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AIMS/HYPOTHESIS: The proinflammatory cytokines IFN-α, IFN-γ, IL-1ß and TNF-α may contribute to innate and adaptive immune responses during insulitis in type 1 diabetes and therefore represent attractive therapeutic targets to protect beta cells. However, the specific role of each of these cytokines individually on pancreatic beta cells remains unknown. METHODS: We used deep RNA-seq analysis, followed by extensive confirmation experiments based on reverse transcription-quantitative PCR (RT-qPCR), western blot, histology and use of siRNAs, to characterise the response of human pancreatic beta cells to each cytokine individually and compared the signatures obtained with those present in islets of individuals affected by type 1 diabetes. RESULTS: IFN-α and IFN-γ had a greater impact on the beta cell transcriptome when compared with IL-1ß and TNF-α. The IFN-induced gene signatures have a strong correlation with those observed in beta cells from individuals with type 1 diabetes, and the level of expression of specific IFN-stimulated genes is positively correlated with proteins present in islets of these individuals, regulating beta cell responses to 'danger signals' such as viral infections. Zinc finger NFX1-type containing 1 (ZNFX1), a double-stranded RNA sensor, was identified as highly induced by IFNs and shown to play a key role in the antiviral response in beta cells. CONCLUSIONS/INTERPRETATION: These data suggest that IFN-α and IFN-γ are key cytokines at the islet level in human type 1 diabetes, contributing to the triggering and amplification of autoimmunity.
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Diabetes Mellitus Tipo 1 , Islotes Pancreáticos , Humanos , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Interferones/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Interferón gamma/metabolismo , Islotes Pancreáticos/metabolismoRESUMEN
PURPOSE: Familial neurohypophyseal diabetes insipidus (FNDI) is a rare disorder characterized by childhood-onset progressive polyuria and polydipsia due to mutations in the arginine vasopressin (AVP) gene. The aim of the study was to describe the clinical and molecular characteristics of families with neurohypophyseal diabetes insipidus. METHODS: Five Portuguese families with autosomal dominant FNDI underwent sequencing of the AVP gene and the identified mutations were functionally characterized by in vitro studies. RESULTS: Three novel and two recurrent heterozygous mutations were identified in the AVP gene. These consisted of one initiation codon mutation in the signal peptide coding region (c.2T > C, p.Met1?), three missense mutations in the neurophysin II (NPII) coding region (c.154T > C, p.Cys52Arg; c.289C > G, p.Arg97Gly; and c.293G > C, p.Cys98Ser), and one nonsense mutation in the NPII coding region (c.343G > T, p.Glu115Ter). In vitro transfection of neuronal cells with expression vectors containing each mutation showed that the mutations resulted in intracellular retention of the vasopressin prohormone. Patients showed progressive symptoms of polyuria and polydipsia, but with wide variability in severity and age at onset. No clear genotype-phenotype correlation was observed. CONCLUSION: The intracellular accumulation of mutant vasopressin precursors supports the role of cellular toxicity of the mutant proteins in the etiology of the disorder and explains the progressive onset of the disorder. These findings further expand the AVP mutational spectrum in FNDI and contribute to the understanding of the molecular pathogenic mechanisms involved in FNDI.
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Diabetes Insípida Neurogénica , Diabetes Insípida , Diabetes Mellitus , Arginina Vasopresina/genética , Diabetes Insípida Neurogénica/genética , Humanos , Mutación/genética , Neurofisinas/genética , Linaje , Polidipsia , Poliuria , Vasopresinas/genéticaRESUMEN
Pancreatic beta cell failure is the central event leading to diabetes. Beta cells share many phenotypic traits with neurons, and proper beta cell function relies on the activation of several neuron-like transcription programs. Regulation of gene expression by alternative splicing plays a pivotal role in brain, where it affects neuronal development, function, and disease. The role of alternative splicing in beta cells remains unclear, but recent data indicate that splicing alterations modulated by both inflammation and susceptibility genes for diabetes contribute to beta cell dysfunction and death. Here we used RNA sequencing to compare the expression of splicing-regulatory RNA-binding proteins in human islets, brain, and other human tissues, and we identified a cluster of splicing regulators that are expressed in both beta cells and brain. Four of them, namely Elavl4, Nova2, Rbox1, and Rbfox2, were selected for subsequent functional studies in insulin-producing rat INS-1E, human EndoC-ßH1 cells, and in primary rat beta cells. Silencing of Elavl4 and Nova2 increased beta cell apoptosis, whereas silencing of Rbfox1 and Rbfox2 increased insulin content and secretion. Interestingly, Rbfox1 silencing modulates the splicing of the actin-remodeling protein gelsolin, increasing gelsolin expression and leading to faster glucose-induced actin depolymerization and increased insulin release. Taken together, these findings indicate that beta cells share common splicing regulators and programs with neurons. These splicing regulators play key roles in insulin release and beta cell survival, and their dysfunction may contribute to the loss of functional beta cell mass in diabetes.
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Células Secretoras de Insulina/citología , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo , Animales , Apoptosis , Línea Celular , Supervivencia Celular , Células Cultivadas , Proteína 4 Similar a ELAV/genética , Proteína 4 Similar a ELAV/metabolismo , Regulación de la Expresión Génica , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Proteínas de Unión al ARN/genética , RatasRESUMEN
Pancreatic ß-cell dysfunction and death are determinant events in type 1 diabetes (T1D), but the molecular mechanisms behind ß-cell fate remain poorly understood. Alternative splicing is a post-transcriptional mechanism by which a single gene generates different mRNA and protein isoforms, expanding the transcriptome complexity and enhancing protein diversity. Neuron-specific and certain serine/arginine-rich RNA binding proteins (RBP) are enriched in ß-cells, playing crucial roles in the regulation of insulin secretion and ß-cell survival. Moreover, alternative exon networks, regulated by inflammation or diabetes susceptibility genes, control key pathways and processes for the correct function and survival of ß-cells. The challenge ahead of us is to understand the precise role of alternative splicing regulators and splice variants on ß-cell function, dysfunction and death and develop tools to modulate it.
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Empalme Alternativo/fisiología , Células Secretoras de Insulina/fisiología , Empalme Alternativo/genética , Autoinmunidad/genética , Autoinmunidad/fisiología , Secuencia de Bases/genética , Secuencia de Bases/fisiología , Muerte Celular/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 1/prevención & control , Expresión Génica/genética , Humanos , Neuronas/metabolismo , Fosfoproteínas/genética , Proteínas de Unión al ARN/fisiología , Análisis de Secuencia de ARN , Factores de Empalme Serina-Arginina/genéticaRESUMEN
High-throughput omics technologies have generated a wealth of large protein, gene, and transcript datasets that have exacerbated the need for new methods to analyse and compare big datasets. Rank-rank hypergeometric overlap is an important threshold-free method to combine and visualize two ranked lists of P-values or fold-changes, usually from differential gene expression analyses. Here, we introduce a new rank-rank hypergeometric overlap-based method aimed at gene level and alternative splicing analyses at transcript or exon level, hitherto unreachable as transcript numbers are an order of magnitude larger than gene numbers. We tested the tool on synthetic and real datasets at gene and transcript levels to detect correlation and anticorrelation patterns and found it to be fast and accurate, even on very large datasets thanks to an evolutionary algorithm-based minimal P-value search. The tool comes with a ready-to-use permutation scheme allowing the computation of adjusted P-values at low time cost. The package compatibility mode is a drop-in replacement to previous packages. RedRibbon holds the promise to accurately extricate detailed information from large comparative analyses.
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Algoritmos , Perfilación de la Expresión Génica , Perfilación de la Expresión Génica/métodos , Exones/genética , Empalme Alternativo/genéticaRESUMEN
Tyrosine protein-kinase 2 (TYK2), a member of the Janus kinase family, mediates inflammatory signaling through multiple cytokines, including interferon-α (IFNα), interleukin (IL)-12, and IL-23. Missense mutations in TYK2 are associated with protection against type 1 diabetes (T1D), and inhibition of TYK2 shows promise in the management of other autoimmune conditions. Here, we evaluated the effects of specific TYK2 inhibitors (TYK2is) in pre-clinical models of T1D. First, human ß cells, cadaveric donor islets, and iPSC-derived islets were treated in vitro with IFNα in combination with a small molecule TYK2i (BMS-986165 or a related molecule BMS-986202). TYK2 inhibition prevented IFNα-induced ß cell HLA class I up-regulation, endoplasmic reticulum stress, and chemokine production. In co-culture studies, pre-treatment of ß cells with a TYK2i prevented IFNα-induced activation of T cells targeting an epitope of insulin. In vivo administration of BMS-986202 in two mouse models of T1D (RIP-LCMV-GP mice and NOD mice) reduced systemic and tissue-localized inflammation, prevented ß cell death, and delayed T1D onset. Transcriptional phenotyping of pancreatic islets, pancreatic lymph nodes (PLN), and spleen during early disease pathogenesis highlighted a role for TYK2 inhibition in modulating signaling pathways associated with inflammation, translational control, stress signaling, secretory function, immunity, and diabetes. Additionally, TYK2i treatment changed the composition of innate and adaptive immune cell populations in the blood and disease target tissues, resulting in an immune phenotype with a diminished capacity for ß cell destruction. Overall, these findings indicate that TYK2i has beneficial effects in both the immune and endocrine compartments in models of T1D, thus supporting a path forward for testing TYK2 inhibitors in human T1D.
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IFNα is a key regulator of the dialogue between pancreatic ß cells and the immune system in early type 1 diabetes (T1D). IFNα up-regulates HLA class I expression in human ß cells, fostering autoantigen presentation to the immune system. We observed by bulk and single-cell RNA sequencing that exposure of human induced pluripotent-derived islet-like cells to IFNα induces expression of HLA class I and of other genes involved in antigen presentation, including the transcriptional activator NLRC5. We next evaluated the global role of NLRC5 in human insulin-producing EndoC-ßH1 and human islet cells by RNA sequencing and targeted gene/protein determination. NLRC5 regulates expression of HLA class I, antigen presentation-related genes, and chemokines. NLRC5 also mediates the effects of IFNα on alternative splicing, a generator of ß cell neoantigens, suggesting that it is a central player of the effects of IFNα on ß cells that contribute to trigger and amplify autoimmunity in T1D.
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Diabetes Mellitus Tipo 1 , Células Secretoras de Insulina , Islotes Pancreáticos , Empalme Alternativo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Humanos , Interferón-alfa/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Islotes Pancreáticos/metabolismo , Transcripción GenéticaRESUMEN
Completion of the Human Genome Project enabled a novel systems- and network-level understanding of biology, but this remains to be applied for understanding the pathogenesis of type 1 diabetes (T1D). We propose that defining the key gene regulatory networks that drive ß-cell dysfunction and death in T1D might enable the design of therapies that target the core disease mechanism, namely, the progressive loss of pancreatic ß-cells. Indeed, many successful drugs do not directly target individual disease genes but, rather, modulate the consequences of defective steps, targeting proteins located one or two steps downstream. If we transpose this to the T1D situation, it makes sense to target the pathways that modulate the ß-cell responses to the immune assault-in relation to signals that may stimulate the immune response (e.g., HLA class I and chemokine overexpression and/or neoantigen expression) or inhibit the invading immune cells (e.g., PDL1 and HLA-E expression)-instead of targeting only the immune system, as it is usually proposed. Here we discuss the importance of a focus on ß-cells in T1D, lessons learned from other autoimmune diseases, the "alternative splicing connection," data mining, and drug repurposing to protect ß-cells in T1D and then some of the initial candidates under testing for ß-cell protection.
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Diabetes Mellitus Tipo 1/tratamiento farmacológico , Redes Reguladoras de Genes , Células Secretoras de Insulina/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , HumanosRESUMEN
Exposure of human pancreatic beta cells to pro-inflammatory cytokines or metabolic stressors is used to model events related to type 1 and type 2 diabetes, respectively. Quantitative real-time PCR is commonly used to quantify changes in gene expression. The selection of the most adequate reference gene(s) for gene expression normalization is an important pre-requisite to obtain accurate and reliable results. There are no universally applicable reference genes, and the human beta cell expression of commonly used reference genes can be altered by different stressors. Here we aimed to identify the most stably expressed genes in human beta cells to normalize quantitative real-time PCR gene expression.We used comprehensive RNA-sequencing data from the human pancreatic beta cell line EndoC-ßH1, human islets exposed to cytokines or the free fatty acid palmitate in order to identify the most stably expressed genes. Genes were filtered based on their level of significance (adjusted P-value >0.05), fold-change (|fold-change| <1.5) and a coefficient of variation <10%. Candidate reference genes were validated by quantitative real-time PCR in independent samples.We identified a total of 264 genes stably expressed in EndoC-ßH1 cells and human islets following cytokines - or palmitate-induced stress, displaying a low coefficient of variation. Validation by quantitative real-time PCR of the top five genes ARF1, CWC15, RAB7A, SIAH1 and VAPA corroborated their expression stability under most of the tested conditions. Further validation in independent samples indicated that the geometric mean of ACTB and VAPA expression can be used as a reliable normalizing factor in human beta cells.
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Genómica/métodos , Células Secretoras de Insulina , Humanos , Células Secretoras de Insulina/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In pancreatic ß-cells, the expression of the splicing factor SRSF6 is regulated by GLIS3, a transcription factor encoded by a diabetes susceptibility gene. SRSF6 down-regulation promotes ß-cell demise through splicing dysregulation of central genes for ß-cells function and survival, but how RNAs are targeted by SRSF6 remains poorly understood. Here, we define the SRSF6 binding landscape in the human pancreatic ß-cell line EndoC-ßH1 by integrating individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) under basal conditions with RNA sequencing after SRSF6 knockdown. We detect thousands of SRSF6 bindings sites in coding sequences. Motif analyses suggest that SRSF6 specifically recognizes a purine-rich consensus motif consisting of GAA triplets and that the number of contiguous GAA triplets correlates with increasing binding site strength. The SRSF6 positioning determines the splicing fate. In line with its role in ß-cell function, we identify SRSF6 binding sites on regulated exons in several diabetes susceptibility genes. In a proof-of-principle, the splicing of the susceptibility gene LMO7 is modulated by antisense oligonucleotides. Our present study unveils the splicing regulatory landscape of SRSF6 in immortalized human pancreatic ß-cells.
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Empalme Alternativo/genética , Regulación de la Expresión Génica , Células Secretoras de Insulina/metabolismo , Fosfoproteínas/metabolismo , ARN/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Sitios de Unión , Línea Celular , Supervivencia Celular/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 2/genética , Exones , Técnicas de Silenciamiento del Gen , Humanos , Proteínas con Dominio LIM/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapas de Interacción de Proteínas , Factores de Empalme Serina-Arginina/química , Factores de Empalme Serina-Arginina/genética , Factores de Transcripción/genética , Transcriptoma , TransfecciónRESUMEN
The early stages of type 1 diabetes (T1D) are characterized by local autoimmune inflammation and progressive loss of insulin-producing pancreatic ß cells. Here we show that exposure to proinflammatory cytokines reveals a marked plasticity of the ß-cell regulatory landscape. We expand the repertoire of human islet regulatory elements by mapping stimulus-responsive enhancers linked to changes in the ß-cell transcriptome, proteome and three-dimensional chromatin structure. Our data indicate that the ß-cell response to cytokines is mediated by the induction of new regulatory regions as well as the activation of primed regulatory elements prebound by islet-specific transcription factors. We find that T1D-associated loci are enriched with newly mapped cis-regulatory regions and identify T1D-associated variants disrupting cytokine-responsive enhancer activity in human ß cells. Our study illustrates how ß cells respond to a proinflammatory environment and implicate a role for stimulus response islet enhancers in T1D.
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Cromatina/genética , Citocinas/farmacología , Diabetes Mellitus Tipo 1/genética , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Células Secretoras de Insulina/metabolismo , Transcriptoma , Cromatina/química , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/patología , Elementos de Facilitación Genéticos , Estudio de Asociación del Genoma Completo , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/patología , Factores de TranscripciónRESUMEN
Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is overexpressed in numerous types of tumors, especially in prostate cancer. STEAP1 is located in the plasma membrane of epithelial cells and may play an important role in inter- and intracellular communication. Several studies suggest STEAP1 as a potential biomarker and an immunotherapeutic target for prostate cancer. However, the role of STEAP1 in cell proliferation and apoptosis remains unclear. Therefore, the role of STEAP1 in prostate cancer cells proliferation and apoptosis was determined by inducing STEAP1 gene knockdown in LNCaP cells. In addition, the effect of DHT on the proliferation of LNCaP cells knocked down for STEAP1 gene was evaluated. Our results demonstrated that silencing the STEAP1 gene reduces LNCaP cell viability and proliferation, while inducing apoptosis. In addition, we showed that the cellular and molecular effects of STEAP1 gene knockdown may be independent of DHT treatment, and blocking STEAP1 may reveal to be an appropriate strategy to activate apoptosis in cancer cells, as well as to prevent the proliferative and anti-apoptotic effects of DHT in prostate cancer.
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Andrógenos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Oxidorreductasas/antagonistas & inhibidores , Neoplasias de la Próstata/patología , ARN Interferente Pequeño/genética , Antígenos de Neoplasias/genética , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Masculino , Oxidorreductasas/genética , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismoRESUMEN
Progressive failure of insulin-producing ß-cells is the central event leading to diabetes, but the signaling networks controlling ß-cell fate remain poorly understood. Here we show that SRp55, a splicing factor regulated by the diabetes susceptibility gene GLIS3, has a major role in maintaining the function and survival of human ß-cells. RNA sequencing analysis revealed that SRp55 regulates the splicing of genes involved in cell survival and death, insulin secretion, and c-Jun N-terminal kinase (JNK) signaling. In particular, SRp55-mediated splicing changes modulate the function of the proapoptotic proteins BIM and BAX, JNK signaling, and endoplasmic reticulum stress, explaining why SRp55 depletion triggers ß-cell apoptosis. Furthermore, SRp55 depletion inhibits ß-cell mitochondrial function, explaining the observed decrease in insulin release. These data unveil a novel layer of regulation of human ß-cell function and survival, namely alternative splicing modulated by key splicing regulators such as SRp55, that may cross talk with candidate genes for diabetes.
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Empalme Alternativo , Apoptosis , Proteína 11 Similar a Bcl2/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Fosfoproteínas/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Proteína 11 Similar a Bcl2/genética , Línea Celular , Supervivencia Celular , Células Cultivadas , Estrés del Retículo Endoplásmico , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Secreción de Insulina , Células Secretoras de Insulina/citología , Sistema de Señalización de MAP Quinasas , Mitocondrias/enzimología , Mitocondrias/metabolismo , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Interferencia de ARN , Factores de Empalme Serina-Arginina/antagonistas & inhibidores , Factores de Empalme Serina-Arginina/química , Factores de Empalme Serina-Arginina/genética , Proteína X Asociada a bcl-2/genéticaRESUMEN
Hepatocyte nuclear factor 1 beta (HNF1B) plays an important role in embryonic development, namely in the kidney, pancreas, liver, genital tract, and gut. Heterozygous germline mutations of HNF1B are associated with the renal cysts and diabetes syndrome (RCAD). Affected individuals may present a variety of renal developmental abnormalities and/or maturity-onset diabetes of the young (MODY). A Portuguese 19-month-old male infant was evaluated due to hypoplastic glomerulocystic kidney disease and renal dysfunction diagnosed in the neonatal period that progressed to stage 5 chronic renal disease during the first year of life. His mother was diagnosed with a solitary hypoplastic microcystic left kidney at age 20, with stage 2 chronic renal disease established at age 35, and presented bicornuate uterus, pancreatic atrophy, and gestational diabetes. DNA sequence analysis of HNF1B revealed a novel germline frameshift insertion (c.110_111insC or c.110dupC) in both the child and the mother. A review of the literature revealed a total of 106 different HNF1B mutations, in 236 mutation-positive families, comprising gross deletions (34%), missense mutations (31%), frameshift deletions or insertions (15%), nonsense mutations (11%), and splice-site mutations (8%). The study of this family with an unusual presentation of hypoplastic glomerulocystic kidney disease with neonatal renal dysfunction identified a previously unreported mutation of the HNF1B gene, thereby expanding the spectrum of known mutations associated with renal developmental disorders.
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Enfermedades del Sistema Nervioso Central/genética , Esmalte Dental/anomalías , Diabetes Mellitus Tipo 2/genética , Factor Nuclear 1-beta del Hepatocito/genética , Enfermedades Renales Quísticas/genética , Insuficiencia Renal/genética , Enfermedades del Sistema Nervioso Central/fisiopatología , Esmalte Dental/fisiopatología , Diabetes Mellitus Tipo 2/fisiopatología , Progresión de la Enfermedad , Femenino , Mutación de Línea Germinal , Humanos , Lactante , Recién Nacido , Enfermedades Renales Quísticas/fisiopatología , Masculino , Embarazo , Complicaciones del Embarazo/fisiopatología , Insuficiencia Renal/fisiopatología , Análisis de Secuencia de ADNRESUMEN
Resistance to thyroid hormone (RTH) represents a syndrome in which patients present elevated circulating thyroid hormones in the presence of non-suppressed TSH. We report a novel case where a patient with RTH presented a differentiated thyroid cancer. A19 year-old female had been referred due to thyroid disease that disclosed features characteristic of a RTH. During the follow up it was detected a follicular tumor that led to the recommendation for thyroid surgical ablation, where an incidental papillary thyroid microcarcinoma (mPTC) was found. The increase of thyroglobulin (TG) levels following thyroid removal referred the patient for radioiodine treatment. Post-treatment, it was detected jugular adenopathies and the patient was subjected to cervical lymph node drainage where metastases of the mPTC were found. RTH syndrome was confirmed by the detection of a THRB germline mutation. A BRAF mutation was also found in the mPTC but not detected in the follicular adenoma or normal adjacent tissue. The young age of the patient, the rarity of BRAF mutations in childhood and the high dissemination of the malignancy, lead us to the speculation that increased TSH stimulation in a RTH background and oncogenic activation of BRAF could have served as (co) drivers and might have triggered an advanced stage of the neoplastic disease. These findings together with a review of published cases add novel information to the management of RTH patients with differentiated thyroid cancer.
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CONTEXT: The association between selenium and inflammation and the relevance of selenoproteins in follicular thyroid cell physiology have pointed to a putative role of selenoproteins in the pathogenesis of autoimmune thyroid diseases. OBJECTIVE: The aim of this study was to evaluate the role of a promoter variation in SEPS1, the selenoprotein S gene, in the risk for developing Hashimoto's thyroiditis (HT). DESIGN: A case-control study was performed to assess the association of genetic variation in the SEPS1 gene (SEPS1 -105G/A single-nucleotide polymorphism, rs28665122) and HT. SETTING: The study was conducted in north Portugal, Porto, in the period of 2007-2013. PATIENTS OR OTHER PARTICIPANTS: A total of 997 individuals comprising 481 HT patients and 516 unrelated controls were enrolled in the study. MAIN OUTCOME MEASURES: Genetic variants were discriminated by real-time PCR using TaqMan single-nucleotide polymorphism genotyping assays. RESULTS: There is a significant association between the SEPS1 -105 GA and AA genotypes and HT [odds ratio (OR) 2.24, confidence interval (CI) 1.67-3.02, P < 5.0 × 10(-7), and OR 2.08, CI 1.09-3.97, P = .0268, respectively]. The A allele carriers are in higher proportion in the patient group than in the control population (46.2% vs 28.1%, P < 5.0 × 10(-7)) with an OR (CI) of 2.22 (1.67-2.97). The proportion of patients carrying the A allele is significantly higher in male patients with HT, representing a 3.94 times increased risk (P = 7.9 × 10(-3)). CONCLUSION: Our findings support the existence of a link between SEPS1 promoter genetic variation and HT risk.
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Predisposición Genética a la Enfermedad , Enfermedad de Hashimoto/genética , Proteínas de la Membrana/genética , Regiones Promotoras Genéticas/genética , Selenoproteínas/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Adulto JovenRESUMEN
OBJECTIVE: Primary hyperparathyroidism (pHPT) is characterised by an inappropriate over production of parathyroid hormone and it is the most frequent pathological condition of the parathyroid glands. A minority of the cases belong to familial forms, but most of them are sporadic. The genetic alterations underlying the sporadic forms of pHPT remain poorly understood. The main goal of our study is to perform the molecular characterisation of a series of sporadic pHPT cases. DESIGN AND METHODS: We have studied matched blood and tumour from 24 patients with pHPT, who went to a medical appointment in Hospital Pedro Hispano. Informed consent was obtained from all individuals. The MEN1, RET and CDKN1B molecular study was carried out in the germline DNA by PCR/SSCP and direct sequencing. Parathyroid tumours were further analysed by the same methods for MEN1, CDKN1B and CTNNB1 genetic alterations. The multiplex ligation-dependent probe amplification technique enabled the evaluation of MEN1 gene deletions. Protein expression for menin, cyclin D1, parafibromin, p27(Kip1), ß-catenin and Ki-67 was conducted by immunohistochemistry. RESULTS: The study of parathyroid tumours detected two somatic MEN1 mutations (c.249_252delGTCT and c.115_163del49bp) and revealed the presence of MEN1 intragenic deletions in 54% (13/24) of the tumours. In RET and CDKN1B genes only previously described, non-pathogenic variants were found. Cyclin D1 protein was overexpressed in 13% (3/24) of tumours. CONCLUSIONS: These results suggest that MEN1 alterations, remarkably intragenic deletions, may represent the most prevalent genetic alteration in sporadic parathyroid tumours.
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Eliminación de Gen , Hiperparatiroidismo/genética , Neoplasia Endocrina Múltiple Tipo 1/genética , Neoplasias de las Paratiroides/genética , Proteínas Proto-Oncogénicas/genética , Adulto , Anciano , Anciano de 80 o más Años , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Humanos , Pérdida de Heterocigocidad , Masculino , Persona de Mediana Edad , Mutación , Proteínas Proto-Oncogénicas c-ret/genética , Estudios RetrospectivosRESUMEN
Primary hyperparathyroidism (PHPT) is a frequent endocrine disorder characterized by an excessive autonomous production and release of parathyroid hormone (PTH) by the parathyroid glands. This endocrinopathy may result from the development of a benign lesion (adenoma or hyperplasia) or from a carcinoma. Most of the PHPT cases occur sporadically; however, approximately 10% of the patients present a familial form of the disease. The molecular mechanisms underlying the pathogenesis of sporadic PHPT are incompletely understood, even though somatic alterations in MEN1 gene and CCND1 protein overexpression are frequently observed. The MEN1 gene is mutated in about 30% of the parathyroid tumours and the protooncogene CCND1 is implicated in parathyroid neoplasia by rearrangements, leading to an overexpression of CCND1 protein in parathyroid cells. The aim of this work is to briefly update the molecular alterations underlying sporadic primary hyperparathyroidism.