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.

Static Postural Control Deficits in Adults with Myotonic Dystrophy Type 1, Steinert Disease

Background: Myotonic dystrophy type 1 (DM1) is characterized by progressive and predominantly distal muscle atrophy and myotonia. Gait and balance impairments, resulting in falls, are frequently reported in this population. However, the extent to which individuals with DM1 rely more on a specific sensory system for balance than asymptomatic individuals (AI) is unknown. Objective: Evaluate postural control performance in individuals with DM1 and its dependence on vision compared to AI. Methods: 20 participants with DM1, divided into two groups based on their diagnosis, i.e. adult and congenital phenotype, and 12 AI participants were recruited. Quiet standing postural control was assessed in two visual conditions: eyes-open and eyes-closed. The outcomes measures were center of pressure (CoP) mean velocity, CoP range of displacement in anteroposterior and mediolateral axis, and the 95% confidence ellipse’s surface. Friedman and Kruskal-Wallis analysis of variance were used to compare outcomes between conditions and groups, respectively. Results: Significant group effect and condition effect were observed on postural control performance. No significant difference was observed between the two DM1 groups. The significant differences observed between the AI group and the two DM1 groups in the eyes-open condition were also observed in the eyes-closed condition. Conclusions: The result revealed poorer postural control performance in people with DM1 compared to AI. The DM1 group also showed similar decrease in performance than AI in eyes-closed condition, suggesting no excessive visual dependency.

Nuclear Envelope Alterations in Myotonic Dystrophy Type 1 Patient-Derived Fibroblasts

Myotonic dystrophy type 1 (DM1) is a hereditary and multisystemic disease characterized by myotonia, progressive distal muscle weakness and atrophy. The molecular mechanisms underlying this disease are still poorly characterized, although there are some hypotheses that envisage to explain the multisystemic features observed in DM1. An emergent hypothesis is that nuclear envelope (NE) dysfunction may contribute to muscular dystrophies, particularly to DM1. Therefore, the main objective of the present study was to evaluate the nuclear profile of DM1 patient-derived and control fibroblasts and to determine the protein levels and subcellular distribution of relevant NE proteins in these cell lines. Our results demonstrated that DM1 patient-derived fibroblasts exhibited altered intracellular protein levels of lamin A/C, LAP1, SUN1, nesprin-1 and nesprin-2 when compared with the control fibroblasts. In addition, the results showed an altered location of these NE proteins accompanied by the presence of nuclear deformations (blebs, lobes and/or invaginations) and an increased number of nuclear inclusions. Regarding the nuclear profile, DM1 patient-derived fibroblasts had a larger nuclear area and a higher number of deformed nuclei and micronuclei than control-derived fibroblasts. These results reinforce the evidence that NE dysfunction is a highly relevant pathological characteristic observed in DM1.

Molecular and Clinical Implications of Variant Repeats in Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is one of the most variable monogenic diseases at phenotypic, genetic, and epigenetic level. The disease is multi-systemic with the age at onset ranging from birth to late age. The underlying mutation is an unstable expansion of CTG repeats in the DMPK gene, varying in size from 50 to >1000 repeats. Generally, large expansions are associated with an earlier age at onset. Additionally, the most severe, congenital DM1 form is typically associated with local DNA methylation. Genetic variability of DM1 mutation is further increased by its structural variations due to presence of other repeats (e.g., CCG, CTC, CAG). These variant repeats or repeat interruptions seem to confer an additional level of epigenetic variability since local DNA methylation is frequently associated with variant CCG repeats independently of the expansion size. The effect of repeat interruptions on DM1 molecular pathogenesis is not investigated enough. Studies on patients indicate their stabilizing effect on DMPK expansions because no congenital cases were described in patients with repeat interruptions, and the age at onset is frequently later than expected. Here, we review the clinical relevance of repeat interruptions in DM1 and genetic and epigenetic characteristics of interrupted DMPK expansions based on patient studies.

Altered Local Brain Amplitude of Fluctuations in Patients With Myotonic Dystrophy Type 1

This study is aimed at investigating the characteristics of the spontaneous brain activity in patients with myotonic dystrophy type 1 (DM1). A total of 18 patients with DM1 and 18 healthy controls (HCs) were examined by resting-state functional MRI. Combined methods include amplitude of low-frequency fluctuations (ALFFs), the fractional amplitude of low-frequency fluctuations (fALFFs), and Wavelet transform-based ALFFs (Wavelet-ALFFs) with standardization, percent amplitude of fluctuation (PerAF) with/without standardization were applied to evaluate the spontaneous brain activity of patients with DM1. Compared with HCs, patients with DM1 showed decreased ALFFs and Wavelet-ALFFs in the bilateral precuneus (PCUN), angular gyrus (ANG), inferior parietal, but supramarginal and angular gyri (IPL), posterior cingulate gyrus (PCG), superior frontal gyrus, medial (SFGmed), middle occipital gyrus (MOG), which were mainly distributed in the brain regions of default mode network (DMN). Decreased ALFFs and Wavelet-ALFFs were also seen in bilateral middle frontal gyrus (MFG), inferior frontal gyrus, opercular part (IFGoperc), which were the main components of the executive control network (ECN). Patients with DM1 also showed decreased fALFFs in SFGmed.R, the right anterior cingulate and paracingulate gyri (ACGR), bilateral MFG. Reduced PerAF in bilateral PCUN, ANG, PCG, MOG, and IPLL as well as decreased PerAF without standardization in PCUNR and bilateral PCG also existed in patients with DM1. In conclusion, patients with DM1 had decreased activity in DMN and ECN with increased fluctuations in the temporal cortex and cerebellum. Decreased brain activity in DMN was the most repeatable and reliable with PCUN and PCG being the most specific imaging biomarker of brain dysfunction in patients with DM1.

Characterization of RAN Translation and Antisense Transcription in Primary Cell Cultures of Patients with Myotonic Dystrophy Type 1

Myotonic Dystrophy type 1 (DM1) is a muscular dystrophy with a multi-systemic nature. It was one of the first diseases in which repeat associated non-ATG (RAN) translation was described in 2011, but has not been further explored since. In order to enhance our knowledge of RAN translation in DM1, we decided to study the presence of DM1 antisense (DM1-AS) transcripts (the origin of the polyglutamine (polyGln) RAN protein) using RT-PCR and FISH, and that of RAN translation via immunoblotting and immunofluorescence in distinct DM1 primary cell cultures, e.g., myoblasts, skin fibroblasts and lymphoblastoids, from ten patients. DM1-AS transcripts were found in all DM1 cells, with a lower expression in patients compared to controls. Antisense RNA foci were found in the nuclei and cytoplasm of a subset of DM1 cells. The polyGln RAN protein was undetectable in all three cell types with both approaches. Immunoblots revealed a 42 kD polyGln containing protein, which was most likely the TATA-box-binding protein. Immunofluorescence revealed a cytoplasmic aggregate, which co-localized with the Golgi apparatus. Taken together, DM1-AS transcript levels were lower in patients compared to controls and a small portion of the transcripts included the expanded repeat. However, RAN translation was not present in patient-derived DM1 cells, or was in undetectable quantities for the available methods.

Brain Pathogenesis and Potential Therapeutic Strategies in Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy that affects multiple systems including the muscle and heart. The mutant CTG expansion at the 3′-UTR of the DMPK gene causes the expression of toxic RNA that aggregate as nuclear foci. The foci then interfere with RNA-binding proteins, affecting hundreds of mis-spliced effector genes, leading to aberrant alternative splicing and loss of effector gene product functions, ultimately resulting in systemic disorders. In recent years, increasing clinical, imaging, and pathological evidence have indicated that DM1, though to a lesser extent, could also be recognized as true brain diseases, with more and more researchers dedicating to develop novel therapeutic tools dealing with it. In this review, we summarize the current advances in the pathogenesis and pathology of central nervous system (CNS) deficits in DM1, intervention measures currently being investigated are also highlighted, aiming to promote novel and cutting-edge therapeutic investigations.

DMPK hypermethylation in sperm cells of myotonic dystrophy type 1 patients

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.