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Abstract Background The cause of the motor neuron (MN) death that drives terminal pathology in amyotrophic lateral sclerosis (ALS) remains unknown, and it is thought that the cellular environment of the MN may play a key role in MN survival. Several lines of evidence implicate vesicles in ALS, including that extracellular vesicles may carry toxic elements from astrocytes towards MNs, and that pathological proteins have been identified in circulating extracellular vesicles of sporadic ALS patients. Because MN degeneration at the neuromuscular junction is a feature of ALS, and muscle is a vesicle‐secretory tissue, we hypothesized that muscle vesicles may be involved in ALS pathology. Methods Sporadic ALS patients were confirmed to be ALS according to El Escorial criteria and were genotyped to test for classic gene mutations associated with ALS, and physical function was assessed using the ALSFRS‐R score. Muscle biopsies of either mildly affected deltoids of ALS patients (n = 27) or deltoids of aged‐matched healthy subjects (n = 30) were used for extraction of muscle stem cells, to perform immunohistology, or for electron microscopy. Muscle stem cells were characterized by immunostaining, RT‐qPCR, and transcriptomic analysis. Secreted muscle vesicles were characterized by proteomic analysis, Western blot, NanoSight, and electron microscopy. The effects of muscle vesicles isolated from the culture medium of ALS and healthy myotubes were tested on healthy human‐derived iPSC MNs and on healthy human myotubes, with untreated cells used as controls. Results An accumulation of multivesicular bodies was observed in muscle biopsies of sporadic ALS patients by immunostaining and electron microscopy. Study of muscle biopsies and biopsy‐derived denervation‐naïve differentiated muscle stem cells (myotubes) revealed a consistent disease signature in ALS myotubes, including intracellular accumulation of exosome‐like vesicles and disruption of RNA‐processing. Compared with vesicles from healthy control myotubes, when administered to healthy MNs the vesicles of ALS myotubes induced shortened, less branched neurites, cell death, and disrupted localization of RNA and RNA‐processing proteins. The RNA‐processing protein FUS and a majority of its binding partners were present in ALS muscle vesicles, and toxicity was dependent on the expression level of FUS in recipient cells. Toxicity to recipient MNs was abolished by anti‐CD63 immuno‐blocking of vesicle uptake. Conclusions ALS muscle vesicles are shown to be toxic to MNs, which establishes the skeletal muscle as a potential source of vesicle‐mediated toxicity in ALS.

Abstract Background Due to their health condition, patients with neuromuscular diseases (NMD) are at greater risk of developing serious complications with COVID-19. The objective of this study was to analyze the prevalence of COVID-19 among NMD patients and the risk factors for its impact and severity during the first wave of the pandemic. Clinical data were collected from NMD-COVID-19 patients, between March 25, 2020 and May 11, 2020 in an anonymous survey carried out by expert physicians from the French Health Care Network Filnemus. Results Physicians reported 84 patients, including: 34 with myasthenia gravis, 27 with myopathy and 23 with neuropathy. COVID-19 had no effect on NMD for 48 (58%) patients and 48 (58%) patients developed low COVID-19 severity. COVID-19 caused the death of 9 (11%) NMD patients. Diabetic patients were at greater risk of dying. Patients with diabetes, hypertension or severe forms of NMD had a higher risk of developing a moderate or severe form of COVID-19. In our cohort, corticosteroids and other immunosuppressants were not significantly associated with higher COVID-19 severity for acquired NMD. Conclusion During this period, a small percentage of French NMD patients was affected by COVID-19 compared to the general French population and COVID-19 had a limited short-term effect on them. Diabetes, hypertension and a severe degree of NMD were identified as risk factors of unfavorable outcome following COVID-19. Conversely, in our cohort of patients with acquired NMD, corticosteroids or other immunosuppressants did not appear to be risk factors for more severe COVID-19.

Autoimmune Myasthenia gravis (MG) is a chronic neuromuscular disease mainly due to antibodies against the acetylcholine receptor (AChR) at the neuromuscular junction that induce invalidating muscle weaknesses. In early-onset MG, the thymus is the effector organ and is often characterized by B-cell infiltrations leading to ectopic germinal center (GC) development. The microRNA miR-150-5p has been previously characterized as a biomarker in MG due to its increase in the serum of patients and its decrease after thymectomy, correlated with an improvement of symptoms. Here, we investigated the causes and consequences of the miR-150 increase in the serum of early-onset MG patients. We observed that miR-150 expression was upregulated in MG thymuses in correlation with the presence of thymic B cells and showed by in situ hybridization experiments, that miR-150 was mainly expressed by cells of the mantle zone of GCs. However, we did not observe any correlation between the degree of thymic hyperplasia and the serum levels in MG patients. In parallel, we also investigated the expression of miR-150 in peripheral blood mononuclear cells (PBMCs) from MG patients. We observed that miR-150 was down-regulated, especially in CD4+ T cells compared to controls. These results suggest that the increased serum levels of miR-150 could result from a release from activated peripheral CD4+ T cells. Next, we demonstrated that the in vitro treatment of PBMCs with miR-150 or antimiR-150 oligonucleotides, respectively, decreased or increased the expression of one of its major target gene: the proto-oncogene MYB, a well-known actor of hematopoiesis. These results revealed that increased serum levels of miR-150 in MG patients could have a functional effect on PBMCs. We also showed that antimiR-150 caused increased cellular death of CD4+ and CD8+ T cells, along with the overexpression of pro-apoptotic genes targeted by miR-150 suggesting that miR-150 controlled the survival of these cells. Altogether, these results showed that miR-150 could play a role in MG both at the thymic level and in periphery by modulating the expression of target genes and peripheral cell survival.

BackgroundIn patients with neuromuscular disorders, assessment of respiratory function relies on forced vital capacity (FVC) measurements. Providing complementary respiratory outcomes may be useful for clinical trials. Diaphragm sniff ultrasound (US) is a noninvasive technique that can assess diaphragm function that may be affected in patients with neuromuscular disorders.PurposeWe aimed to provide normal values of sniff diaphragm ultrasound, to assess the relationship between sniff diaphragm US, vital capacity (VC) and sniff nasal pressure. Additionally, we aimed to evaluate the diagnostic accuracy of sniff diaphragm US for predicting restrictive pulmonary insufficiency.Materials and methodsWe included patients with neuromuscular disorders that had been tested with a sniff diaphragm US and functional respiratory tests. Healthy subjects were also included to obtain normal diaphragm sniff ultrasound. We performed diaphragm tissue Doppler imaging (TDI) and time movement (TM) diaphragm echography combined with sniff maneuver.ResultsA total of 89 patients with neuromuscular diseases and 27 healthy subjects were included in our study. In patients, the median age was 32 years [25; 50] and the median FVC was 34% of predicted [18; 55]. Sniff diaphragm motion using TM ultrasound was significantly associated with sniff nasal pressure, both for the right hemidiaphragm (r = 0.6 p ConclusionSniff diaphragm TM and TDI measures were significantly associated with sniff nasal pressure. Sniff diaphragm TM and TDI had a high level of accuracy to reveal respiratory involvement in patients with neuromuscular disorders. This technique is useful to assess and follow up diaphragm function in patients with neuromuscular disorders. It may be used as a respiratory outcome for clinical trials.

Spinal muscular atrophy (SMA) type III and IV are autosomal recessive, slowly progressive lower motor neuron syndromes. Nevertheless, wider cerebral involvement has been consistently reported in mouse models. The objective of this study is the characterisation of spinal and cerebral pathology in adult forms of SMA using multimodal quantitative imaging. Methods: Twenty-five type III and IV adult SMA patients and 25 age-matched healthy controls were enrolled in a spinal cord and brain imaging study. Structural measures of grey and white matter involvement and diffusion parameters of white matter integrity were evaluated at each cervical spinal level. Whole-brain and region-of-interest analyses were also conducted in the brain to explore cortical thickness, grey matter density and tract-based white matter alterations. Results: In the spinal cord, considerable grey matter atrophy was detected between C2-C6 vertebral levels. In the brain, increased grey matter density was detected in motor and extra-motor regions of SMA patients. No white matter pathology was identified neither at brain and spinal level. Conclusions: Adult forms of SMA are associated with selective grey matter degeneration in the spinal cord with preserved white matter integrity. The observed increased grey matter density in the motor cortex may represent adaptive reorganisation. Keywords: Spinal muscular atrophy, SMA, Multimodal MRI, Spinal cord MRI, Grey matter and white matter degeneration

Duchenne muscular dystrophy (DMD) is an inherited myogenic disorder due to mutations in the dystrophin gene on chromosome Xp21.1. We designed this study to determine the prevalence of left bundle branch block (LBBB), whether there is a relationship between LBBB and genetic pattern, and to assess predictive factors for acute cardiac events and mortality in adult DMD patients.We reviewed the charts of DMD followed at the Home Mechanical Ventilation Unit of the Raymond Poincare University Hospital.A total of 121 patients, aged from 18 to 41 years have been included in our study. Median vital capacity (VC) was 12% [7; 19.5] of predicted. Almost all patients were on home mechanical ventilation (95%). LBBB was present in 15 patients (13%); among them, 10 disclosed exonic deletions. After a median follow up of 6 years, 21 patients (17%) experienced acute heart failure (AHF), 7 patients (6%) supraventricular arrhythmia, 3 patients (2.4%) ventricular tachycardia, 4 patients (3%) significant electrical disturbances. LBBB was significantly associated with cardiac events (OR = 12.7; 95%CI [3.78-42.7]; p

[This corrects the article DOI: 10.1371/journal.pone.0152439.].

PURPOSE:The mechanisms underlying the topography of motor deficits in spinal muscular atrophy (SMA) remain unknown. We investigated the profile of spinal cord atrophy (SCA) in SMN1-linked SMA, and its correlation with the topography of muscle weakness. MATERIALS AND METHODS:Eighteen SMN1-linked SMA patients type III/V and 18 age/gender-matched healthy volunteers were included. Patients were scored on manual muscle testing and functional scales. Spinal cord was imaged using 3T MRI system. Radial distance (RD) and cord cross-sectional area (CSA) measurements in SMA patients were compared to those in controls and correlated with strength and disability scores. RESULTS:CSA measurements revealed a significant cord atrophy gradient mainly located between C3 and C6 vertebral levels with a SCA rate ranging from 5.4% to 23% in SMA patients compared to controls. RD was significantly lower in SMA patients compared to controls in the anterior-posterior direction with a maximum along C4 and C5 vertebral levels (p-values < 10-5). There were no correlations between atrophy measurements, strength and disability scores. CONCLUSIONS:Spinal cord atrophy in adult SMN1-linked SMA predominates in the segments innervating the proximal muscles. Additional factors such as neuromuscular junction or intrinsic skeletal muscle defects may play a role in more complex mechanisms underlying weakness in these patients.

Myotonic Dystrophy type 1 (DM1) is one of the most heterogeneous hereditary disease in terms of age of onset, clinical manifestations, and severity, challenging both medical management and clinical trials. The CTG expansion size is the main factor determining the age of onset although no factor can finely predict phenotype and prognosis. Differences between males and females have not been specifically reported. Our aim is to study gender impact on DM1 phenotype and severity.We first performed cross-sectional analysis of main multiorgan clinical parameters in 1409 adult DM1 patients (>18 y) from the DM-Scope nationwide registry and observed different patterns in males and females. Then, we assessed gender impact on social and economic domains using the AFM-Téléthon DM1 survey (n = 970), and morbidity and mortality using the French National Health Service Database (n = 3301).Men more frequently had (1) severe muscular disability with marked myotonia, muscle weakness, cardiac, and respiratory involvement; (2) developmental abnormalities with facial dysmorphism and cognitive impairment inferred from low educational levels and work in specialized environments; and (3) lonely life. Alternatively, women more frequently had cataracts, dysphagia, digestive tract dysfunction, incontinence, thyroid disorder and obesity. Most differences were out of proportion to those observed in the general population. Compared to women, males were more affected in their social and economic life. In addition, they were more frequently hospitalized for cardiac problems, and had a higher mortality rate.Gender is a previously unrecognized factor influencing DM1 clinical profile and severity of the disease, with worse socio-economic consequences of the disease and higher morbidity and mortality in males. Gender should be considered in the design of both stratified medical management and clinical trials.

BACKGROUND:Type 2C and 2D limb girdle muscular dystrophies (LGMD) are a group of autosomal recessive limb girdle muscular dystrophies manifested by proximal myopathy, impaired respiratory muscle function and cardiomyopathy. The correlation and the prognostic impact of respiratory and heart impairment are poorly described. We aimed to describe the long-term cardiac and respiratory follow-up of these patients and to determine predictive factors of cardio-respiratory events and mortality in LGMD 2C and 2D. METHODS:We reviewed the charts of 34 LGMD patients, followed from 2005 to 2015, to obtain echocardiographic, respiratory function and sleep recording data. We considered respiratory events (acute respiratory failure, pulmonary sepsis, atelectasis or pneumothorax), cardiac events (acute heart failure, significant cardiac arrhythmia or conduction block, ischemic stroke) and mortality as outcomes of interest for the present analysis. RESULTS:A total of 21 patients had type 2C LGMD and 13 patients had type 2D. Median age was 30 years [IQR 24-38]. At baseline, median pulmonary vital capacity (VC) was 31% of predicted value [20-40]. Median maximal inspiratory pressure (MIP) was 31 cmH2O [IQR 20.25-39.75]. Median maximal expiratory pressure (MEP) was 30 cm H2O [20-36]. Median left ventricular ejection fraction (LVEF) was 55% [45-64] with 38% of patients with LVEF

Abstract Background Beyond the observed alterations in cellular structure and mitochondria, the mechanisms linking rare genetic mutations to the development of heart failure in patients affected by desmin mutations remain unclear due in part, to the lack of relevant human cardiomyocyte models. Methods To shed light on the role of mitochondria in these mechanisms, we investigated cardiomyocytes derived from human induced pluripotent stem cells carrying the heterozygous DES E439K mutation that were either isolated from a patient or generated by gene editing. To increase physiological relevance, cardiomyocytes were either cultured on an anisotropic micropatterned surface to obtain elongated and aligned cardiomyocytes, or as a cardiac spheroid to create a micro-tissue. Moreover, when applicable, results from cardiomyocytes were confirmed with heart biopsies of suddenly died patient of the same family harboring DES E439K mutation, and post-mortem heart samples from five control healthy donors. Results The heterozygous DES E439K mutation leads to dramatic changes in the overall cytoarchitecture of cardiomyocytes, including cell size and morphology. Most importantly, mutant cardiomyocytes display altered mitochondrial architecture, mitochondrial respiratory capacity and metabolic activity reminiscent of defects observed in patient’s heart tissue. Finally, to challenge the pathological mechanism, we transferred normal mitochondria inside the mutant cardiomyocytes and demonstrated that this treatment was able to restore mitochondrial and contractile functions of cardiomyocytes. Conclusions This work highlights the deleterious effects of DES E439K mutation, demonstrates the crucial role of mitochondrial abnormalities in the pathophysiology of desmin-related cardiomyopathy, and opens up new potential therapeutic perspectives for this disease.

Myofibrillar myopathy (MFM) is a rare genetic disorder characterized by muscular dystrophy that is often associated with cardiac disease. This disease is caused by mutations in several genes, among them DES (encoding desmin) is the most frequently affected. Peripheral blood mononuclear cells from 5 different MFM patients with different DES mutations were reprogrammed into induced pluripotent stem cells (IPSC) using non-integrative vectors. For each patient, one IPSC clone was selected and demonstrated pluripotency hallmarks without genomic abnormalities. SNP profiles were identical to the cells of origin and all the clones have the capacity to differentiate into all three germ layers.

Abstract Myasthenia gravis (MG) is an autoimmune disease characterized by muscle fatigability due to acetylcholine receptor (AChR) autoantibodies. To better characterize juvenile MG (JMG), we analyzed 85 pre- and 132 post-pubescent JMG (with a cutoff age of 13) compared to 721 adult MG patients under 40 years old using a French database. Clinical data, anti-AChR antibody titers, thymectomy, and thymic histology were analyzed. The proportion of females was higher in each subgroup. No significant difference in the anti-AChR titers was observed. Interestingly, the proportion of AChR+ MG patients was notably lower among adult MG patients aged between 30 and 40 years, at 69.7%, compared to over 82.4% in the other subgroups. Thymic histological data were examined in patients who underwent thymectomy during the year of MG onset. Notably, in pre-JMG, the percentage of thymectomized patients was significantly lower (32.9% compared to more than 42.5% in other subgroups), and the delay to thymectomy was twice as long. We found a positive correlation between anti-AChR antibodies and germinal center grade across patient categories. Additionally, only females, particularly post-JMG patients, exhibited the highest rates of lymphofollicular hyperplasia (95% of cases) and germinal center grade. These findings reveal distinct patterns in JMG patients, particularly regarding thymic follicular hyperplasia, which appears to be exacerbated in females after puberty.

Abstract Background In 2017, a new treatment by nusinersen, an antisense oligonucleotide delivered by repeated intrathecal injections, became available for patients with spinal muscular atrophy (SMA), whereas clinical trials had mainly involved children. Since 2020, the oral, selective SMN2-splicing modifier risdiplam has been available with restrictions evolving with time. In this peculiar context of lack of data regarding adult patients, many questions were raised to define the indications of treatment and the appropriate follow-up in this population. To homogenize access to treatment in France, a national multidisciplinary team meeting dedicated to adult SMA patients, named SMA multidisciplinary team meeting, (SMDTs) was created in 2018. Our objective was to analyze the value of SMDTs in the decision-making process in SMA adult patients and to provide guidelines about treatment. Methods From October 2020 to September 2021, data extracted from the SMDT reports were collected. The primary outcome was the percentage of cases in which recommendations on validating treatment plans were given. The secondary outcomes were type of treatment requested, description of expectations regarding treatment and description of recommendations or follow-up and discontinuation. Data were analyzed using descriptive statistics. Comparisons between the type of treatment requested were performed using Mann–Whitney test or the Student t test for quantitative data and the Fisher’s exact test or the χ2 test for qualitative data. Results Cases of 107 patients were discussed at the SMDTs with a mean age of 35.3 (16–62). Forty-seven were SMA type 2, and 57 SMA type 3. Twelve cases were presented twice. Out of 122 presentations to the SMDTs, most of requests related to the initiation of a treatment (nusinersen (n = 46), risdiplam (n = 54), treatment without mentioning preferred choice (n = 5)) or a switch of treatment (n = 12). Risdiplam requests concerned significantly older patients (p = 0.002), mostly SMA type 2 (p

Abstract Congenital myasthenic syndromes (CMS) are a clinically and genetically heterogeneous group of rare diseases due to mutations in neuromuscular junction (NMJ) protein-coding genes. Until now, many mutations encoding postsynaptic proteins as Agrin, MuSK and LRP4 have been identified as responsible for increasingly complex CMS phenotypes. The majority of mutations identified in LRP4 gene causes bone diseases including CLS and sclerosteosis-2 and rare cases of CMS with mutations in LRP4 gene has been described so far. In the French cohort of CMS patients, we identified a novel LRP4 homozygous missense mutation (c.1820A > G; p.Thy607Cys) within the β1 propeller domain in a patient presenting CMS symptoms, including muscle weakness, fluctuating fatigability and a decrement in compound muscle action potential in spinal accessory nerves, associated with congenital agenesis of the hands and feet and renal malformation. Mechanistic expression studies show a significant decrease of AChR aggregation in cultured patient myotubes, as well as altered in vitro binding of agrin and Wnt11 ligands to the mutated β1 propeller domain of LRP4 explaining the dual phenotype characterized clinically and electoneuromyographically in the patient. These results expand the LRP4 mutations spectrum associated with a previously undescribed clinical association involving impaired neuromuscular transmission and limb deformities and highlighting the critical role of a yet poorly described domain of LRP4 at the NMJ. This study raises the question of the frequency of this rare neuromuscular form and the future diagnosis and management of these cases.

Background Chronic respiratory failure and heart involvement may occur in Duchenne muscular dystrophy. We aimed to assess the prognostic value of the right ventricular (RV) systolic dysfunction in patients with Duchenne muscular dystrophy. Methods and Results We studied 90 genetically proven patients with Duchenne muscular dystrophy from 2010 to 2019, to obtain respiratory function and Doppler echocardiographic RV systolic function. Prognostic value was assessed in terms of death and cardiac events. The median age was 27.5 years, and median forced vital capacity was at 10% of the predicted value: 83 patients (92%) were on home mechanical ventilation. An RV systolic dysfunction was found in 46 patients (51%). In patients without RV dysfunction at inclusion, a left ventricular systolic dysfunction at inclusion was associated with a higher risk of developing RV dysfunction during follow‐up with an odds ratio of 4.5 (P=0.03). RV systolic dysfunction was significantly associated with cardiac events, mainly acute heart failure (62%) and cardiogenic shock (23%). In a multivariable Cox model, the adjusted hazard ratio was 4.96 (95% CI [1.09–22.6]; P=0.04). In terms of death, we found a significant difference between patients with RV dysfunction versus patients without RV dysfunction in the Kaplan–Meier curves (log‐rank P=0.045). Conclusions RV systolic dysfunction is frequently present in patients with Duchenne muscular dystrophy and is associated with increased risk of cardiac events, irrespective of left ventricular dysfunction and mechanical ventilation. Registration URL: https://www.clinicaltrials.org; unique identifier: NCT02501083.

Myasthenia Gravis (MG) is a neurological autoimmune disease characterized by disabling muscle weaknesses due to anti-acetylcholine receptor (AChR) autoantibodies. To gain insight into immune dysregulation underlying early-onset AChR+ MG, we performed an in-depth analysis of peripheral mononuclear blood cells (PBMCs) using mass cytometry. PBMCs from 24 AChR+ MG patients without thymoma and 16 controls were stained with a panel of 37 antibodies. Using both unsupervised and supervised approaches, we observed a decrease in monocytes, for all subpopulations: classical, intermediate, and non-classical monocytes. In contrast, an increase in innate lymphoid cells 2 (ILC2s) and CD27- γδ T cells was observed. We further investigated the dysregulations affecting monocytes and γδ T cells in MG. We analyzed CD27- γδ T cells in PBMCs and thymic cells from AChR+ MG patients. We detected the increase in CD27- γδ T cells in thymic cells of MG patients suggesting that the inflammatory thymic environment might affect γδ T cell differentiation. To better understand changes that might affect monocytes, we analyzed RNA sequencing data from CD14+ PBMCs and showed a global decrease activity of monocytes in MG patients. Next, by flow cytometry, we especially confirmed the decrease affecting non-classical monocytes. In MG, as for other B-cell mediated autoimmune diseases, dysregulations are well known for adaptive immune cells, such as B and T cells. Here, using single-cell mass cytometry, we unraveled unexpected dysregulations for innate immune cells. If these cells are known to be crucial for host defense, our results demonstrated that they could also be involved in autoimmunity.

Myofibrillar myopathies (MFM) are a group of heterogenic muscular disorders characterized by histological disorders with accumulation of myofibrillar degradation products and providing disorganization of the myofibril network. Respiratory muscles may be involved in this disease. Ultrasound is used to assess not only the diaphragm but also the parasternal intercostal muscle. Parasternal intercostal muscle thickening may be used as an index of respiratory effort particularly in patients with diaphragm failure. We report the case of high parasternal intercostal muscle thickening associated with diaphragm dysfunction in a patient with MFM.