RESUMEN
The minor spliceosome regulates the removal of a conserved subset of introns present in genes with regulatory functions. In this issue of Molecular Cell, Augspach et al.1 report that elevated levels of U6atac snRNA, a key minor spliceosome component, contribute to prostate cancer cell growth and can be a novel therapeutic target.
Asunto(s)
Neoplasias , Empalmosomas , Empalmosomas/genética , Empalmosomas/metabolismo , Empalme del ARN , Secuencia de Bases , Intrones , ARN Nuclear Pequeño/genética , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
BACKGROUND: Lipids are regulators of insulitis and ß-cell death in type 1 diabetes development, but the underlying mechanisms are poorly understood. Here, we investigated how the islet lipid composition and downstream signaling regulate ß-cell death. METHODS: We performed lipidomics using three models of insulitis: human islets and EndoC-ßH1 ß cells treated with the pro-inflammatory cytokines interlukine-1ß and interferon-γ, and islets from pre-diabetic non-obese mice. We also performed mass spectrometry and fluorescence imaging to determine the localization of lipids and enzyme in islets. RNAi, apoptotic assay, and qPCR were performed to determine the role of a specific factor in lipid-mediated cytokine signaling. RESULTS: Across all three models, lipidomic analyses showed a consistent increase of lysophosphatidylcholine species and phosphatidylcholines with polyunsaturated fatty acids and a reduction of triacylglycerol species. Imaging assays showed that phosphatidylcholines with polyunsaturated fatty acids and their hydrolyzing enzyme phospholipase PLA2G6 are enriched in islets. In downstream signaling, omega-3 fatty acids reduce cytokine-induced ß-cell death by improving the expression of ADP-ribosylhydrolase ARH3. The mechanism involves omega-3 fatty acid-mediated reduction of the histone methylation polycomb complex PRC2 component Suz12, upregulating the expression of Arh3, which in turn decreases cell apoptosis. CONCLUSIONS: Our data provide insights into the change of lipidomics landscape in ß cells during insulitis and identify a protective mechanism by omega-3 fatty acids. Video Abstract.
Asunto(s)
Ácidos Grasos Omega-3 , Islotes Pancreáticos , N-Glicosil Hidrolasas , Ratones , Animales , Humanos , Islotes Pancreáticos/metabolismo , Muerte Celular , Citocinas/metabolismo , Ácidos Grasos Omega-3/metabolismo , Ácidos Grasos Insaturados , Fosfatidilcolinas/metabolismoRESUMEN
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.
Asunto(s)
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.
Asunto(s)
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
Pancreatic islets control glucose homeostasis by the balanced secretion of insulin and other hormones, and their abnormal function causes diabetes or hypoglycaemia. Here we uncover a conserved programme of alternative microexons included in mRNAs of islet cells, particularly in genes involved in vesicle transport and exocytosis. Islet microexons (IsletMICs) are regulated by the RNA binding protein SRRM3 and represent a subset of the larger neural programme that are particularly sensitive to SRRM3 levels. Both SRRM3 and IsletMICs are induced by elevated glucose levels, and depletion of SRRM3 in human and rat beta cell lines and mouse islets, or repression of particular IsletMICs using antisense oligonucleotides, leads to inappropriate insulin secretion. Consistently, mice harbouring mutations in Srrm3 display defects in islet cell identity and function, leading to hyperinsulinaemic hypoglycaemia. Importantly, human genetic variants that influence SRRM3 expression and IsletMIC inclusion in islets are associated with fasting glucose variation and type 2 diabetes risk. Taken together, our data identify a conserved microexon programme that regulates glucose homeostasis.
Asunto(s)
Diabetes Mellitus Tipo 2 , Hipoglucemia , Células Secretoras de Insulina , Ratas , Ratones , Humanos , Animales , Células Secretoras de Insulina/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Secreción de Insulina , Glucosa/metabolismo , Hipoglucemia/metabolismo , Homeostasis/fisiologíaRESUMEN
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.
Asunto(s)
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
Interferon-α (IFNα), a type I interferon, is expressed in the islets of type 1 diabetic individuals, and its expression and signaling are regulated by T1D genetic risk variants and viral infections associated with T1D. We presently characterize human beta cell responses to IFNα by combining ATAC-seq, RNA-seq and proteomics assays. The initial response to IFNα is characterized by chromatin remodeling, followed by changes in transcriptional and translational regulation. IFNα induces changes in alternative splicing (AS) and first exon usage, increasing the diversity of transcripts expressed by the beta cells. This, combined with changes observed on protein modification/degradation, ER stress and MHC class I, may expand antigens presented by beta cells to the immune system. Beta cells also up-regulate the checkpoint proteins PDL1 and HLA-E that may exert a protective role against the autoimmune assault. Data mining of the present multi-omics analysis identifies two compound classes that antagonize IFNα effects on human beta cells.
Asunto(s)
Empalme Alternativo , Células Secretoras de Insulina/fisiología , Interferón-alfa/metabolismo , Interferón-alfa/farmacología , Empalme Alternativo/efectos de los fármacos , Células Cultivadas , Cromatina/efectos de los fármacos , Cromatina/metabolismo , Minería de Datos , Diabetes Mellitus Tipo 1/genética , Regulación de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Mapas de Interacción de Proteínas , Proteómica , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
miRNAs are a class of small non-coding RNAs that regulate gene expression. Type 1 diabetes is an autoimmune disease characterized by insulitis (islets inflammation) and pancreatic beta cell destruction. The pro-inflammatory cytokines interleukin 1 beta (IL1B) and interferon gamma (IFNG) are released during insulitis and trigger endoplasmic reticulum (ER) stress and expression of pro-apoptotic members of the BCL2 protein family in beta cells, thus contributing to their death. The nature of miRNAs that regulate ER stress and beta cell apoptosis remains to be elucidated. We have performed a global miRNA expression profile on cytokine-treated human islets and observed a marked downregulation of miR-211-5p. By real-time PCR and Western blot analysis, we confirmed cytokine-induced changes in the expression of miR-211-5p and the closely related miR-204-5p and downstream ER stress related genes in human beta cells. Blocking of endogenous miRNA-211-5p and miR-204-5p by the same inhibitor (it is not possible to block separately these two miRs) increased human beta cell apoptosis, as measured by Hoechst/propidium Iodide staining and by determination of cleaved caspase-3 activation. Interestingly, miRs-211-5p and 204-5p regulate the expression of several ER stress markers downstream of PERK, particularly the pro-apoptotic protein DDIT3 (also known as CHOP). Blocking CHOP expression by a specific siRNA partially prevented the increased apoptosis observed following miR-211-5p/miR-204-5p inhibition. These observations identify a novel crosstalk between miRNAs, ER stress and beta cell apoptosis in early type 1 diabetes.
Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Células Secretoras de Insulina/metabolismo , MicroARNs/metabolismo , Apoptosis/fisiología , Biomarcadores/metabolismo , Caspasa 3/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Humanos , Factor de Transcripción CHOP/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
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
Type 1 diabetes (T1D) is an autoimmune disease leading to ß-cell destruction. MicroRNAs (miRNAs) are small noncoding RNAs that control gene expression and organ formation. They participate in the pathogenesis of several autoimmune diseases, but the nature of miRNAs contributing to ß-cell death in T1D and their target genes remain to be clarified. We performed an miRNA expression profile on human islet preparations exposed to the cytokines IL-1ß plus IFN-γ. Confirmation of miRNA and target gene modification in human ß-cells was performed by real-time quantitative PCR. Single-stranded miRNAs inhibitors were used to block selected endogenous miRNAs. Cell death was measured by Hoechst/propidium iodide staining and activation of caspase-3. Fifty-seven miRNAs were detected as modulated by cytokines. Three of them, namely miR-23a-3p, miR-23b-3p, and miR-149-5p, were downregulated by cytokines and selected for further studies. These miRNAs were found to regulate the expression of the proapoptotic Bcl-2 proteins DP5 and PUMA and consequent human ß-cell apoptosis. These results identify a novel cross talk between a key family of miRNAs and proapoptotic Bcl-2 proteins in human pancreatic ß-cells, broadening our understanding of cytokine-induced ß-cell apoptosis in early T1D.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Células Secretoras de Insulina/metabolismo , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Anciano , Proteínas Reguladoras de la Apoptosis/genética , Western Blotting , Femenino , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Masculino , MicroARNs/genética , Proteínas Proto-Oncogénicas/genética , Interferencia de ARNRESUMEN
Type 1 diabetes (T1D) is a chronic autoimmune disease in which pancreatic ß cells are killed by infiltrating immune cells and by cytokines released by these cells. This takes place in the context of a dysregulated dialogue between invading immune cells and target ß cells, but the intracellular signals that decide ß cell fate remain to be clarified. Alternative splicing (AS) is a complex post-transcriptional regulatory mechanism affecting gene expression. It regulates the inclusion/exclusion of exons into mature mRNAs, allowing individual genes to produce multiple protein isoforms that expand the proteome diversity. Functionally related transcript populations are co-ordinately spliced by master splicing factors, defining regulatory networks that allow cells to rapidly adapt their transcriptome in response to intra and extracellular cues. There is a growing interest in the role of AS in autoimmune diseases, but little is known regarding its role in T1D. In this review, we discuss recent findings suggesting that splicing events occurring in both immune and pancreatic ß cells contribute to the pathogenesis of T1D. Splicing switches in T cells and in lymph node stromal cells are involved in the modulation of the immune response against ß cells, while ß cells exposed to pro-inflammatory cytokines activate complex splicing networks that modulate ß cell viability, expression of neoantigens and susceptibility to immune-induced stress. Unveiling the role of AS in ß cell functional loss and death will increase our understanding of T1D pathogenesis and may open new avenues for disease prevention and therapy.
Asunto(s)
Empalme Alternativo , Diabetes Mellitus Tipo 1 , Empalme Alternativo/inmunología , Linfocitos B/inmunología , Diabetes Mellitus Tipo 1/inmunología , Humanos , Células Secretoras de Insulina/inmunologíaRESUMEN
Recent advances in molecular therapies for Duchenne muscular dystrophy (DMD) require precise genetic diagnosis because most therapeutic strategies are mutation-specific. To understand more about the genotype-phenotype correlations of the DMD gene we performed a comprehensive analysis of the DMD mutational spectrum in a large series of families. Here we provide the clinical, pathological and genetic features of 576 dystrophinopathy patients. DMD gene analysis was performed using the MLPA technique and whole gene sequencing in blood DNA and muscle cDNA. The impact of the DNA variants on mRNA splicing and protein functionality was evaluated by in silico analysis using computational algorithms. DMD mutations were detected in 576 unrelated dystrophinopathy families by combining the analysis of exonic copies and the analysis of small mutations. We found that 471 of these mutations were large intragenic rearrangements. Of these, 406 (70.5%) were exonic deletions, 64 (11.1%) were exonic duplications, and one was a deletion/duplication complex rearrangement (0.2%). Small mutations were identified in 105 cases (18.2%), most being nonsense/frameshift types (75.2%). Mutations in splice sites, however, were relatively frequent (20%). In total, 276 mutations were identified, 85 of which have not been previously described. The diagnostic algorithm used proved to be accurate for the molecular diagnosis of dystrophinopathies. The reading frame rule was fulfilled in 90.4% of DMD patients and in 82.4% of Becker muscular dystrophy patients (BMD), with significant differences between the mutation types. We found that 58% of DMD patients would be included in single exon-exon skipping trials, 63% from strategies directed against multiexon-skipping exons 45 to 55, and 14% from PTC therapy. A detailed analysis of missense mutations provided valuable information about their impact on the protein structure.
Asunto(s)
Distrofina/genética , Genotipo , Distrofia Muscular de Duchenne/genética , Mutación , Linaje , Fenotipo , Distrofina/química , Distrofina/metabolismo , Femenino , Humanos , MasculinoRESUMEN
DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements.
Asunto(s)
Distrofina/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Fenotipo , Mutación Puntual , Empalme del ARN , Distrofina/metabolismo , Exones , Femenino , Mutación del Sistema de Lectura , Genotipo , Humanos , Masculino , Distrofia Muscular de Duchenne/patología , Mutación Missense , Sitios de Empalme de ARNRESUMEN
BACKGROUND: Between 8% and 22% of female carriers of DMD mutations exhibit clinical symptoms of variable severity. Development of symptoms in DMD mutation carriers without chromosomal rearrangements has been attributed to skewed X-chromosome inactivation (XCI) favouring predominant expression of the DMD mutant allele. However the prognostic use of XCI analysis is controversial. We aimed to evaluate the correlation between X-chromosome inactivation and development of clinical symptoms in a series of symptomatic female carriers of dystrophinopathy. METHODS: We reviewed the clinical, pathological and genetic features of twenty-four symptomatic carriers covering a wide spectrum of clinical phenotypes. DMD gene analysis was performed using MLPA and whole gene sequencing in blood DNA and muscle cDNA. Blood and muscle DNA was used for X-chromosome inactivation (XCI) analysis thought the AR methylation assay in symptomatic carriers and their female relatives, asymptomatic carriers as well as non-carrier females. RESULTS: Symptomatic carriers exhibited 49.2% more skewed XCI profiles than asymptomatic carriers. The extent of XCI skewing in blood tended to increase in line with the severity of muscle symptoms. Skewed XCI patterns were found in at least one first-degree female relative in 78.6% of symptomatic carrier families. No mutations altering XCI in the XIST gene promoter were found. CONCLUSIONS: Skewed XCI is in many cases familial inherited. The extent of XCI skewing is related to phenotype severity. However, the assessment of XCI by means of the AR methylation assay has a poor prognostic value, probably because the methylation status of the AR gene in muscle may not reflect in all cases the methylation status of the DMD gene.
Asunto(s)
Distrofina/genética , Tamización de Portadores Genéticos , Distrofias Musculares/genética , Inactivación del Cromosoma X , Adolescente , Adulto , Anciano , Biopsia , Niño , Preescolar , Femenino , Humanos , Músculo Esquelético/patología , Distrofias Musculares/patología , Mutación , Pronóstico , Regiones Promotoras Genéticas , Adulto JovenRESUMEN
No disponible