Does quantitative EMG differ myotonic dystrophy type 2 and type 1?

Genetic testing is considered the only reliable diagnostic approach in myotonic dystrophy. However it has recently been reported that a considerable number of patients with genetically proven types of the disease have unusual phenotypic presentation. The aim of our study was to evaluate motor unit reorganization reflected by various electrophysiological abnormalities in myotonic dystrophies and to compare findings between type 1 (DM 1) and type 2 myotonic dystrophy (DM2). Quantitative electromyography (EMG) recordings in 63 patients (33 with DM1 and 30 with DM2) from the biceps brachii (BB), rectus femoris (RF), first dorsal interosseus (FDI), and tibialis anterior (TA) muscles were analyzed. Mean amplitude and size index (SI) of motor unit potentials recorded in TA and RF muscles, mean potential duration in TA, and mean SI and the number of outliers with amplitude above the normal range in BB were significantly increased in DM2 as compared to DM1. Myotonic discharges were recorded more frequently in DM1 than in DM2. EMG findings significantly differ between DM1 and DM2. The presence of high amplitude potentials in lower limb muscles in DM2 patients, atypical for myogenic muscle lesions, could be explained by muscle fiber hypertrophy observed in muscle biopsies.     

Alternative splicing of PDLIM3/ALP, for α-actinin-associated LIM protein 3, is aberrant in persons with myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is an autosomal dominant disorder of muscular dystrophy characterized by muscle weakness and wasting. DM1 is caused by expansion of CTG repeats in the 3′-untranslated region (3′-UTR) of DM protein kinase (DMPK) gene. Since CUG-repeat RNA transcribed from the expansion of CTG repeats traps RNA-binding proteins that regulate alternative splicing, several abnormalities of alternative splicing are detected in DM1, and the abnormal splicing of important genes results in the appearance of symptoms. In this study, we identify two abnormal splicing events for actinin-associated LIM protein 3 (PDLIM3/ALP) and fibronectin 1 (FN1) in the skeletal muscles of DM1 patients. From the analysis of the abnormal PDLIM3 splicing, we propose that ZASP-like motif-deficient PDLIM3 causes the muscular symptoms in DM. PDLIM3 binds α-actinin 2 in the Z-discs of muscle, and the ZASP-like motif is needed for this interaction. Moreover, in adult humans, PDLIM3 expression is highest in skeletal muscles, and PDLIM3 splicing in skeletal muscles is regulated during human development.     

Overexpression of CUGBP1 in Skeletal Muscle from Adult Classic Myotonic Dystrophy Type 1 but Not from Myotonic Dystrophy Type 2

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are progressive multisystemic disorders caused by similar mutations at two different genetic loci. The common key feature of DM pathogenesis is nuclear accumulation of mutant RNA which causes aberrant alternative splicing of specific pre-mRNAs by altering the functions of two RNA binding proteins, MBNL1 and CUGBP1. However, DM1 and DM2 show disease-specific features that make them clearly separate diseases suggesting that other cellular and molecular pathways may be involved. In this study we have analysed the histopathological, and biomolecular features of skeletal muscle biopsies from DM1 and DM2 patients in relation to presenting phenotypes to better define the molecular pathogenesis. Particularly, the expression of CUGBP1 protein has been examined to clarify if this factor may act as modifier of disease-specific manifestations in DM. The results indicate that the splicing and muscle pathological alterations observed are related to the clinical phenotype both in DM1 and in DM2 and that CUGBP1 seems to play a role in classic DM1 but not in DM2. In conclusion, our results indicate that multisystemic disease spectrum of DM pathologies may not be explained only by spliceopathy thus confirming that the molecular pathomechanism of DM is more complex than that actually suggested.     

Development of a Drosophila melanogaster spliceosensor system for in vivo high-throughput screening in myotonic dystrophy type 1

Alternative splicing of pre-mRNAs is an important mechanism that regulates cellular function in higher eukaryotes. A growing number of human genetic diseases involve splicing defects that are directly connected to their pathology. In myotonic dystrophy type 1 (DM1), several clinical manifestations have been proposed to be the consequence of tissue-specific missplicing of numerous genes. These events are triggered by an RNA gain-of-function and resultant deregulation of specific RNA-binding factors, such as the nuclear sequestration of muscleblind-like family factors (MBNL1–MBNL3). Thus, the identification of chemical modulators of splicing events could lead to the development of the first valid therapy for DM1 patients. To this end, we have generated and validated transgenic flies that contain a luciferase-reporter-based system that is coupled to the expression of MBNL1-reliant splicing (spliceosensor flies), to assess events that are deregulated in DM1 patients in a relevant disease tissue. We then developed an innovative 96-well plate screening platform to carry out in vivo high-throughput pharmacological screening (HTS) with the spliceosensor model. After a large-scale evaluation (>16,000 chemical entities), several reliable splicing modulators (hits) were identified. Hit validation steps recognized separate DM1-linked therapeutic traits for some of the hits, which corroborated the feasibility of the approach described herein to reveal promising drug candidates to correct missplicing in DM1. This powerful Drosophila-based screening tool might also be applied in other disease models displaying abnormal alternative splicing, thus offering myriad uses in drug discovery.KEY WORDS: Myotonic dystrophy, Splicing, Luciferase, In vivo screening, Minigene     

Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing

Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder caused by a CTG expansion in the 3' untranslated region of the DMPK gene. A predominant characteristic of DM1 is myotonia resulting from skeletal muscle membrane hyperexcitability. Here we demonstrate loss of the muscle-specific chloride channel (ClC-1) mRNA and protein in DM1 skeletal muscle tissue due to aberrant splicing of the ClC-1 pre-mRNA. The splicing regulator, CUG binding protein (CUG-BP), which is elevated in DM1 striated muscle, binds to the ClC-1 pre-mRNA, and overexpression of CUG-BP in normal cells reproduces the aberrant pattern of ClC-1 splicing observed in DM1 skeletal muscle. We propose that disruption of alternative splicing regulation causes a predominant pathological feature of DM1.     

Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy

Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate-binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.     

Alternative splicing of human insulin receptor gene (INSR) in type I and type II skeletal muscle fibers of patients with myotonic dystrophy type 1 and type 2

INSR, one of those genes aberrantly expressed in myotonic dystrophy type 1 (DM1) and type 2 (DM2) due to a toxic RNA effect, encodes for the insulin receptor (IR). Its expression is regulated by alternative splicing generating two isoforms: IR-A, which predominates in embryonic tissue, and IR-B, which is highly expressed in adult, insulin-responsive tissues (skeletal muscle, liver, and adipose tissue). The aberrant INSR expression detected in DM1 and DM2 muscles tissues, characterized by a relative increase of IR-A versus IR-B, was pathogenically related to the insulin resistance occurring in DM patients. To assess if differences in the aberrant splicing of INSR could underlie the distinct fiber type involvement observed in DM1 and DM2 muscle tissues, we have used laser capture microdissection (LCM) and RT-PCR, comparing the alternative splicing of INSR in type I and type II muscle fibers isolated from muscle biopsies of DM1, DM2 patients and controls. In the controls, the relative amounts of IR-A and IR-B showed no obvious differences between type I and type II fibers, as in the whole muscle tissue. In DM1 and DM2 patients, both fiber types showed a similar, relative increase of IR-A versus IR-B, as also evident in the whole muscle tissue. Our data suggest that the distinct fiber type involvement in DM1 and DM2 muscle tissues would not be related to qualitative differences in the expression of INSR. LCM can represent a powerful tool to give a better understanding of the pathogenesis of myotonic dystrophies, as well as other myopathies.     

TWEAK Regulates Muscle Functions in a Mouse Model of RNA Toxicity

Myotonic dystrophy type 1 (DM1), the most common form of muscular dystrophy in adults, is caused by toxic RNAs produced from the mutant DM protein kinase (DMPK) gene. DM1 is characterized by progressive muscle wasting and weakness. Therapeutic strategies have mainly focused on targeting the toxic RNA. Previously, we found that fibroblast growth factor-inducible 14 (Fn14), the receptor for TWEAK, is induced in skeletal muscles and hearts of mouse models of RNA toxicity and that blocking TWEAK/Fn14 signaling improves muscle function and histology. Here, we studied the effect of Tweak deficiency in a RNA toxicity mouse model. The genetic deletion of Tweak in these mice significantly reduced muscle damage and improved muscle function. In contrast, administration of TWEAK in the RNA toxicity mice impaired functional outcomes and worsened muscle histopathology. These studies show that signaling via TWEAK is deleterious to muscle in RNA toxicity and support the demonstrated utility of anti-TWEAK therapeutics.     

Myotonic dystrophy: RNA pathogenesis comes into focus

Myotonic dystrophy (DM)--the most common form of muscular dystrophy in adults, affecting 1/8000 individuals--is a dominantly inherited disorder with a peculiar and rare pattern of multisystemic clinical features affecting skeletal muscle, the heart, the eye, and the endocrine system. Two genetic loci have been associated with the DM phenotype: DM1, on chromosome 19, and DM2, on chromosome 3. In 1992, the mutation responsible for DM1 was identified as a CTG expansion located in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK). How this untranslated CTG expansion causes myotonic dystrophy type 1(DM1) has been controversial. The recent discovery that myotonic dystrophy type 2 (DM2) is caused by an untranslated CCTG expansion, along with other discoveries on DM1 pathogenesis, indicate that the clinical features common to both diseases are caused by a gain-of-function RNA mechanism in which the CUG and CCUG repeats alter cellular function, including alternative splicing of various genes. We discuss the pathogenic mechanisms that have been proposed for the myotonic dystrophies, the clinical and molecular features of DM1 and DM2, and the characterization of murine and cell-culture models that have been generated to better understand these diseases.     

Six Serum miRNAs Fail to Validate as Myotonic Dystrophy Type 1 Biomarkers

Myotonic dystrophy type 1 (DM1) is an autosomal dominant genetic disease caused by expansion of a CTG microsatellite in the 3’ untranslated region of the DMPK gene. Despite characteristic muscular, cardiac, and neuropsychological symptoms, CTG trinucleotide repeats are unstable both in the somatic and germinal lines, making the age of onset, clinical presentation, and disease severity very variable. A molecular biomarker to stratify patients and to follow disease progression is, thus, an unmet medical need. Looking for a novel biomarker, and given that specific miRNAs have been found to be misregulated in DM1 heart and muscle tissues, we profiled the expression of 175 known serum miRNAs in DM1 samples. The differences detected between patients and controls were less than 2.6 fold for all of them and a selection of six candidate miRNAs, miR-103, miR-107, miR-21, miR-29a, miR-30c, and miR-652 all failed to show consistent differences in serum expression in subsequent validation experiments.     

DDX6 regulates sequestered nuclear CUG-expanded DMPK-mRNA in dystrophia myotonica type 1

Myotonic dystrophy type 1 (DM1) is caused by CUG triplet expansions in the 3′ UTR of dystrophia myotonica protein kinase (DMPK) messenger ribonucleic acid (mRNA). The etiology of this multi-systemic disease involves pre-mRNA splicing defects elicited by the ability of the CUG-expanded mRNA to ‘sponge’ splicing factors of the muscleblind family. Although nuclear aggregation of CUG-containing mRNPs in distinct foci is a hallmark of DM1, the mechanisms of their homeostasis have not been completely elucidated. Here we show that a DEAD-box helicase, DDX6, interacts with CUG triplet-repeat mRNA in primary fibroblasts from DM1 patients and with CUG–RNA in vitro. DDX6 overexpression relieves DM1 mis-splicing, and causes a significant reduction in nuclear DMPK-mRNA foci. Conversely, knockdown of endogenous DDX6 leads to a significant increase in DMPK-mRNA foci count and to increased sequestration of MBNL1 in the nucleus. While the level of CUG-expanded mRNA is unaffected by increased DDX6 expression, the mRNA re-localizes to the cytoplasm and its interaction partner MBNL1 becomes dispersed and also partially re-localized to the cytoplasm. Finally, we show that DDX6 unwinds CUG-repeat duplexes in vitro in an adenosinetriphosphate-dependent manner, suggesting that DDX6 can remodel and release nuclear DMPK messenger ribonucleoprotein foci, leading to normalization of pathogenic alternative splicing events.     

RBFOX1 Cooperates with MBNL1 to Control Splicing in Muscle,Including Events Altered in Myotonic Dystrophy Type 1

With the goal of identifying splicing alterations in myotonic dystrophy 1 (DM1) tissues that may yield insights into targets or mechanisms, we have surveyed mis-splicing events in three systems using a RT-PCR screening and validation platform. First, a transgenic mouse model expressing CUG-repeats identified splicing alterations shared with other mouse models of DM1. Second, using cell cultures from human embryonic muscle, we noted that DM1-associated splicing alterations were significantly enriched in cytoskeleton (e.g. SORBS1, TACC2, TTN, ACTN1 and DMD) and channel (e.g. KCND3 and TRPM4) genes. Third, of the splicing alterations occurring in adult DM1 tissues, one produced a dominant negative variant of the splicing regulator RBFOX1. Notably, half of the splicing events controlled by MBNL1 were co-regulated by RBFOX1, and several events in this category were mis-spliced in DM1 tissues. Our results suggest that reduced RBFOX1 activity in DM1 tissues may amplify several of the splicing alterations caused by the deficiency in MBNL1.