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Enzyme replacement therapy (ERT) is the only approved disease-modifying treatment modality for Pompe disease, a rare, inherited metabolic disorder caused by a deficiency in the acid α-glucosidase (GAA) enzyme that catabolizes lysosomal glycogen. First-generation recombinant human GAA (rhGAA) ERT (alglucosidase alfa) can slow the progressive muscle degeneration characteristic of the disease. Still, most patients experience diminished efficacy over time, possibly because of poor uptake into target tissues. Next-generation ERTs aim to address this problem by increasing bis-phosphorylated high mannose (bis-M6P) N-glycans on rhGAA as these moieties have sufficiently high receptor binding affinity at the resultant low interstitial enzyme concentrations after dosing to drive uptake by the cation-independent mannose 6-phosphate receptor on target cells. However, some approaches introduce bis-M6P onto rhGAA via non-natural linkages that cannot be hydrolyzed by natural human enzymes and thus inhibit the endolysosomal glycan trimming necessary for complete enzyme activation after cell uptake. Furthermore, all rhGAA ERTs face potential inactivation during intravenous delivery (and subsequent non-productive clearance) as GAA is an acid hydrolase that is rapidly denatured in the near-neutral pH of the blood. One new therapy, cipaglucosidase alfa plus miglustat, is hypothesized to address these challenges by combining an enzyme enriched with naturally occurring bis-M6P N-glycans with a small-molecule stabilizer. Here, we investigate this hypothesis by analyzing published and new data related to the mechanism of action of the enzyme and stabilizer molecule. Based on an extensive collection of in vitro, preclinical, and clinical data, we conclude that cipaglucosidase alfa plus miglustat successfully addresses each of these challenges to offer meaningful advantages in terms of pharmacokinetic exposure, target-cell uptake, endolysosomal processing, and clinical benefit.

Abstract Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common autosomal dominant muscle disorders, yet no cure or amelioration exists. The clinical presentation is diverse, making it difficult to identify the actual driving pathomechanism among many downstream events. To unravel this complexity, we performed a meta-analysis of 13 original omics datasets (in total 171 FSHD and 129 control samples). Our approach confirmed previous findings about the disease pathology and specified them further. We confirmed increased expression of former proposed DUX4 biomarkers, and furthermore impairment of the respiratory chain. Notably, the meta-analysis provides insights about so far not reported pathways, including misregulation of neuromuscular junction protein encoding genes, downregulation of the spliceosome, and extensive alterations of nuclear envelope protein expression. Finally, we developed a publicly available shiny app to provide a platform for researchers who want to search our analysis for genes of interest in the future.

Abstract Background The minimal clinically important difference (MCID) is the smallest change in outcome that physicians or patients would consider meaningful and is relevant when evaluating disease progression or the efficacy of interventions. Studies of patients with late-onset Pompe disease (LOPD) have used the 6-min walk distance (6MWD) as an endpoint to assess motor function. However, an MCID for 6MWD (% predicted and meters) has yet to be established in LOPD. The objective of the study was to derive 6MWD MCID (% predicted and meters) with different analysis methods and for subgroups of different disease severity for LOPD. Methods Data from the PROPEL trial were used to calculate 6MWD MCID in the overall PROPEL population and subgroups of baseline severity as assessed by walking distance and body mass index (BMI), using anchor- and distribution-based approaches. Results The 6MWD MCIDs varied widely, depending on the method and subgroup, ranging from 2.27%-8.11% predicted for the overall LOPD population (23.7 m-57.2 m). For patients with baseline 6MWD

IntroductionChronic muscle pain is common in myotonic dystrophies (DM). Little is known about its pathophysiology. We aimed to investigate the characteristics of the neuropathic pain component contributing contributes to the pathogenesis of chronic pain in DM.MethodsTwenty-one DM1 and 32 DM2 patients completed pain questionnaires (Brief pain inventory–BPI, PAIN-DETECT, pain disability index–PDI) and underwent neurological examination, nerve conduction studies (NCS), quantitative sensory testing (QST, dorsum of the right hand and right thigh) and skin biopsy to determine the intraepidermal nerve fiber density (IENFD, distal and proximal site of lower extremity). NCS and QST results at the thigh were compared to 27 healthy controls and IENFD and QST at the dorsum of the hand to published reference values.ResultsThe sensory profile of DM2 patients was characterized by a loss in thermal and mechanical detection, while DM1 patients showed reduced mechanical and heat pain thresholds and higher mechanical pain sensitivity. Both DM groups showed pressure hyperalgesia. IENFD was reduced in 63% of DM1 patients and 50% of DM2. The slightly higher pain interference and disability found in DM2 was rather due to age difference than disease.ConclusionSimilar pain mechanisms likely occur in both DM1 and DM2, even though a tendency toward more pain sensitivity was observed in DM1 and more sensory loss in DM2. Both QST and reduced IENFD highlight the presence of peripheral nerve damage in DM. This must be considered for the best pain management strategies.

Pompe disease is a rare genetic disorder characterized by a deficiency of acid α-glucosidase (GAA), leading to the accumulation of glycogen in various tissues, especially in skeletal muscles. The disease manifests as a large spectrum of phenotypes from infantile-onset Pompe disease (IOPD) to late-onset Pompe disease (LOPD), depending on the age of symptoms onset. Quantifying GAA activity and glycogen content in skeletal muscle provides important information about the disease severity. However, the distribution of GAA and glycogen levels in skeletal muscles from healthy individuals and those impacted by Pompe disease remains poorly understood, and there is currently no universally accepted standard assay for GAA activity measurement. This systematic literature review aims to provide an overview of the available information on GAA activity and glycogen content levels in skeletal muscle biopsies from patients with Pompe disease.A structured review of PubMed and Google Scholar literature (with the latter used to check that no additional publications were identified) was conducted to identify peer-reviewed publications on glycogen storage disease type II [MeSH term] + GAA, protein human (supplementary concept), Pompe, muscle; and muscle, acid alpha-glucosidase. A limit of English language was applied. Results were grouped by methodologies used to quantify GAA activity and glycogen content in skeletal muscle. The search and selection strategy were devised and carried out in line with Preferred Reporting of Items in Systematic Reviews and Meta-Analysis guidelines and documented using a flowchart. Bibliographies of papers included in the analysis were reviewed and applicable publications not already identified in the search were included.Of the 158 articles retrieved, 24 (comprising >100 muscle biopsies from >100 patients) were included in the analysis, with four different assays. Analysis revealed that patients with IOPD exhibited markedly lower GAA activity in skeletal muscles than those with LOPD, regardless of the measurement method employed. Additionally, patients with IOPD had notably higher glycogen content levels in skeletal muscles than those with LOPD. In general, however, it was difficult to fully characterize GAA activity because of the different methods used. The findings underscore the challenges in the interpretation and comparison of the results across studies because of the substantial methodological variations. There is a need to establish standardized reference ranges of GAA activity and glycogen content in healthy individuals and in Pompe disease patients based on globally standardized methods to improve comparability and reliability in assessing this rare disease.

Skeletal muscle MRI studies in limb-girdle muscular dystrophy (LGMD) have increased over the past decades, improving the utility of MRI as a differential diagnostic tool. Nevertheless, the relative rarity of individual genotypes limits the scope of what each study can address, making it challenging to obtain a comprehensive overview of the MRI image of this splintered group. Furthermore, MRI studies have varied in their methods for assessing fat infiltration, which is essential in skeletal muscle MRI evaluation. It stayed problematic and impeded attempts to integrate multiple studies to cover the core MRI features of a distinct LGMD. In this study, we conducted a systematic review of LGMD in adults published until April 2023; 935 references were screened in PubMed and EMBASE, searches of the gray literature, and additional records were added during the screening process. Finally, 39 studies were included in our final analysis. We attempted to quantitatively synthesize the MRI data sets from the 39 individual studies. Finally, we illustrated ideal and simple MRI muscle involvement patterns of six representative LGMD genotypes. Our summary synthesis reveals a distinct distribution pattern of affected muscles by LGMD genotypes, which may be helpful for a quick first-tier differential diagnosis guiding genetic diagnostics.

Abstract Background Causal therapies are not yet available for most neuromuscular diseases. Additionally, data on the use of complementary or alternative therapies (CAM) in patients groups with a variety of different neuromuscular diseases are rare. This retrospective cross-sectional study aims to record the frequency of use and satisfaction of conventional therapies and complementary or alternative medicine (CAM) in patients with neuromuscular disorders in order to compare them afterwards. Methods Patients from the University of Leipzig (Department and Outpatient Department of Neurology), the Friedrich-Baur-Institute (Department of Neurology), the Hoher Meißner Clinic (Department of Neurology) and the German Society for Muscular Diseases (DGM e.V.) were included. The focus of this study has been on patients with chronic neuromuscular diseases. Our data are based on standardised questionnaires in analogue form from three German neuromuscular centres and in digital form from the official website of the German Society for Muscular Diseases. Therapy satisfaction was assessed with the Patient Evaluation of Global Response (PEGR) ranking scale (very satisfactory + 2 to very unsatisfactory − 2). Results Of 192 questionnaires analysed, the most common diagnoses were spinal muscular atrophy (n = 42; 21.9%), muscular dystrophies (n = 41; 21.4%) and myotonic dystrophies (n = 38; 19.8%). More than half (n = 112; 58.3%) used both conventional and CAM treatments. Physiotherapy (n = 165) was used most frequently within all treatments with conventional manual (PEGR 1.25, p = 0.013; CI 95%) and aquatic therapy (PEGR 1.3, p = 0.038) showing significantly higher satisfaction compared to therapy on training equipment. Less-used therapies such as psychotherapy (n = 53; PEGR 1.2) were also satisfactory. Within CAM, massages (n = 96) were the most reported and meditation (PEGR 1.5) was best rated. Massage therapy was significantly more satisfactory than progressive muscle relaxation (p = 0.003) and chiropractic treatment (p = 0.036). Chiropractic treatment (PEGR − 0.1) was rated most negatively. No significant differences were found between the group of conventional therapies and CAM users regarding social and disease-dependent factors. Conclusions Treatment with conventional therapy (manual, aquatic therapy) and some CAM therapies can be recommended if adequately indicated.

Abstract Introduction In 2021, the Deutsche Gesellschaft für Neurology published a new guideline on diagnosis and therapy of motor neuron disorders. Motor neuron disorders affect upper motor neurons in the primary motor cortex and/or lower motor neurons in the brain stem and spinal cord. The most frequent motor neuron disease amyotrophic lateral sclerosis (ALS) is a rapidly progressive disease with an average life expectancy of 2–4 years with a yearly incidence of 3.1/100,000 in Central Europe (Rosenbohm et al. in J Neurol 264(4):749–757, 2017. https://doi.org/10.1007/s00415-017-8413-3 ). It is considered a rare disease mainly due to its low prevalence as a consequence of short disease duration. Recommendations These guidelines comprise recommendations regarding differential diagnosis, neuroprotective therapies and multidisciplinary palliative care including management of respiration and nutrition as well as provision of assistive devices and end-of-life situations. Conclusion Diagnostic and therapeutic guidelines are necessary due the comparatively high number of cases and the aggressive disease course. Given the low prevalence and the severe impairment of patients, it is often impossible to generate evidence-based data so that ALS guidelines are partially dependent on expert opinion.

Myasthenia gravis (MG), Lambert-Eaton myasthenic syndrome (LEMS), and congenital myasthenic syndromes (CMS) represent an etiologically heterogeneous group of (very) rare chronic diseases. MG and LEMS have an autoimmune-mediated etiology, while CMS are genetic disorders. A (strain dependent) muscle weakness due to neuromuscular transmission disorder is a common feature. Generalized MG requires increasingly differentiated therapeutic strategies that consider the enormous therapeutic developments of recent years. To include the newest therapy recommendations, a comprehensive update of the available German-language guideline ‘Diagnostics and therapy of myasthenic syndromes’ has been published by the German Neurological society with the aid of an interdisciplinary expert panel. This paper is an adapted translation of the updated and partly newly developed treatment guideline. It defines the rapid achievement of complete disease control in myasthenic patients as a central treatment goal. The use of standard therapies, as well as modern immunotherapeutics, is subject to a staged regimen that takes into account autoantibody status and disease activity. With the advent of modern, fast-acting immunomodulators, disease activity assessment has become pivotal and requires evaluation of the clinical course, including severity and required therapies. Applying MG-specific scores and classifications such as Myasthenia Gravis Activities of Daily Living, Quantitative Myasthenia Gravis, and Myasthenia Gravis Foundation of America allows differentiation between mild/moderate and (highly) active (including refractory) disease. Therapy decisions must consider age, thymic pathology, antibody status, and disease activity. Glucocorticosteroids and the classical immunosuppressants (primarily azathioprine) are the basic immunotherapeutics to treat mild/moderate to (highly) active generalized MG/young MG and ocular MG. Thymectomy is indicated as a treatment for thymoma-associated MG and generalized MG with acetylcholine receptor antibody (AChR-Ab)-positive status. In (highly) active generalized MG, complement inhibitors (currently eculizumab and ravulizumab) or neonatal Fc receptor modulators (currently efgartigimod) are recommended for AChR-Ab-positive status and rituximab for muscle-specific receptor tyrosine kinase (MuSK)-Ab-positive status. Specific treatment for myasthenic crises requires plasmapheresis, immunoadsorption, or IVIG. Specific aspects of ocular, juvenile, and congenital myasthenia are highlighted. The guideline will be further developed based on new study results for other immunomodulators and biomarkers that aid the accurate measurement of disease activity.

Abstract Pompe disease is a debilitating medical condition caused by a functional deficiency of lysosomal acid alpha-glucosidase (GAA). In addition to muscle weakness, people living with Pompe disease experience motor coordination deficits including an instable gait and posture. We reasoned that an impaired muscle spindle function might contribute to these deficiencies and therefore analyzed proprioception as well as muscle spindle structure and function in 4- and 8-month-old Gaa −/− mice. Gait analyses showed a reduced inter-limb and inter-paw coordination in Gaa −/− mice. Electrophysiological analyses of single-unit muscle spindle proprioceptive afferents revealed an impaired sensitivity of the dynamic and static component of the stretch response. Finally, a progressive degeneration of the sensory neuron and of the intrafusal fibers was detectable in Gaa −/− mice. We observed an increased abundance and size of lysosomes, a fragmentation of the inner and outer connective tissue capsule and a buildup of autophagic vacuoles in muscle spindles from 8-month-old Gaa −/− mice, indicating lysosomal defects and an impaired autophagocytosis. These results demonstrate a structural and functional degeneration of muscle spindles and an altered motor coordination in Gaa −/− mice. Similar changes could contribute to the impaired motor coordination in patients living with Pompe disease.

Abstract Background Pain occurs in the majority of patients with late onset Pompe disease (LOPD) and is associated with a reduced quality of life. The aim of this study was to analyse the pain characteristics and its relation to a small nerve fiber involvement in LOPD patients. Methods In 35 patients with LOPD under enzyme replacement therapy without clinical signs of polyneuropathy (19 females; 51 ± 15 years), pain characteristics as well as depressive and anxiety symptoms were assessed using the PainDetect questionnaire (PDQ) and the hospital anxiety and depression scale (HADS), respectively. Distal skin biopsies were analysed for intraepidermal nerve fiber density (IENFD) and compared to age- and gender-matched reference data. Skin biopsies from 20 healthy subjects served as controls to assure validity of the morphometric analysis. Results Pain was reported in 69% of the patients with an average intensity of 4.1 ± 1.1 on the numeric rating scale (NRS; anchors: 0–10). According to PDQ, neuropathic pain was likely in one patient, possible in 29%, and unlikely in 67%. Relevant depression and anxiety symptoms occurred in 31% and 23%, respectively, and correlated with pain intensity. Distal IENFD (3.98 ± 1.95 fibers/mm) was reduced in 57% of the patients. The degree of IENFD reduction did not correlate with the durations of symptoms to ERT or duration of ERT to biopsy. Conclusions Pain is a frequent symptom in treated LOPD on ERT, though a screening questionnaire seldom indicated neuropathic pain. The high frequency of small nerve fiber pathology in a treated LOPD cohort was found regardless of the presence of pain or comorbid risk factors for SFN and needs further exploration in terms of clinical context, exact mechanisms and when developing novel therapeutic options for LOPD.

Myotonic dystrophy type 1 is a multisystemic disorder with predominant muscle and neurological involvement. Despite a well described pathomechanism, which is primarily a global missplicing due to sequestration of RNA-binding proteins, there are still many unsolved questions. One such question is the disease etiology in the different affected tissues. We observed alterations at the nuclear envelope in primary muscle cell cultures before. This led us to reanalyze a published RNA-sequencing dataset of DM1 and control muscle biopsies regarding the misregulation of NE proteins. We could identify several muscle NE protein encoding genes to be misregulated depending on the severity of the muscle phenotype. Among these misregulated genes were NE transmembrane proteins (NETs) involved in nuclear-cytoskeletal coupling as well as genome organization. For selected genes, we could confirm that observed gene-misregulation led to protein expression changes. Furthermore, we investigated if genes known to be under expression-regulation by genome organization NETs were also misregulated in DM1 biopsies, which revealed that misregulation of two NETs alone is likely responsible for differential expression of about 10% of all genes being differentially expressed in DM1. Notably, the majority of NETs identified here to be misregulated in DM1 muscle are mutated in Emery-Dreifuss muscular dystrophy or clinical similar muscular dystrophies, suggesting a broader similarity on the molecular level for muscular dystrophies than anticipated. This shows not only the importance of muscle NETs in muscle health and disease, but also highlights the importance of the NE in DM1 disease progression.

Abstract Background Previous research has described a neuroprotective effect of IGF-I, supporting neuronal survival, axon growth and proliferation of muscle cells. Therefore, the association between IGF-I concentration, muscle histology and electrophysiological markers in a cohort of patients with sarcopenia dares investigation. Methods Measurement of serum concentrations of IGF-I and binding partners, electromyographic measurements with the MUNIX (Motor Unit Number Index) method and muscle biopsies were performed in 31 patients with acute hip fracture older age 60 years. Molecular markers for denervation (neural cell adhesion molecule NCAM) and proliferation markers (Ki67) were assessed by immunofluorescence staining of muscle biopsy tissue. Skeletal muscle mass by bioelectrical impedance analysis and hand-grip strength were measured to assess sarcopenia status according to EWGSOP2 criteria. Results Thirty-one patients (20 women) with a mean age of 80.6 ± 7.4 years were included. Concentrations of IGF-I and its binding partners were significantly associated with sarcopenia (ß = − 0.360; p = 0.047) and MUNIX (ß = 0.512; p = 0.005). Further, expression of NCAM (ß = 0.380; p = 0.039) and Ki67 (ß = 0.424; p = 0.022) showed significant associations to IGF-I concentrations. Conclusions The findings suggest a pathogenetic role of IGF-I in sarcopenia based on muscle denervation.

IntroductionTo date, a few studies have used somatosensory evoked potentials (SEP) to demonstrate cortical sensory changes among healthy subjects or to estimate cortical plasticity and rehabilitation prognosis in stroke patients after peripheral electrical stimulation (PES) intervention. The primary aim was to systematically review whether PES has a role in changing latencies and amplitudes of SEPs in healthy subjects and stroke patients. Moreover, we searched for a correlation between sensory and motor function assessments and changes in SEP components of included studies.MethodsThe following databases were searched: Pubmed/MEDLINE, Scopus/ScienceDirect, Web of Science/Clarivate, Cochrane Library, The Physiotherapy Evidence Database (PEDro), and ClinicalTrials.gov. Titles and abstracts, as well as full-text reports, were screened for eligibility by two independent reviewers according to a priori defined eligibility criteria. There were no study limitations concerning the treatment of the upper limb, lower limb, or torso with PES.ResultsThe final systematic search resulted in 11,344 records, however only 10 were evaluated. We could not find enough evidence to confirm use of SEP as a predictor to estimate the rehabilitation prognosis after stroke. However, we found a correlation between different sensory and motor function assessments and changes in SEP components. The stroke studies involving PES that initiate a voluntary contraction used for a specific movement or task indicate a positive relationship and correlation to assessments of motor function. It could be indicated that PES have a predictive impact of sensory reorganization, as mirrored by the change in SEP amplitude and latency. However, it is not possible to verify the degree of connectivity between SEP and cortical plasticity. To confirm this hypothesis, we propose the conduction of randomized controlled trials in healthy volunteers and stroke patients.Systematic review registrationhttps://doi.org/10.17605/OSF.IO/U7PSY.

Severe cases of age-related loss of muscle function and mass are clinically unique to sarcopenia. Mitochondrial dysfunction has been associated with aging and sarcopenia, but the causal connection in this context is not well eluded. Here we investigated different aspects of mitochondrial respiration in sarcopenia. Open muscle biopsies were taken from a total of 31 hip fracture patients, older than 70 years. Patients were assigned a sarcopenia Z-score based on EWGSOP2 criteria. Primary myoblast cultures were generated from the muscle tissue samples and used for real time metabolic measurement. Muscle and serum samples showed correlation of high Z-scores with reduced mitochondrial complex I activity, increased tricarboxylic acid cycle (TCA) metabolites, reduced vitamin D3 levels, and signs of an altered iron metabolism. Primary myoblast cultures gained from the same muscle biopsies did not show significant mitochondrial defects. We hypothesize that a sum of external consequences, including vitamin D3 deficiency and iron deficiency caused by disturbances in the iron metabolism, result in complex I deficiency, which in turn affects the TCA and contributes to muscle weakness and loss.

Mutations in the DMD gene can cause Duchenne or Becker muscular dystrophy (DMD/BMD) by affecting the giant isoform of dystrophin, a protein encoded by the DMD gene. The role of small dystrophin isoforms is not well investigated yet, and they may play a role in muscle development and molecular pathology. Here, we investigated the nuclear localization of short carboxy-terminal dystrophin isoforms during the in vitro differentiation of human, porcine, and murine myoblast cultures. We could not only confirm the presence of Dp71 in the nucleoplasm and at the nuclear envelope, but we could also identify the Dp40 isoform in muscle nuclei. The localization of both isoforms over the first six days of differentiation was similar between human and porcine myoblasts, but murine myoblasts behaved differently. This highlights the importance of the porcine model in investigating DMD. We could also detect a wave-like pattern of nuclear presence of both Dp71 and Dp40, indicating a direct or indirect involvement in gene expression control during muscle differentiation.

Abstract Background Sarcopenia is the age-related loss of muscle mass and strength. Undiagnosed late-onset neuromuscular disorders need to be considered in the differential diagnosis of sarcopenia. Aim Based on emblematic case reports and current neuromuscular diagnostic guidelines for three common late-onset neuromuscular disorders, a differential diagnostic approach for geriatric patients presenting with a sarcopenic phenotype is given. Methods Patients over 65 years of age with sarcopenia, amyotrophic lateral sclerosis, inclusion body myositis and myotonic dystrophy type 2 were recruited. All patients were assessed for sarcopenia based on the revised European consensus definition. Patients with neuromuscular diseases were diagnosed according to the revised El Escorial criteria and the European neuromuscular centre criteria. Phenotypes and diagnostic criteria for all patients were summarized including their specific histopathological findings. Results All patients with neuromuscular diseases were positively screened for sarcopenia and classified as severe sarcopenic by means of assessment. The clinical phenotype, the evolution pattern of weakness and muscle atrophy combined with laboratory finding including electromyography could unquestionably distinguish the diseases. Discussion Neuromuscular disorders can manifest beyond the age of 65 years and misdiagnosed as sarcopenia. The most common diseases are inclusion body myositis, amyotrophic lateral sclerosis and myotonic dystrophy type 2. A diagnostic work-up for neuromuscular diseases ensures their correct diagnosis by clinical-, electrophysiological, histopathological, and genetic work-up. Conclusions In geriatric patients with a focal or asymmetrical muscular weakness and atrophy, sarcopenia assessment should be extended with patient’s history of disease course. Furthermore, concomitant diseases, analysis of serum creatine kinase, electrophysiological examination, and in selected patients muscle biopsy and gene analysis is needed to rule out a late-onset neuromuscular disorder.

Abstract Pompe disease, an autosomal recessive lysosomal storage disorder, is caused by deficiency of lysosomal acid alpha‐glucosidase (GAA). On cellular level, there is lysosomal‐bound and free accumulation of glycogen and subsequent damage of organelles and organs. The most severe affected tissues are skeletal muscles and heart. The only available treatment to date is an enzyme replacement therapy (ERT) with alglucosidase alfa, a recombinant human GAA (rhGAA) modified with mannose‐6‐phosphate (M6P), which is internalized via M6P‐mediated endocytosis. There is an unmet need to improve this type of therapy, especially in regard to skeletal muscle. Using different tissue culture models, we recently provided evidence that a moss‐derived nonphosphorylated rhGAA (moss‐GAA), carrying a glycosylation with terminal N‐acetylglucosamine residues (GnGn), might have the potential to improve targeting of skeletal muscle. Now, we present a pilot treatment of Gaa−/− mice with moss‐GAA. We investigated general effects as well as the uptake into different organs following short‐term treatment. Our results do confirm that moss‐GAA reaches the target disease organs and thus might have the potential to be an alternative or complementary ERT to the existing one.

Abstract Infection with the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) leads to a previously unknown clinical picture, which is known as COVID-19 (COrona VIrus Disease-2019) and was first described in the Hubei region of China. The SARS-CoV-2 pandemic has implications for all areas of medicine. It directly and indirectly affects the care of neurological diseases. SARS-CoV-2 infection may be associated with an increased incidence of neurological manifestations such as encephalopathy and encephalomyelitis, ischemic stroke and intracerebral hemorrhage, anosmia and neuromuscular diseases. In October 2020, the German Society of Neurology (DGN, Deutsche Gesellschaft für Neurologie) published the first guideline on the neurological manifestations of the new infection. This S1 guideline provides guidance for the care of patients with SARS-CoV-2 infection regarding neurological manifestations, patients with neurological disease with and without SARS-CoV-2 infection, and for the protection of healthcare workers. This is an abbreviated version of the guideline issued by the German Neurological society and published in the Guideline repository of the AWMF (Working Group of Scientific Medical Societies; Arbeitsgemeinschaft wissenschaftlicher Medizinischer Fachgesellschaften).

Duchenne muscular dystrophy (DMD) is the most frequent genetic myopathy in childhood and leads to progressive muscle atrophy, weakness, and premature death. So far, there is no curative treatment available. Therapeutic development from bench to bedside takes time, and promising therapies need to be tested in suitable preclinical animal models prior to clinical trials in DMD patients. Existing mouse and dog models are limited with regard to the comparability of the clinical phenotype and the underlying mutation. Therefore, our group established a tailored large animal model of DMD, the DMD pig, mirroring the human size, anatomy, and physiology. For testing novel approaches, we developed a corresponding in vitro model, facilitating preclinical testing for toxicity, dosing, and efficacy, which we describe here. We first extracted primary muscle cells from wild-type and DMD pigs of different age groups and characterized those cells, then improved their differentiation process for identification of dystrophin and utrophin in myotubes. Our porcine in vitro model represents an important step for the development of novel therapeutic approaches, which should be validated further to minimize the need for living animals for bioassays, and thereby support the ‘3R’ (replace, reduce, refine) principle, as fewer animals have to be raised and treated for preclinical trials.

Dominant VCP–mutations cause a variety of neurological manifestations including inclusion body myopathy with early–onset Paget disease and frontotemporal dementia 1 (IBMPFD). VCP encodes a ubiquitously expressed multifunctional protein that is a member of the AAA+ protein family, implicated in multiple cellular functions ranging from organelle biogenesis to ubiquitin–dependent protein degradation. The latter function accords with the presence of protein aggregates in muscle biopsy specimens derived from VCP–patients. Studying the proteomic signature of VCP–mutant fibroblasts, we identified a (pathophysiological) increase of FYCO1, a protein involved in autophagosome transport. We confirmed this finding applying immunostaining also in muscle biopsies derived from VCP–patients. Treatment of fibroblasts with arimoclomol, an orphan drug thought to restore physiologic cellular protein repair pathways, ameliorated cellular cytotoxicity in VCP–patient derived cells. This finding was accompanied by increased abundance of proteins involved in immune response with a direct impact on protein clearaqnce as well as by elevation of pro–survival proteins as unravelled by untargeted proteomic profiling. Hence, the combined results of our study reveal a dysregulation of FYCO1 in the context of VCP–etiopathology, highlight arimoclomol as a potential drug and introduce proteins targeted by the pre–clinical testing of this drug in fibroblasts.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disorder caused by unstable CTG-repeat expansions in the DMPK gene. Tissue mosaicism has been described for the length of these repeat expansions. The most obvious affected tissue is skeletal muscle, making it the first target for therapy development. To date there is no approved therapy despite some existing approaches. Thus, there is the demand to further advance therapeutic developments, which will in return require several well-characterized preclinical tools and model systems. Here we describe a modified method to identify the CTG-repeat length in primary human myoblasts isolated from DM1 patients that requires less genomic DNA and avoids radioactive labeling. Using this method, we show that primary human DM1 myoblast cultures represent a population of cells with different CTG-repeat length. Comparing DNA from the identical muscle biopsy specimen, the range of CTG-repeat length in the myoblast culture is within the same range of the muscle biopsy specimen. In conclusion, primary human DM1 myoblast cultures are a well-suited model to investigate certain aspects of the DM1 pathology. They are a useful platform to perform first-line investigations of preclinical therapies.

Abstract Glycogen storage disorder type III (GSDIII), or debranching enzyme (GDE) deficiency, is a rare metabolic disorder characterized by variable liver, cardiac, and skeletal muscle involvement. GSDIII manifests with liver symptoms in infancy and muscle involvement during early adulthood. Muscle biopsy is mainly performed in patients diagnosed in adulthood, as routine diagnosis relies on blood or liver GDE analysis, followed by AGL gene sequencing. The GSDIII mouse model recapitulate the clinical phenotype in humans, and a nearly full rescue of muscle function was observed in mice treated with the dual AAV vector expressing the GDE transgene. In order to characterize GSDIII muscle morphological spectrum and identify novel disease markers and pathways, we performed a large international multicentric morphological study on 30 muscle biopsies from GSDIII patients. Autophagy flux studies were performed in human muscle biopsies and muscles from GSDIII mice. The human muscle biopsies revealed a typical and constant vacuolar myopathy, characterized by multiple and variably sized vacuoles filled with PAS-positive material. Using electron microscopy, we confirmed the presence of large non-membrane bound sarcoplasmic deposits of normally structured glycogen as well as smaller rounded sac structures lined by a continuous double membrane containing only glycogen, corresponding to autophagosomes. A consistent SQSTM1/p62 decrease and beclin-1 increase in human muscle biopsies suggested an enhanced autophagy. Consistent with this, an increase in the lipidated form of LC3, LC3II was found in patients compared to controls. A decrease in SQSTM1/p62 was also found in the GSDIII mouse model. In conclusion, we characterized the morphological phenotype in GSDIII muscle and demonstrated dysfunctional autophagy in GSDIII human samples. These findings suggest that autophagic modulation combined with gene therapy might be considered as a novel treatment for GSDIII.

Abstract Background The protein O-mannosyltransferase 1, encoded by the POMT1 gene, is a key enzyme in the glycosylation of α-dystroglycan. POMT1–related disorders belong to the group of dystroglycanopathies characterized by a proximally pronounced muscular dystrophy with structural or functional involvement of the brain and/or the eyes. The phenotypic spectrum ranges from the severe Walker-Warburg syndrome (WWS) to milder forms of limb girdle muscular dystrophy (LGMD). The phenotypic severity of POMT1-related dystroglycanopathies depends on the residual enzyme activity. A genotype-phenotype correlation can be assumed. Results The clinical, neuroradiological, and genetic findings of 35 patients with biallelic POMT1 mutations (15 WWS, 1 MEB (muscle-eye-brain disease), 19 LGMD) from 27 independent families are reported. The representative clinical course of an infant with WWS and the long-term course of a 32 years old patient with LGMD are described in more detail. Specific features of 15 patients with the homozygous founder mutation p.Ala200Pro are defined as a distinct and mildly affected LGMD subgroup. Ten previously reported and 8 novel POMT1 mutations were identified. Type and location of each of the POMT1 mutations are evaluated in detail and a list of all POMT1 mutations reported by now is provided. Patients with two mutations leading to premature protein termination had a WWS phenotype, while the presence of at least one missense mutation was associated with milder phenotypes. In the patient with MEB-like phenotype two missense mutations were observed within the catalytic active domain of the enzyme. Conclusions Our large cohort confirms the importance of type and location of each POMT1 mutation for the individual clinical manifestation and thereby expands the knowledge on the genotype-phenotype correlation in POMT1-related dystroglycanopathies. This genotype-phenotype correlation is further supported by the observation of an intrafamiliar analogous clinical manifestation observed in all affected 13 siblings from 5 independent families. Our data confirm the progressive nature of the disease also in milder LGMD phenotypes, ultimately resulting in loss of ambulation at a variable age. Our data define two major clinical POMT1 phenotypes, which should prompt genetic testing including the POMT1 gene: patients with a severe WWS manifestation predominantly present with profound neonatal muscular hypotonia and a severe and progressive hydrocephalus with involvement of brainstem and/or cerebellum. The presence of an occipital encephalocele in a WWS patient might point to POMT1 as causative gene within the different genes associated with WWS. The milder LGMD phenotypes constantly show markedly elevated creatine kinase values in combination with microcephaly and cognitive impairment.

Background: Neonatal screening for Pompe disease is complicated by difficulties in predicting symptom onset in patients with the common c.-32-13T>G (IVS1) variant/null (i.e. fully deleterious) acid α-glucosidase (GAA) genotype. This splicing variant occurs in 90% of Caucasian late onset patients, and is associated with a broad range of symptom onset. Methods: We analyzed a cohort of 143 compound heterozygous and 10 homozygous IVS1 patients, and we assessed ages at symptom onset, the presence of cis-acting single nucleotide variants (SNVs), and performed splicing analysis and enzyme activity assays. Findings: In compound heterozygous IVS1 patients, the synonymous variant c.510C>T was uniquely present on the IVS1 allele in 9/33 (27%) patients with childhood onset, but was absent from 110 patients with onset in adulthood. GAA enzyme activity was lower in fibroblasts from patients who contained c.510C>T than it was in patients without c.510C>T. By reducing the extent of leaky wild-type splicing, c.510C>T modulated aberrant splicing caused by the IVS1 variant. The deleterious effect of c.510C>T was also found in muscle cells, the main target cells in Pompe disease. In homozygous IVS1 patients, the c.510C>T variant was absent in 4/4 (100%) asymptomatic individuals and present in 3/6 (50%) symptomatic patients. In cells from homozygous IVS1 patients, c.510C>T caused reduced leaky wild-type splicing. Interpretation: c.510C>T is a genetic modifier in compound heterozygous and homozygous IVS1 patients. This finding is important for neonatal screening programs for Pompe disease. Fund: This work was funded by grants from Sophia Children's Hospital Foundation (SSWO, grant S17–32) and Metakids (2016–063). Keywords: Modifying factor, Pompe disease, Lysosomal storage disease, Pre-mRNA splicing, c.510C>T

Introduction: Myotonic dystrophy type 2 (DM2) lacks disease-specific, validated, motor outcome measures (OMs), and patients' reported outcomes (PROs). This represents a limit for the monitoring of disease progression and treatment response. Our aim was to identify the most appropriate OMs to be translated in clinical practice and clinical trials on DM2. This study has been registered on clinicaltrials.gov NCT03603171 (https://clinicaltrials.gov/ct2/show/NCT03603171).Methods: Sixty-six patients with genetically confirmed DM2 underwent a baseline and a follow-up visit after 1 year. The tested OMs included: hand opening time, pressure pain threshold (PPT), manual muscle testing (MMT), hand held dynamometry (HHD), scale for the assessment and rating of ataxia (SARA), quantitative motor function test (QMFT), gait stairs Gowers chair (GSGC), 30-s sit to stand test, functional index 2 (FI-2) and 6MWT. The PROs included DM1-Active-C, Rasch-built Pompe-specific activity scale (R-Pact), fatigue and daytime sleepiness (FDSS), brief pain inventory short form (BPI-sf), myotonia behavior scale (MBS), and the McGill pain questionnaire.Results: All patients completed the MBS and the results correlated well with the hand-opening time. The PPT showed a low reliability, no correlation with pain questionnaires, and did not differentiate patients with or without myalgia. Both muscle strength assessments, MMT and HHD, showed good construct validity. The QMFT showed an acceptable ceiling effect (14.5%), good convergent and differential validity and performed overall better than GSGC. The SARA score showed high flooring effect and is not useful in DM2. 6MWT proved a valid outcome measure in DM2. The 30-s sit to stand is a feasible test with good convergent validity, showing a flooring effect of 20% as it cannot be used in more severely affected patients. The FI-2 is time-consuming and has a high ceiling effect. At the 1-year visit the only assessments able to detect a worsening of DM2 were HHD, QMFT, and 6MWT, which are the most sensitive to change, and therefore clinically meaningful OMs in DM2.Conclusion: The clinical meaningful motor outcome measures that best depict the multifaceted phenotype of DM2 and its slow progression are MBS, MMT, or HHD (depending on the clinical setting), QMFT, and the 6MWT.

Myotonic dystrophy type 1 (DM1) is a rare genetic disorder, characterised by muscular dystrophy, myotonia, and other symptoms. DM1 is caused by the expansion of a CTG repeat in the 3'-untranslated region of DMPK. Longer CTG expansions are associated with greater symptom severity and earlier age at onset. The primary mechanism of pathogenesis is thought to be mediated by a gain of function of the CUG-containing RNA, that leads to trans-dysregulation of RNA metabolism of many other genes. Specifically, the alternative splicing (AS) and alternative polyadenylation (APA) of many genes is known to be disrupted. In the context of clinical trials of emerging DM1 treatments, it is important to be able to objectively quantify treatment efficacy at the level of molecular biomarkers. We show how previously described candidate mRNA biomarkers can be used to model an effective reduction in CTG length, using modern high-dimensional statistics (machine learning), and a blood and muscle mRNA microarray dataset. We show how this model could be used to detect treatment effects in the context of a clinical trial.

Background: As genome-wide approaches prove difficult with genetically heterogeneous orphan diseases, we developed a new approach to identify candidate genes. We applied this to Emery-Dreifuss muscular dystrophy (EDMD), characterised by early onset contractures, slowly progressive muscular wasting, and life-threatening heart conduction disturbances with wide intra- and inter-familial clinical variability. Roughly half of EDMD patients are linked to six genes encoding nuclear envelope proteins, but the disease mechanism remains unclear because the affected proteins function in both cell mechanics and genome regulation. Methods: A primer library was generated to test for mutations in 301 genes from four categories: (I) all known EDMD-linked genes; (II) genes mutated in related muscular dystrophies; (III) candidates generated by exome sequencing in five families; (IV) functional candidates — other muscle nuclear envelope proteins functioning in mechanical/genome processes affected in EDMD. This was used to sequence 56 unlinked patients with EDMD-like phenotype. Findings: Twenty-one patients could be clearly assigned: 18 with mutations in genes of similar muscular dystrophies; 3 with previously missed mutations in EDMD-linked genes. The other categories yielded novel candidate genes, most encoding nuclear envelope proteins with functions in gene regulation. Interpretation: Our multi-pronged approach identified new disease alleles and many new candidate EDMD genes. Their known functions strongly argue the EDMD pathomechanism is from altered gene regulation and mechanotransduction due to connectivity of candidates from the nuclear envelope to the plasma membrane. This approach highlights the value of testing for related diseases using primer libraries and may be applied for other genetically heterogeneous orphan diseases. Funding: The Wellcome Trust, Muscular Dystrophy UK, Medical Research Council, European Community's Seventh Framework Programme “Integrated European –omics research project for diagnosis and therapy in rare neuromuscular and neurodegenerative diseases (NEUROMICS)”. Keywords: Emery-Dreifuss muscular dystrophy, Nuclear envelope, Nuclear envelope transmembrane protein, primer library, Orphan disease

Abstract Background Myotonic Dystrophy is the most common form of muscular dystrophy in adults, affecting an estimated 10 per 100,000 people. It is a multisystemic disorder affecting multiple generations with increasing severity. There are currently no licenced therapies to reverse, slow down or cure its symptoms. In 2009 TREAT-NMD (a global alliance with the mission of improving trial readiness for neuromuscular diseases) and the Marigold Foundation held a workshop of key opinion leaders to agree a minimal dataset for patient registries in myotonic dystrophy. Eight years after this workshop, we surveyed 22 registries collecting information on myotonic dystrophy patients to assess the proliferation and utility the dataset agreed in 2009. These registries represent over 10,000 myotonic dystrophy patients worldwide (Europe, North America, Asia and Oceania). Results The registries use a variety of data collection methods (e.g. online patient surveys or clinician led) and have a variety of budgets (from being run by volunteers to annual budgets over €200,000). All registries collect at least some of the originally agreed data items, and a number of additional items have been suggested in particular items on cognitive impact. Conclusions The community should consider how to maximise this collective resource in future therapeutic programmes.

Myotonic dystrophy (DM) type 2 is a neuromuscular pathology caused by large expansions of CCTG repeats. Here the authors find that rbFOX1 RNA binding protein binds to CCUG RNA repeats and competes with MBNL1 for the binding to CCUG repeats, releasing MBNL1 from sequestration in DM2 muscle cells.

Disorders of the peripheral nerves can be caused by a broad spectrum of acquired or hereditary aetiologies. The objective of these practice guidelines is to provide the reader with information about the differential diagnostic workup for a target-oriented diagnosis. Following an initiative of the German-speaking Society of Neuropaediatrics, delegates from 10 German societies dedicated to neuroscience worked in close co-operation to write this guideline. Applying the Delphi methodology, the authors carried out a formal consensus process to develop practice recommendations. These covered the important diagnostic steps both for acquired neuropathies (traumatic, infectious, inflammatory) and the spectrum of hereditary Charcot–Marie–Tooth (CMT) diseases. Some of our most important recommendations are that: (i) The indication for further diagnostics must be based on the patient’s history and clinical findings; (ii) Potential toxic neuropathy also has to be considered; (iii) For focal and regional neuropathies of unknown aetiology, nerve sonography and MRI should be performed; and (iv) For demyelinated hereditary neuropathy, genetic diagnostics should first address PMP22 gene deletion: once that has been excluded, massive parallel sequencing including an analysis of relevant CMT-genes should be performed. This article contains a short version of the guidelines. The full-length text (in German) can be found at the Website of the “Arbeitsgemeinschaft der Wissenschftlichen Medizinischen Fachgesellschaften e.V. (AWMF), Germany.

Myotonic dystrophy type 1 (DM1) is caused by CTG-repeat expansions leading to a complex pathology with a multisystemic phenotype that primarily affects the muscles and brain. Despite a multitude of information, especially on the alternative splicing of several genes involved in the pathology, information about additional factors contributing to the disease development is still lacking. We performed RNAseq and gene expression analyses on proliferating primary human myoblasts and differentiated myotubes. GO-term analysis indicates that in myoblasts and myotubes, different molecular pathologies are involved in the development of the muscular phenotype. Gene set enrichment for splicing reveals the likelihood of whole, differentiation stage specific, splicing complexes that are misregulated in DM1. These data add complexity to the alternative splicing phenotype and we predict that it will be of high importance for therapeutic interventions to target not only mature muscle, but also satellite cells.

(1) Background and Purpose: The skeletal muscles of patients suffering from neuromuscular diseases (NMD) are affected by atrophy, hypertrophy, fatty infiltration, and edematous changes. Magnetic resonance imaging (MRI) is an important tool for diagnosis and monitoring. Concerning fatty infiltration, T1-weighted or T2-weighted DIXON turbo spin echo (TSE) sequences enable a qualitative assessment of muscle involvement. To achieve higher comparability, semi-quantitative grading scales, such as the four-point Mercuri scale, are commonly applied. However, the evaluation remains investigator-dependent. Therefore, effort is being invested to develop quantitative MRI techniques for determination of imaging markers such as the proton density fat fraction (PDFF). The present work aims to assess the diagnostic value of PDFF in correlation to Mercuri grading and clinically determined muscle strength in patients with myotonic dystrophy type 2 (DM2), limb girdle muscular dystrophy type 2A (LGMD2A), and adult Pompe disease. (2) Methods: T2-weighted two-dimensional (2D) DIXON TSE and chemical shift encoding-based water-fat MRI were acquired in 13 patients (DM2: n = 5; LGMD2A: n = 5; Pompe disease: n = 3). Nine different thigh muscles were rated in all patients according to the Mercuri grading and segmented to extract PDFF values. Muscle strength was assessed according to the British Medical Research Council (BMRC) scale. For correlation analyses between Mercuri grading, muscle strength, and PDFF, the Spearman correlation coefficient (rs) was computed. (3) Results: Mean PDFF values ranged from 7% to 37% in adults with Pompe disease and DM2 and up to 79% in LGMD2A patients. In all three groups, a strong correlation of the Mercuri grading and PDFF values was observed for almost all muscles (rs > 0.70, p < 0.05). PDFF values correlated significantly to muscle strength for muscle groups responsible for knee flexion (rs = −0.80, p < 0.01). (4) Conclusion: In the small, investigated patient cohort, PDFF offers similar diagnostic precision as the clinically established Mercuri grading. Based on these preliminary data, PDFF could be further considered as an MRI-based biomarker in the assessment of fatty infiltration of muscle tissue in NMD. Further studies with larger patient cohorts are needed to advance PDFF as an MRI-based biomarker in NMD, with advantages such as its greater dynamic range, enabling the assessment of subtler changes, the amplified objectivity, and the potential of direct correlation to muscle function for selected muscles.

Abstract Background Humanistic burden considers the impact of an illness on a patient’s health-related quality of life (HRQoL), activities of daily living (ADL), caregiver health, and caregiver QoL. Humanistic burden also considers treatment satisfaction and adherence to treatment regimens. Pompe disease is an autosomal recessive, progressive, multisystemic neuromuscular disease. Approval of enzyme-replacement therapy (ERT) markedly improved prognosis for patients, but considerable morbidity and a substantial humanistic burden remain. This article characterizes the humanistic burden of Pompe disease through a systematic literature review. Methods A systematic search of MEDLINE® and Embase® with back-referencing and supplementary literature searches was performed to retrieve data from interventional and non-interventional studies on the humanistic burden of Pompe disease. Publications were screened according to predefined criteria, extracted, and assessed for quality. Extracted data were narratively synthesized. Results No publications on the humanistic burden of infantile-onset Pompe disease (IOPD) were identified. As such, of 17 publications included here, all are in patients with late-onset Pompe disease (LOPD). Thirteen publications were initiated after approval of ERT, two were initiated before, and two overlapped the approval of ERT. The review shows that LOPD patients have a significantly lower HRQoL than the general population, even if treated with ERT. On transitioning to ERT, treatment was associated with improvement in the physical component score of the SF-36 and fatigue, although the SF-36 mental component score remained stable. Physical HRQoL remained below population norms after 4 years of ERT. Significantly more ERT-treated patients reported pain than controls, and bodily pain worsened in later years following ERT initiation. Treatment-naïve LOPD patients had significantly poorer ADL functioning compared with the general population, although ERT stabilized deteriorating functioning impairment. ERT studies showed caregivers provide 17.7 h/week informal care on average. Fifty percent, 40% and

IntroductionThe system of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (cas) is a new technology that allows easier manipulation of the genome. Its potential to edit genes opened a new door in treatment development for incurable neurological monogenic diseases (NMGDs). The aim of this systematic review was to summarise the findings on the current development of CRISPR-cas for therapeutic purposes in the most frequent NMGDs and provide critical assessment.Methods and data acquisitionWe searched the MEDLINE and EMBASE databases, looking for original studies on the use of CRISPR-cas to edit pathogenic variants in models of the most frequent NMGDs, until end of 2017. We included all the studies that met the following criteria: 1. Peer-reviewed study report with explicitly described experimental designs; 2. In vitro, ex vivo, or in vivo study using human or other animal biological systems (including cells, tissues, organs, organisms); 3. focusing on CRISPR as the gene-editing method of choice; and 5. featured at least one NMGD.ResultsWe obtained 404 papers from MEDLINE and 513 from EMBASE. After removing the duplicates, we screened 490 papers by title and abstract and assessed them for eligibility. After reading 50 full-text papers, we finally selected 42 for the review.DiscussionHere we give a systematic summary on the preclinical development of CRISPR-cas for therapeutic purposes in NMGDs. Furthermore, we address the clinical interpretability of the findings, giving a comprehensive overview of the current state of the art. Duchenne's muscular dystrophy (DMD) paves the way forward, with 26 out of 42 studies reporting different strategies on DMD gene editing in different models of the disease. Most of the strategies aimed for permanent exon skipping by deletion with CRISPR-cas. Successful silencing of the mHTT gene with CRISPR-cas led to successful reversal of the neurotoxic effects in the striatum of mouse models of Huntington's disease. Many other strategies have been explored, including epigenetic regulation of gene expression, in cellular and animal models of: myotonic dystrophy, Fraxile X syndrome, ataxias, and other less frequent dystrophies. Still, before even considering the clinical application of CRISPR-cas, three major bottlenecks need to be addressed: efficacy, safety, and delivery of the systems. This requires a collaborative approach in the research community, while having ethical considerations in mind.

Pompe disease is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of lysosomal acid alpha-glucosidase (GAA). The result of the GAA deficiency is a ubiquitous lysosomal and non-lysosomal accumulation of glycogen. The most affected tissues are heart, skeletal muscle, liver, and the nervous system. Replacement therapy with the currently approved enzyme relies on M6P-mediated endocytosis. However, therapeutic outcomes still leave room for improvement, especially with regard to skeletal muscles. We tested the uptake, activity, and effect on glucose metabolism of a non-phosphorylated recombinant human GAA produced in moss (moss-GAA). Three variants of moss-GAA differing in glycosylation pattern have been analyzed: two with terminal mannose residues in a paucimannosidic (Man3) or high-mannose (Man 5) configuration and one with terminal N-acetylglucosamine residues (GnGn). Compared to alglucosidase alfa the moss-GAA GnGn variant showed increased uptake in differentiated myotubes. Moreover, incubation of immortalized muscle cells of Gaa−/− mice with moss-GAA GnGn led to similarly efficient clearance of accumulated glycogen as with alglucosidase alfa. These initial data suggest that M6P-residues might not always be necessary for the cellular uptake in enzyme replacement therapy (ERT) and indicate the potential of moss-GAA GnGn as novel alternative drug for targeting skeletal muscle in Pompe patients.

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder with predominant myotonia and muscular dystrophy which is caused by CTG-repeat expansions in the DMPK gene. These repeat expansions are transcribed and the resulting mRNA accumulates RNA-binding proteins involved in splicing, resulting in a general splicing defect. We observed nuclear envelope (NE) alterations in DM1 primary myoblasts. These included invaginations of the NE as well as an altered composition of the nuclear lamina. Specifically, we investigated NE transmembrane proteins (NETs) in DM1 primary myoblasts, staining to determine if their distribution was altered compared to controls and if this could contribute to these structural defects. We also tested the expression of these NETs in muscle and how localization changes in the DM1 primary myoblasts undergoing differentiation in vitro to myotubes. We found no changes in the localization of the tested NETs, but most tended to exhibit reduced expression with increasing DMPK-repeat length. Nonetheless, the DM1 patient expression range was within the expression range of the controls. Additionally, we found a down-regulation of the possible nesprin 1 giant isoform in DM1 primary myoblasts which could contribute to the increased NE invaginations. Thus, nesprin 1 may be an interesting target for further investigation in DM1 disease pathology.

Myotonic dystrophies (DM) are slowly progressing multisystemic disorders caused by repeat expansions in the DMPK or CNBP genes. The multisystemic involvement in DM patients often reflects the appearance of accelerated aging. This is partly due to visible features such as cataracts, muscle weakness, and frontal baldness, but there are also less obvious features like cardiac arrhythmia, diabetes or hypogammaglobulinemia. These aging features suggest the hypothesis that DM could be a segmental progeroid disease. To identify the molecular cause of this characteristic appearance of accelerated aging we compare clinical features of DM to “typical” segmental progeroid disorders caused by mutations in DNA repair or nuclear envelope proteins. Furthermore, we characterize if this premature aging effect is also reflected on the cellular level in DM and investigate overlaps with “classical” progeroid disorders. To investigate the molecular similarities at the cellular level we use primary DM and control cell lines. This analysis reveals many similarities to progeroid syndromes linked to the nuclear envelope. Our comparison on both clinical and molecular levels argues for qualification of DM as a segmental progeroid disorder.

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) represent the most frequent multisystemic muscular dystrophies in adulthood. They are progressive, autosomal dominant diseases caused by an abnormal expansion of an unstable nucleotide repeat located in the non-coding region of their respective genes DMPK for DM1 and CNBP in DM2. Clinically, these multisystemic disorders are characterized by a high variability of muscular and extramuscular symptoms, often causing a delay in diagnosis. For both subtypes, many symptoms overlap, but some differences allow their clinical distinction. This article highlights the clinical core features of myotonic dystrophies, thus facilitating their early recognition and diagnosis. Particular attention will be given to signs and symptoms of muscular involvement, to issues related to respiratory impairment, and to the multiorgan involvement. This article is part of a Special Issue entitled “Beyond Borders: Myotonic Dystrophies—A European Perception.”

Magnetic resonance imaging (MRI) can non-invasively assess muscle anatomy, exercise effects and pathologies with different underlying causes such as neuromuscular diseases (NMD). Quantitative MRI including fat fraction mapping using chemical shift encoding-based water-fat MRI has emerged for reliable determination of muscle volume and fat composition. The data analysis of water-fat images requires segmentation of the different muscles which has been mainly performed manually in the past and is a very time consuming process, currently limiting the clinical applicability. An automatization of the segmentation process would lead to a more time-efficient analysis. In the present work, the manually segmented thigh magnetic resonance imaging database MyoSegmenTUM is presented. It hosts water-fat MR images of both thighs of 15 healthy subjects and 4 patients with NMD with a voxel size of 3.2x2x4 mm3 with the corresponding segmentation masks for four functional muscle groups: quadriceps femoris, sartorius, gracilis, hamstrings. The database is freely accessible online at https://osf.io/svwa7/?view_only=c2c980c17b3a40fca35d088a3cdd83e2. The database is mainly meant as ground truth which can be used as training and test dataset for automatic muscle segmentation algorithms. The segmentation allows extraction of muscle cross sectional area (CSA) and volume. Proton density fat fraction (PDFF) of the defined muscle groups from the corresponding images and quadriceps muscle strength measurements/neurological muscle strength rating can be used for benchmarking purposes.

The original version of this article [1] unfortunately included an error to an author’s name. Author Jordi Díaz-Manera was erroneously presented as Jorge Alberto Diaz Manera. The correct author name has been included in the author list of this Correction article.

Myogenesis is defined as growth, differentiation and repair of muscles where cell fusion of myoblasts to multinucleated myofibers is one major characteristic. Other cell fusion events in humans are found with bone resorbing osteoclasts and placental syncytiotrophoblasts. No unifying gene regulation for natural cell fusions has been found. We analyzed skeletal muscle biopsies of competitive cyclists for muscle-specific attributes and expression of human endogenous retrovirus (ERV) envelope genes due to their involvement in cell fusion of osteoclasts and syncytiotrophoblasts. Comparing muscle biopsies from post- with the pre-competitive seasons a significant 2.25-fold increase of myonuclei/mm fiber, a 2.38-fold decrease of fiber area/nucleus and a 3.1-fold decrease of satellite cells (SCs) occurred. We propose that during the pre-competitive season SC proliferation occurred following with increased cell fusion during the competitive season. Expression of twenty-two envelope genes of muscle biopsies demonstrated a significant increase of putative muscle-cell fusogenic genes Syncytin-1 and Syncytin-3, but also for the non-fusogenic erv3. Immunohistochemistry analyses showed that Syncytin-1 mainly localized to the sarcolemma of myofibers positive for myosin heavy-chain isotypes. Cellular receptors SLC1A4 and SLC1A5 of Syncytin-1 showed significant decrease of expression in post-competitive muscles compared with the pre-competitive season, but only SLC1A4 protein expression localized throughout the myofiber. Erv3 protein was strongly expressed throughout the myofiber, whereas envK1-7 localized to SC nuclei and myonuclei. Syncytin-1 transcription factors, PPARγ and RXRα, showed no protein expression in the myofiber, whereas the pCREB-Ser133 activator of Syncytin-1 was enriched to SC nuclei and myonuclei. Syncytin-1, Syncytin-3, SLC1A4 and PAX7 gene regulations along with MyoD1 and myogenin were verified during proliferating or actively-fusing human primary myoblast cell cultures, resembling muscle biopsies of cyclists. Myoblast treatment with anti-Synycytin-1 abrogated cell fusion in vitro. Our findings support functional roles for ERV envelope proteins, especially Syncytin-1, contributing to cell fusion of myotubes.