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Most expression and splicing changes in myotonic dystrophy type 1 and type 2 skeletal muscle are shared with other muscular dystrophies.

ABSTRACT: The prevailing pathomechanistic paradigm for myotonic dystrophy (DM) is that aberrant expression of embryonic/fetal mRNA/protein isoforms accounts for most aspects of the pleiotropic phenotype. To identify aberrant isoforms in skeletal muscle of DM1 and DM2 patients, we performed exon-array profiling and RT-PCR validation on the largest DM sample set to date, including Duchenne, Becker and tibial muscular dystrophy (NMD) patients as disease controls, and non-disease controls. Strikingly, most expression and splicing changes in DM patients were shared with NMD controls. Comparison between DM and NMD identified almost no significant differences. We conclude that DM1 and DM2 are essentially identical for dysregulation of gene expression, and DM expression changes represent a subset of broader spectrum dystrophic changes. We found no evidence for qualitative splicing differences between DM1 and DM2. While some DM-specific splicing differences exist, most of the DM splicing differences were also seen in NMD controls. SSBP3 exon 6 missplicing was observed in all diseased muscle and led to reduced protein. We conclude there is no widespread DM-specific spliceopathy in skeletal muscle and suggest that missplicing in DM (and NMD) may not be the driving mechanism for the muscle pathology, since the same pathways show expression changes unrelated to splicing.

SUBMITTER: Bachinski LL

PROVIDER: S-EPMC3943873 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Project description:Cardiac involvement is one of the most important manifestations of the multisystemic phenotype of patients affected by myotonic dystrophy (DM) and represents the second cause of premature death. Molecular mechanisms responsible for DM cardiac defects are still unclear; however, missplicing of the cardiac isoform of troponin T (TNNT2) and of the cardiac sodium channel (SCN5A) genes might contribute to the reduced myocardial function and conduction abnormalities seen in DM patients. Since, in DM skeletal muscle, the TNNT2 gene shows the same aberrant splicing pattern observed in cardiac muscle, the principal aim of this work was to verify if the TNNT2 aberrant fetal isoform expression could be secondary to myopathic changes or could reflect the DM cardiac phenotype. Analysis of alternative splicing of TNNT2 and of several genes involved in DM pathology has been performed on muscle biopsies from patients affected by DM type 1 (DM1) or type 2 (DM2) with or without cardiac involvement. Our analysis shows that missplicing of muscle-specific genes is higher in DM1 and DM2 than in regenerating control muscles, indicating that these missplicing could be effectively important in DM skeletal muscle pathology. When considering the TNNT2 gene, missplicing appears to be more evident in DM1 than in DM2 muscles since, in DM2, the TNNT2 fetal isoform appears to be less expressed than the adult isoform. This evidence does not seem to be related to less severe muscle histopathological alterations that appear to be similar in DM1 and DM2 muscles. These results seem to indicate that the more severe TNNT2 missplicing observed in DM1 could not be related only to myopathic changes but could reflect the more severe general phenotype compared to DM2, including cardiac problems that appear to be more severe and frequent in DM1 than in DM2 patients. Moreover, TNNT2 missplicing significantly correlates with the QRS cardiac parameter in DM1 but not in DM2 patients, indicating that this splicing event has good potential to function as a biomarker of DM1 severity and it should be considered in pharmacological clinical trials to monitor the possible effects of different therapeutic approaches on skeletal muscle tissues.

Project description:MicroRNAs are highly conserved, noncoding RNAs involved in post-transcriptional gene silencing. They have been shown to participate in a wide range of biological processes, including myogenesis and muscle regeneration. The goal of this study is to test the hypothesis that myo-miRs (myo = muscle + miR = miRNA) expression is altered in muscle from patients affected by myotonic dystrophy type 1 (DM1), the most frequently inherited neuromuscular disease in adults. In order to gain better insights about the role of miRNAs in the DM1 pathogenesis, we have also analyzed the muscular expression of miR-103 and miR-107, which have been identified in silico as attractive candidates for binding to the DMPK mRNA.To this aim, we have profiled the expression of miR-133 (miR-133a, miR-133b), miR-1, miR-181 (miR-181a, miR-181b, miR-181c) and miR-206, that are specifically induced during myogenesis in cardiac and skeletal muscle tissues. miR-103 and miR-107, highly expressed in brain, heart and muscle have also been included in this study. QRT-PCR experiments have been performed on RNA from vastus lateralis biopsies of DM1 patients (n = 7) and control subjects (n = 4). Results of miRNAs expression have been confirmed by Northern blot, whereas in situ hybridization technique have been performed to localize misexpressed miRNAs on muscle sections from DM1 and control individuals.Only miR-206 showed an over-expression in 5 of 7 DM1 patients (threshold = 2, fold change between 1.20 and 13.22, average = 5.37) compared to the control group. This result has been further confirmed by Northern blot analysis (3.37-fold overexpression, R2 = 0.89). In situ hybridization localized miR-206 to nuclear site both in normal and DM1 tissues. Cellular distribution in DM1 tissues includes also the nuclear regions of centralized nuclei, with a strong signal corresponding to nuclear clumps.This work provides, for the first time, evidences about miRNAs misexpression in DM1 muscle tissues, adding a new element in the pathogenesis of this complex genetic disease.

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Most expression and splicing changes in myotonic dystrophy type 1 and type 2 skeletal muscle are shared with other muscular dystrophies.

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