso/genética , Factor de Transcripción PAX6 , Proglucagón/metabolismo , Proteínas Proto-Oncogénicas c-maf/genética , Activación TranscripcionalRESUMEN
OBJECTIVE: Chromosome 15q11-q13 has been proposed to harbor a gene for autism susceptibility because deletions of the region lead to Prader-Willi syndrome and Angelman syndrome, whose phenotypes overlap with autism. These deletions generally occur with the use of three commonly recognized breakpoints (BP1, BP2, and BP3); therefore, it may be possible that genes located in the breakpoints are impaired and contribute to autism susceptibility. No study, however, has investigated the genetic association between the breakpoints and autism, to our knowledge. Here, we investigated the association between the common breakpoints of chromosome 15q11-q13 and autism in a Japanese population. METHODS: We genotyped 12 single nucleotide polymorphisms (SNPs) in 166 patients with autistic disorder and 415 healthy controls. The SNPs are located in two additional distal breakpoints (BP4 and BP5), involved in duplications and triplications of the region, as well as in BP1 and BP3. RESULTS: No significant difference was observed between the controls and patients in allelic frequencies or genotypic distributions of the 12 SNPs. In the analyses of the suggested five haplotypes, no significant difference between the controls and patients was observed in the distributions of any estimated haplotypes. When confining the patients to only males, a difference was observed in a two-marker haplotype in BP3 between the controls and patients (global permutation P value=0.006), although the statistical level became insignificant after correction for multiple testing. CONCLUSION: This study provides no positive evidence of the association between the common breakpoints of chromosome 15q11-q13 and autism in the Japanese population.
Asunto(s)
Pueblo Asiatico/genética , Trastorno Autístico/genética , Rotura Cromosómica , Cromosomas Humanos Par 15/genética , Predisposición Genética a la Enfermedad , Adulto , Cadherinas/genética , Proteínas Portadoras/genética , Estudios de Casos y Controles , Femenino , Frecuencia de los Genes , Haplotipos , Humanos , Japón , Desequilibrio de Ligamiento/genética , Masculino , Proteínas del Tejido Nervioso/genética , Proteína 7B2 Secretora Neuroendocrina/genética , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
BACKGROUND: The identification of novel genes is critical to understanding the molecular basis of body weight. Towards this goal, we have identified secretogranin V (Scg5; also referred to as Sgne1), as a candidate gene for growth traits. RESULTS: Through a combination of DNA microarray analysis and quantitative PCR we identified a strong expression quantitative trait locus (eQTL) regulating Scg5 expression in two mouse chromosome 2 congenic strains and three additional F2 intercrosses. More importantly, the eQTL was coincident with a body weight QTL in congenic mice and Scg5 expression was negatively correlated with body weight in two of the F2 intercrosses. Analysis of haplotype blocks and genomic sequencing of Scg5 in high (C3H/HeJ, DBA/2J, BALB/cByJ, CAST/EiJ) and low (C57BL/6J) expressing strains revealed mutations unique to C57BL/6J and possibly responsible for the difference in mRNA abundance. To evaluate the functional consequence of Scg5 overexpression we measured the pituitary levels of 7B2 protein and PCSK2 activity and found both to be increased. In spite of this increase, the level of pituitary alpha-MSH, a PCSK2 processing product, was unaltered. CONCLUSION: Together, these data support a role for Scg5 in the modulation of body weight.
Asunto(s)
Peso Corporal/genética , Regulación Enzimológica de la Expresión Génica , Proteína 7B2 Secretora Neuroendocrina/genética , Proproteína Convertasa 2/genética , Sitios de Carácter Cuantitativo , Animales , Perfilación de la Expresión Génica , Ratones , Ratones Congénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Regulación hacia ArribaRESUMEN
Prohormone convertases (PCs) are proteinases that cleave inactive prohormones to biologically active peptides. Seven PCs have been identified; two of them, PC1/3 and PC2, have only been localized in neuroendocrine (NE) tissues; a third, furin, in both endocrine and exocrine tissues. We have studied the immunoreactivity of PC1/3, PC2 and furin in the four major NE cell types of the human pancreas by using double immunofluorescence techniques. The study also included the expression of NE secretory protein 7B2 (secretogranin V), a member of the granin family, which influences the function of PC2. The results showed that the three PCs and 7B2 were expressed only in endocrine pancreas, furin also in exocrine cells. Insulin (B) cells harboured PC1/3 and PC2, but not furin. Glucagon (A) cells were immunoreactive to all three PCs; all glucagon cells expressed PC2, but one subpopulation showed PC1/3 immunoreactivity and another furin. Only a few somatostatin (D) cells contained PC2, but no other proconvertase. Pancreatic polypeptide (PP) cells were non-reactive to all three PCs. 7B2 occurred only in insulin and glucagon cells. A varying co-localization pattern was observed between PCs and between PCs and 7B2, with the exception of PC1/3 and furin which were not co-localized. In conclusion, our study shows that PCs are localized in insulin and glucagon cells and do seem to be important in these cell types for processing of hormone and other protein precursors, especially chromogranins, but for the two other major cell types probably other enzymes are of importance.
Asunto(s)
Furina/metabolismo , Islotes Pancreáticos/fisiología , Proteína 7B2 Secretora Neuroendocrina/metabolismo , Páncreas/metabolismo , Proproteína Convertasa 1/metabolismo , Proproteína Convertasa 2/metabolismo , Células Cultivadas , Furina/genética , Humanos , Proteína 7B2 Secretora Neuroendocrina/genética , Páncreas/citología , Páncreas/enzimología , Proproteína Convertasa 1/genética , Proproteína Convertasa 2/genéticaRESUMEN
Cellular synthesis of naturally occurring, bioactive peptides requires the proprotein convertase PC2/EGL-3 for cleavage from the larger peptide precursors. A neuroendocrine chaperone 7B2 is needed for the proteolytical activation of proPC2, as extensively studied in mouse models. To determine the role of its orthologue in Caenorhabditis elegans, we analyzed wild-type and 7B2-null strains by HPLC and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, which allowed the identification of a novel neuropeptide gene, flp-33. The presence and/or absence of some neuropeptides in 7B2-null animals strongly differs form the peptide profile in wild-type, suggesting a specific and determined action of 7B2 in C. elegans.
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Caenorhabditis elegans/metabolismo , Proteína 7B2 Secretora Neuroendocrina/deficiencia , Neuropéptidos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Cromatografía Líquida de Alta Presión , Genes de Helminto , Datos de Secuencia Molecular , Mutación/genética , Proteína 7B2 Secretora Neuroendocrina/química , Proteína 7B2 Secretora Neuroendocrina/genética , Neuropéptidos/química , Proteómica , Alineación de Secuencia , Análisis de Secuencia de ProteínaRESUMEN
BACKGROUND: 7B2 is a regulator/activator of the prohormone convertase 2 which is involved in the processing of numerous neuropeptides, including insulin, glucagon and pro-opiomelanocortin. We have previously described a suggestive genetic linkage peak with childhood obesity on chr15q12-q14, where the 7B2 encoding gene, SGNE1 is located. The aim of this study is to analyze associations of SGNE1 genetic variation with obesity and metabolism related quantitative traits. METHODS: We screened SGNE1 for genetic variants in obese children and genotyped 12 frequent single nucleotide polymorphisms (SNPs). Case control analyses were performed in 1,229 obese (534 children and 695 adults), 1,535 individuals with type 2 diabetes and 1,363 controls, all French Caucasians. We also studied 4,922 participants from the D.E.S.I.R prospective population-based cohort. RESULTS: We did not find any association between SGNE1 SNPs and childhood or adult obesity. However, the 5' region SNP -1,701A>G associated with higher area under glucose curve after oral glucose tolerance test (p = 0.0005), higher HOMA-IR (p = 0.005) and lower insulinogenic index (p = 0.0003) in obese children. Similar trends were found in obese adults. SNP -1,701A>G did not associate with risk of T2D but tends to associate with incidence of type 2 diabetes (HR = 0.75 95%CI [0.55-1.01]; p = 0.06) in the prospective cohort. CONCLUSION: SGNE1 genetic variation does not contribute to obesity and common forms of T2D but may worsen glucose intolerance and insulin resistance, especially in the background of severe and early onset obesity. Further molecular studies are required to understand the molecular bases involved in this process.
Asunto(s)
Variación Genética , Intolerancia a la Glucosa/genética , Proteína 7B2 Secretora Neuroendocrina/genética , Obesidad/genética , Adolescente , Adulto , Estudios de Casos y Controles , Niño , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/genética , Femenino , Francia , Genotipo , Humanos , Resistencia a la Insulina/genética , Masculino , Persona de Mediana Edad , Obesidad/complicaciones , Polimorfismo de Nucleótido SimpleRESUMEN
In a genome-wide screen using differential methylation hybridization (DMH), we have identified a CpG island within the 5' region and untranslated first exon of the secretory granule neuroendocrine protein 1 gene (SGNE1/7B2) that showed hypermethylation in medulloblastomas compared to fetal cerebellum. Bisulfite sequencing and combined bisulfite restriction assay were performed to confirm the methylation status of this CpG island in primary medulloblastomas and medulloblastoma cell lines. Hypermethylation was detected in 16/23 (70%) biopsies and 7/8 (87%) medulloblastoma cell lines, but not in non-neoplastic fetal (n=8) cerebellum. Expression of SGNE1 was investigated by semi-quantitative competitive reverse transcription-polymerase chain reaction and found to be significantly downregulated or absent in all, but one primary medulloblastomas and all cell lines compared to fetal cerebellum. After treatment of medulloblastoma cell lines with 5-aza-2'-deoxycytidine, transcription of SGNE1 was restored. No mutation was found in the coding region of SGNE1 by single-strand conformation polymorphism analysis. Reintroduction of SGNE1 into the medulloblastoma cell line D283Med led to a significant growth suppression and reduced colony formation. In summary, we have identified SGNE1 as a novel epigenetically silenced gene in medulloblastomas. Its frequent inactivation, as well as its inhibitory effect on tumor cell proliferation and focus formation strongly argues for a significant role in medulloblastoma development.
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Neoplasias Cerebelosas/patología , Metilación de ADN , Meduloblastoma/patología , Proteína 7B2 Secretora Neuroendocrina/genética , Azacitidina/análogos & derivados , Azacitidina/farmacología , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular , Neoplasias Cerebelosas/genética , Islas de CpG/genética , Metilasas de Modificación del ADN/antagonistas & inhibidores , Decitabina , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Meduloblastoma/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Transcripción Genética/efectos de los fármacosRESUMEN
C57BL/6 (B6) mice develop glucose intolerance with age, whereas C3H/He (C3H) mice do not. In this study, we examined whether this differential glucose homeostasis was associated with differences of proteolytic activation of pancreatic prohormones. Radioimmunoassays showed comparable levels of fasting plasma insulin between the two strains but a significantly lower glucagon level in B6 mice. Pulse-chase analysis of glucagon biosynthesis in isolated pancreatic islets revealed that proglucagon was less efficiently processed in B6 mice. Because proprotein convertase (PC)2 and its 7B2 helper protein are required for this processing, we quantified islet mRNA levels by RT-PCR and protein levels by immunoblotting. The levels of proPC2 mRNA were similar between the two strains, but B6 protein extracts contained less of the mature PC2. In contrast, 7B2 mRNA and protein levels were both significantly lower in B6 pancreas. Sequencing of the 7B2 gene promoter and cDNA in the two strains revealed seven single nucleotide polymorphisms and one dinucleotide insertion/deletion in the cDNA as well as a single nucleotide polymorphism and two insertions/deletions in the promoter. Differential expression of 7B2 may contribute to the difference between B6 and C3H mice not only in glucagon production and secretion but also in glucose tolerance.