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Type 1 diabetes (T1D) is an autoimmune disease leading to β-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 β-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-1β plus IFN-γ. Confirmation of miRNA and target gene modification in human β-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 β-cell apoptosis. These results identify a novel cross talk between a key family of miRNAs and proapoptotic Bcl-2 proteins in human pancreatic β-cells, broadening our understanding of cytokine-induced β-cell apoptosis in early T1D. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.