The CTG repeat expansion size correlates with the splicing defects observed in muscles from myotonic dystrophy type 1 patients

Botta, Annalisa; Rinaldi, Fabrizio; Catalli, Claudio; Vergani, Lodovica; Bonifazi, E.; Romeo, Vincenzo; Loro, Emanuele; Viola, Antonella; Angelini, C.; Novelli, Giuseppe

doi:10.1136/jmg.2008.058909

Cited by 53 publications

(52 citation statements)

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“…8 To assess the in trans effect of the CTG repetition on the splicing regulation in DM1 differentiating myoblasts, we analyzed the expression of IR and MBNL1 gene isoforms at different times of differentiation (stages T0, T4 and T10). RT-PCR amplification showed that in DM1 cells the ratios of the IR-A/TOT ( Figure 2b Catabolic pathways in DM1 muscle cells.…”

Section: Resultsmentioning

confidence: 99%

“…RNA fluorescence in situ hybridization was performed as described. 8 TUNEL and cytochrome c release detection. Apoptosis detection in 0-, 4-, 10-and 15-day myotubes was performed using TUNEL and cytochrome c release assays.…”

Section: Discussionmentioning

confidence: 99%

“…The PCR splicing assays for IR and MBNL1 genes were performed as described earlier. 8 Total PCR products, obtained within the linear range of amplification, were electrophoresed on 3.5% agarose gel. Quantitative analysis of the amplified products was performed using ethidium bromide staining.…”

Section: Discussionmentioning

confidence: 99%

“…2,6 It has been reported that (CTG) n trinucleotide repeat length in muscle directly correlates with both frequency of severe cDM1 7 and rate of splicing impairment typical of DM. 8 The pathology of skeletal muscle, including both myotonic and dystrophic features, has been reproduced in different mouse models by the following different strategies: random insertion of a genomic fragment carrying the expanded sequence in its human DM1 context 9 or in human skeletal actin (HSA) gene, 10 or insertion of a humanized DMPK locus containing a human expanded sequence . 11 An inducible model for the pathology carrying 960 (CTG) n and showing severe muscle wasting was also recently created.…”

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Normal myogenesis and increased apoptosis in myotonic dystrophy type-1 muscle cells

et al. 2010

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Myotonic dystrophy (DM) is caused by a (CTG) n expansion in the 3 0 -untranslated region of DMPK gene. Mutant transcripts are retained in nuclear RNA foci, which sequester RNA binding proteins thereby misregulating the alternative splicing. Controversy still surrounds the pathogenesis of the DM1 muscle distress, characterized by myotonia, weakness and wasting with distal muscle atrophy. Eight primary human cell lines from adult-onset (DM1) and congenital (cDM1) patients, (CTG) n range 90-1800, were successfully differentiated into aneural-immature and contracting-innervated-mature myotubes. Morphological, immunohistochemical, RT-PCR and western blotting analyses of several markers of myogenesis indicated that in vitro differentiation-maturation of DM1 myotubes was comparable to age-matched controls. In all pathological muscle cells, (CTG) n expansions were confirmed by long PCR and RNA fluorescence in situ hybridization. Moreover, the DM1 myotubes showed the splicing alteration of insulin receptor and muscleblind-like 1 (MBNL1) genes associated with the DM1 phenotype. Considerable myotube loss and atrophy of 15-day-differentiated DM1 myotubes indicated activated catabolic pathways, as confirmed by the presence of apoptotic (caspase-3 activation, cytochrome c release, chromatin fragmentation) and autophagic (P62/LC3) markers. Z-VAD treatment significantly reduced the decrease in myonuclei number and in average width in 15-day-differentiated DM1 myotubes. We thus propose that the muscle wasting typical in DM1 is due to impairment of muscle mass maintenanceregeneration, through premature apoptotic-autophagic activation, rather than altered myogenesis. Myotonic dystrophy (DM) is a multi-systemic disorder caused by two different microsatellite expansions in non-coding regions. Together, these two mutations affect 1 out of 8000 individuals and represent the most common form of muscular dystrophy in adults. DM1 and DM2 have common symptoms such as myotonia, muscle weakness and early cataract development. 1,2 Although DM1 and DM2 initially affect different muscles (distal versus proximal), histological analysis of the muscular tissues shows common aspects such as central nucleation. The classic form of DM1 is characterized by muscle distress with myotonia, progressive muscle weakness and wasting. Atrophy has also been reported, occurring preferentially in type-1 fibers in DM1 and in type-2 in DM2. 3 DM1 but not DM2 also presents a congenital form (cDM1), characterized by a high neonatal mortality and symptoms such as hypotonia, mental retardation and respiratory distress. 4,5 DM1 is associated with an unstable (CTG) n trinucleotide expansion located in the 3 0 -untranslated (3 0 -UTR) region of the DM protein kinase (DMPK) gene on chromosome 19q13.3. The mutant DMPK transcript, containing the expanded (CTG) n sequence, accumulates in discrete nuclear foci able to sequester various nuclear factors such as RNAbinding proteins or splicing regulators, causing different and highly variable downstream deleterious effects. 2,6...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Normal myogenesis and increased apoptosis in myotonic dystrophy type-1 muscle cells

et al. 2010

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“…1A, and contains the open reading frame of lacZ, 24 MS2 stem-loop sequences, and the 3ЈUTR of DMPK containing either five or 145 CUG-triplet repeats (lacZ-MS2-5CUG or lacZ-MS2-145CUG). Although five CUG repeats are insufficient to promote RNA aggregation, 145 CUG repeats are known to promote RNA foci formation in DM1 patients (Botta et al, 2008). We chose this CUG-repeat expansion size so that the transcripts would not all be aggregated and we would be able to detect and track single CUG-rich mRNAs (see below).…”

Section: The Ms2-gfp System For Visualization Of Transcripts In Livinmentioning

confidence: 99%

Stochastic and reversible aggregation of mRNA with expanded CUG-triplet repeats

Querido

Gallardo

Beaudoin

et al. 2011

Journal of Cell Science

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SummaryTranscripts containing expanded CNG repeats, which are found in several neuromuscular diseases, are not exported from the nucleus and aggregate as ribonuclear inclusions by an unknown mechanism. Using the MS2-GFP system, which tethers fluorescent proteins to a specific mRNA, we followed the dynamics of single CUG-repeat transcripts and RNA aggregation in living cells. Single transcripts with 145 CUG repeats from the dystrophia myotonica-protein kinase (DMPK) gene had reduced diffusion kinetics compared with transcripts containing only five CUG repeats. Fluorescence recovery after photobleaching (FRAP) experiments showed that CUGrepeat RNAs display a stochastic aggregation behaviour, because individual RNA foci formed at different rates and displayed different recoveries. Spontaneous clustering of CUG-repeat RNAs was also observed, confirming the stochastic aggregation revealed by FRAP. The splicing factor Mbnl1 colocalized with individual CUG-repeat transcripts and its aggregation with RNA foci displayed the same stochastic behaviour as CUG-repeat mRNAs. Moreover, depletion of Mbnl1 by RNAi resulted in decreased aggregation of CUG-repeat transcripts after FRAP, supporting a direct role for Mbnl1 in CUG-rich RNA foci formation. Our data reveal that nuclear CUG-repeat RNA aggregates are labile, constantly forming and disaggregating structures, and that the Mbnl1 splicing factor is directly involved in the aggregation process.

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Therapeutic advances in muscular dystrophy

Leung

Wagner

2013

Annals of Neurology

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The muscular dystrophies comprise a heterogeneous group of genetic disorders that produce progressive skeletal muscle weakness and wasting. There has been rapid growth and change in our understanding of these disorders in recent years, and advances in basic science are being translated into increasing numbers of clinical trials. This review will discuss therapeutic developments in 3 of the most common forms of muscular dystrophy: Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, and myotonic dystrophy. Each of these disorders represents a different class of genetic disease (monogenic, epigenetic, and repeat expansion disorders), and the approach to therapy addresses the diverse and complex molecular mechanisms involved in these diseases. The large number of novel pharmacologic agents in development with good biologic rationale and strong proof of concept suggests there will be an improved quality of life for individuals with muscular dystrophy.

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The CTG repeat expansion size correlates with the splicing defects observed in muscles from myotonic dystrophy type 1 patients

Abstract: The CTG repeat length plays a key role in the extent of splicing misregulation and foci formation, thus providing a useful link between the genotype and the molecular cellular phenotype in DM1.

Cited by 53 publications

References 25 publications

Normal myogenesis and increased apoptosis in myotonic dystrophy type-1 muscle cells

Normal myogenesis and increased apoptosis in myotonic dystrophy type-1 muscle cells

Stochastic and reversible aggregation of mRNA with expanded CUG-triplet repeats

Therapeutic advances in muscular dystrophy

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