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Myotonic dystrophy type 1 embryonic stem cells show decreased myogenic potential, increased CpG methylation at the DMPK locus and RNA mis-splicing.

ABSTRACT: Skeletal muscle tissue is severely affected in myotonic dystrophy type 1 (DM1) patients, characterised by muscle weakness, myotonia and muscle immaturity in the most severe congenital form of the disease. Previously, it was not known at what stage during myogenesis the DM1 phenotype appears. In this study we differentiated healthy and DM1 human embryonic stem cells to myoblasts and myotubes and compared their differentiation potential using a comprehensive multi-omics approach. We found myogenesis in DM1 cells to be abnormal with altered myotube generation compared to healthy cells. We did not find differentially expressed genes between DM1 and non-DM1 cell lines within the same developmental stage. However, during differentiation we observed an aberrant inflammatory response and increased CpG methylation upstream of the CTG repeat at the myoblast level and RNA mis-splicing at the myotube stage. We show that early myogenesis modelled in hESC reiterates the early developmental manifestation of DM1.

SUBMITTER: Franck S

PROVIDER: S-EPMC8764412 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Furamidine Rescues Myotonic Dystrophy Type I Associated Mis-Splicing through Multiple Mechanisms.

Project description:Myotonic dystrophy type 1 (DM1) is an autosomal dominant, CTG•CAG microsatellite expansion disease. Expanded CUG repeat RNA sequester the muscleblind-like (MBNL) family of RNA-binding proteins, thereby disrupting their normal cellular function which leads to global mis-regulation of RNA processing. Previously, the small molecule furamidine was shown to reduce CUG foci and rescue mis-splicing in a DM1 HeLa cell model and to rescue mis-splicing in the HSALR DM1 mouse model, but furamidine's mechanism of action was not explored. Here we use a combination of biochemical, cell toxicity, and genomic studies in DM1 patient-derived myotubes and the HSALR DM1 mouse model to investigate furamidine's mechanism of action. Mis-splicing rescue was observed in DM1 myotubes and the HSALR DM1 mouse with furamidine treatment. Interestingly, while furamidine was found to bind CTG•CAG repeat DNA with nanomolar affinity, a reduction in expanded CUG repeat transcript levels was observed in the HSALR DM1 mouse but not DM1 patient-derived myotubes. Further investigation in these cells revealed that furamidine treatment at nanomolar concentrations led to up-regulation of MBNL1 and MBNL2 protein levels and a reduction of ribonuclear foci. Additionally, furamidine was shown to bind CUG RNA with nanomolar affinity and disrupted the MBNL1 -CUG RNA complex in vitro at micromolar concentrations. Furamidine's likely promiscuous interactions in vitro and in vivo appear to affect multiple pathways in the DM1 mechanism to rescue mis-splicing, yet surprisingly furamidine was shown globally to rescue many mis-splicing events with only modest off-target effects on gene expression in the HSALR DM1 mouse model. Importantly, over 20% of the differentially expressed genes were shown to be returned, to varying degrees, to wild-type expression levels.

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Epigenetics of the myotonic dystrophy-associated DMPK gene neighborhood.

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Muscular myopathies other than myotonic dystrophy also associated with (CTG)n expansion at the DMPK locus.

Project description:OBJECTIVE:Assess triplet repeat expansion (CTG)n at the 'dystrophia-myotonica protein kinase' (DMPK) locus in muscular myopathies to elucidate its role in myopathic symptoms and enable genetic counseling and prenatal diagnosis in families. METHODS AND RESULTS:Individuals with symptoms of myopathy, hypotonia and controls selected randomly from the population were evaluated for triplet repeat expansion of (CTG)n repeats in the 3'untranslated region (UTR) of DMPK gene, the causative mutation in myotonic dystrophy (DM). DNA was isolated from peripheral blood of 40 individuals; they presented symptoms of muscle myopathy (n = 11), muscle hypotonia (n = 4), members of their families (n = 5) and control individuals from random population (n = 20). Molecular analysis of genomic DNA by polymerase chain reaction (PCR) using primers specific for the DMPK gene encompassing the triplet repeat expansion, showed that all controls (n = 20) gave a 2.1 kb band indicating normal triplet repeat number. Three out of 11 cases (two clinically diagnosed DM and one muscular dystrophy) had an expansion of the (CTG)n repeat in the range of 1000-2100 repeats corresponding to the repeat number in cases of severe DM. Other two of these 11 cases, showed a mild expansion of ~ 66 repeats. Three samples, which included two cases of hypotonia and the father of a subject with muscular dystrophy, also gave a similar repeat expansion (~66 repeats). CONCLUSION:Results suggest a role of (CTG)n expansion at the DMPK locus in unexplained hypotonias and muscular myopathies other than DM. This calls for screening of the triplet repeat expansion at the DMPK locus in cases of idiopathic myopathies and hypotonia.

| S-EPMC3611902 | biostudies-literature

DMPK hypermethylation in sperm cells of myotonic dystrophy type 1 patients.

Project description:Myotonic dystrophy type 1 (DM1) is an autosomal dominant muscular dystrophy that results from a CTG expansion (50-4000 copies) in the 3' UTR of the DMPK gene. The disease is classified into four or five somewhat overlapping forms, which incompletely correlate with expansion size in somatic cells of patients. With rare exception, it is affected mothers who transmit the congenital (CDM1) and most severe form of the disease. Why CDM1 is hardly ever transmitted by fathers remains unknown. One model to explain the almost exclusive transmission of CDM1 by affected mothers suggests a selection against hypermethylated large expansions in the germline of male patients. By assessing DNA methylation upstream to the CTG expansion in motile sperm cells of four DM1 patients, together with availability of human embryonic stem cell (hESCs) lines with paternally inherited hypermethylated expansions, we exclude the possibility that DMPK hypermethylation leads to selection against viable sperm cells (as indicated by motility) in DM1 patients.

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Stable Longitudinal Methylation Levels at the CpG Sites Flanking the CTG Repeat of DMPK in Patients with Myotonic Dystrophy Type 1.

Project description:Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystem disorder mainly characterized by gradual muscle loss, weakness, and delayed relaxation after muscle contraction. It is caused by an expanded CTG repeat in the 3' UTR of DMPK, which is transcribed into a toxic gain-of-function mRNA that affects the splicing of a range of other genes. The repeat is unstable, with a bias towards expansions both in somatic cells and in the germline, which results in a tendency for earlier onset with each generation, as longer repeat lengths generally correlate with earlier onset. Previous studies have found hypermethylation in the regions flanking the repeat in congenital onset DM1 and in some patients with non-congenital DM1. We used pyrosequencing to investigate blood methylation levels in 68 patients with non-congenital DM1, compare the methylation levels between the blood and muscle, and assess whether methylation levels change over time in the blood. We found higher methylation levels in the blood of DM1 patients than in healthy controls and especially in the patients who had inherited the disease allele maternally. The methylation levels remained relatively stable over time and are a strong biomarker of the disease, as well as of the maternal inheritance of the disease.

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Stable Longitudinal Methylation Levels at the CpG Sites Flanking the CTG Repeat of DMPK in Patients with Myotonic Dystrophy Type 1.

| S-EPMC7465187 | biostudies-literature

Modifications to toxic CUG RNAs induce structural stability, rescue mis-splicing in a myotonic dystrophy cell model and reduce toxicity in a myotonic dystrophy zebrafish model.

Project description:CUG repeat expansions in the 3' UTR of dystrophia myotonica protein kinase (DMPK) cause myotonic dystrophy type 1 (DM1). As RNA, these repeats elicit toxicity by sequestering splicing proteins, such as MBNL1, into protein-RNA aggregates. Structural studies demonstrate that CUG repeats can form A-form helices, suggesting that repeat secondary structure could be important in pathogenicity. To evaluate this hypothesis, we utilized structure-stabilizing RNA modifications pseudouridine (Ψ) and 2'-O-methylation to determine if stabilization of CUG helical conformations affected toxicity. CUG repeats modified with Ψ or 2'-O-methyl groups exhibited enhanced structural stability and reduced affinity for MBNL1. Molecular dynamics and X-ray crystallography suggest a potential water-bridging mechanism for Ψ-mediated CUG repeat stabilization. Ψ modification of CUG repeats rescued mis-splicing in a DM1 cell model and prevented CUG repeat toxicity in zebrafish embryos. This study indicates that the structure of toxic RNAs has a significant role in controlling the onset of neuromuscular diseases.

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Choroid plexus mis-splicing and altered cerebrospinal fluid composition in myotonic dystrophy type 1

Project description:This SuperSeries is composed of the SubSeries listed below.

2023-07-03 | GSE228459 | GEO

Variant repeats within the DMPK CTG expansion protect function in myotonic dystrophy type 1.

Project description:Objective:We tested the hypothesis that variant repeat interruptions (RIs) within the DMPK CTG repeat tract lead to milder symptoms compared with pure repeats (PRs) in myotonic dystrophy type 1 (DM1). Methods:We evaluated motor, neurocognitive, and behavioral outcomes in a group of 6 participants with DM1 with RI compared with a case-matched sample of 12 participants with DM1 with PR and a case-matched sample of 12 unaffected healthy comparison participants (UA). Results:In every measure, the RI participants were intermediate between UA and PR participants. For muscle strength, the RI group was significantly less impaired than the PR group. For measures of Full Scale IQ, depression, and sleepiness, all 3 groups were significantly different from each other with UA > RI > PR in order of impairment. The RI group was different from unaffected, but not significantly different from PR (UA > RI = PR) in apathy and working memory. Finally, in finger tapping and processing speed, RI did not differ from UA comparisons, but PR had significantly lower scores than the UA comparisons (UA = RI > PR). Conclusions:Our results support the notion that patients affected by DM1 with RI demonstrate a milder phenotype with the same pattern of deficits as those with PR indicating a similar disease process.

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Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice.

Project description:Myotonic dystrophy type 1 (DM1), a dominant hereditary muscular dystrophy, is caused by an abnormal expansion of a (CTG)n trinucleotide repeat in the 3' UTR of the human dystrophia myotonica protein kinase (DMPK) gene. As a consequence, mutant transcripts containing expanded CUG repeats are retained in nuclear foci and alter the function of splicing regulatory factors members of the MBNL and CELF families, resulting in alternative splicing misregulation of specific transcripts in affected DM1 tissues. In the present study, we treated DMSXL mice systemically with a 2'-4'-constrained, ethyl-modified (ISIS 486178) antisense oligonucleotide (ASO) targeted to the 3' UTR of the DMPK gene, which led to a 70% reduction in CUGexp RNA abundance and foci in different skeletal muscles and a 30% reduction in the heart. Furthermore, treatment with ISIS 486178 ASO improved body weight, muscle strength, and muscle histology, whereas no overt toxicity was detected. This is evidence that the reduction of CUGexp RNA improves muscle strength in DM1, suggesting that muscle weakness in DM1 patients may be improved following elimination of toxic RNAs.

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