A humanized knock-in Col6a1 mouse recapitulates a deep-intronic splice-activating variant.

Antisense therapeutics such as splice-modulating antisense oligonucleotides (ASOs) are promising tools to treat diseases caused by splice-altering intronic variants. However, their testing in animal models is hampered by the generally poor sequence conservation of the intervening sequences between human and other species. Here we aimed to model in the mouse a recurrent, deep-intronic, splice-activating, COL6A1 variant, associated with a severe form of Collagen VI-related muscular dystrophies (COL6-RDs), for the purpose of testing human-ready antisense therapeutics in vivo. The variant, c.930+189C>T, creates a donor splice site and inserts a 72-nt-long pseudoexon, which, when translated, acts in a dominant-negative manner, but which can be skipped with ASOs. We created a unique humanized mouse allele (designated as "h"), in which a 1.9 kb of the mouse genomic region encoding the amino-terminus (N-) of the triple helical (TH) domain of collagen a1(VI) was swapped for the human orthologous sequence. In addition, we also created an allele that carries the c.930+189C>T variant on the same humanized knock-in sequence (designated as "h+189T"). We show that in both models, the human exons are spliced seamlessly with the mouse exons to generate a chimeric mouse-human collagen a1(VI) protein. In homozygous Col6a1 h+189T/h+189T mice, the pseudoexon is expressed at levels comparable to those observed in heterozygous patients' muscle biopsies. While Col6a1h/h mice do not show any phenotype compared to wildtype animals, Col6a1 h/h+189T and Col6a1 h+189T/h+189T mice have smaller muscle masses and display grip strength deficits detectable as early as 4 weeks of age. The pathogenic h+189T humanized knock-in mouse allele thus recapitulates the pathogenic splicing defects seen in patients' biopsies and allows testing of human-ready precision antisense therapeutics aimed at skipping the pseudoexon. Given that the COL6A1 N-TH region is a hot-spot for COL6-RD variants, the humanized knock-in mouse model can be utilized as a template to introduce other COL6A1 pathogenic variants. This unique humanized mouse model thus represents a valuable tool for the development of antisense therapeutics for COL6-RDs.

The Role of ß-Dystroglycan in Nuclear Dynamics.

Dystroglycan is a ubiquitously expressed heterodimeric cell-surface laminin receptor with roles in cell adhesion, signalling, and membrane stabilisation. More recently, the transmembrane ß-subunit of dystroglycan has been shown to localise to both the nuclear envelope and the nucleoplasm. This has led to the hypothesis that dystroglycan may have a structural role at the nuclear envelope analogous to its role at the plasma membrane. The biochemical fraction of myoblast cells clearly supports the presence of dystroglycan in the nucleus. Deletion of the dystroglycan protein by disruption of the DAG1 locus using CRISPR/Cas9 leads to changes in nuclear size but not overall morphology; moreover, the Young's modulus of dystroglycan-deleted nuclei, as determined by atomic force microscopy, is unaltered. Dystroglycan-disrupted myoblasts are also no more susceptible to nuclear stresses including chemical and mechanical, than normal myoblasts. Re-expression of dystroglycan in DAG1-disrupted myoblasts restores nuclear size without affecting other nuclear parameters.

The Impact of miR-155-5p on Myotube Differentiation: Elucidating Molecular Targets in Skeletal Muscle Disorders.

MicroRNAs are small regulatory molecules that control gene expression. An emerging property of muscle miRNAs is the cooperative regulation of transcriptional and epitranscriptional events controlling muscle phenotype. miR-155 has been related to muscular dystrophy and muscle cell atrophy. However, the function of miR-155 and its molecular targets in muscular dystrophies remain poorly understood. Through in silico and in vitro approaches, we identify distinct transcriptional profiles induced by miR-155-5p in muscle cells. The treated myotubes changed the expression of 359 genes (166 upregulated and 193 downregulated). We reanalyzed muscle transcriptomic data from dystrophin-deficient patients and detected overlap with gene expression patterns in miR-155-treated myotubes. Our analysis indicated that miR-155 regulates a set of transcripts, including Aldh1l, Nek2, Bub1b, Ramp3, Slc16a4, Plce1, Dync1i1, and Nr1h3. Enrichment analysis demonstrates 20 targets involved in metabolism, cell cycle regulation, muscle cell maintenance, and the immune system. Moreover, digital cytometry confirmed a significant increase in M2 macrophages, indicating miR-155's effects on immune response in dystrophic muscles. We highlight a critical miR-155 associated with disease-related pathways in skeletal muscle disorders.

Induced degeneration and regeneration in aged muscle reduce tubular aggregates but not muscle function.

Introduction: Tubular aggregates (TA) are skeletal muscle structures that arise from the progressive accumulation of sarcoplasmic reticulum proteins. Cytoplasmic aggregates in muscle fibers have already been observed in mice and humans, mainly during aging and muscle disease processes. However, the effects of muscle regeneration on TA formation have not yet been reported. This study aimed to investigate the relationship between degeneration/regeneration and TA in aged murine models. We investigated the presence and quantity of TA in old males from two murine models with intense muscle degeneration and regeneration. Methods: One murine lineage was a Dmdmdx model of Duchenne muscular dystrophy (n = 6). In the other model, muscle damage was induced by electroporation in C57BL/6J wild-type mice, and analyzed after 5, 15, and 30 days post-electroporation (dpe; n = 15). Regeneration was evaluated based on the quantity of developmental myosin heavy chain (dMyHC)-positive fibers. Results: The frequency of fibers containing TA was higher in aged C57BL/6J (26 ± 8.3%) than in old dystrophic Dmdmdx mice (2.4 ± 2%). Comparing the data from induced degeneration/regeneration in normal mice revealed a reduced proportion of TA-containing fibers after 5 and 30 dpe. Normal aged muscle was able to regenerate and form dMyHC+ fibers, mainly at 5 dpe (0.1 ± 0.1 vs. 16.5 ± 2.6%). However, there was no difference in force or resistance between normal and 30 dpe animals, except for the measurements by the Actimeter device, which showed the worst parameters in the second group. Discussion: Our results suggest that TA also forms in the Dmdmdx muscle but in smaller amounts. The intense degeneration and regeneration of the old dystrophic model resulted in the generation of new muscle fibers with a lower quantity of TA. Data from electroporated wild-type mice support the idea that muscle regeneration leads to a reduction in the amount of TA. We suggest that TA accumulates in muscle fibers throughout physiological aging and that regeneration leads to the formation of new fibers without these structures. In addition, these new fibers do not confer functional benefits to the muscle.

Hospital admissions from the emergency department of adult patients affected by myopathies.

BACKGROUND AND PURPOSE: Myopathies are associated with classic signs and symptoms, but also with possible life-threatening complications that may require assistance in an emergency setting. This phenomenon is understudied in the literature. We aimed to assess the presentation, management, and outcomes of clinical manifestations potentially related to a muscle disorder requiring referral to the adult emergency department (ED) and hospitalization. METHODS: Anonymized patient data retrieved using the International Classification of Diseases, Ninth Revision codes related to muscle disorders over 4 years were retrospectively analyzed. Medical reports were evaluated to extract demographic and clinical variables, along with outcomes. Two groups were defined based on the presence (known diagnosis [KD] group) or absence (unknown diagnosis [UD] group) of a diagnosed muscle disorder at arrival. RESULTS: A total of 244 patients were included, 51% of whom were affected by a known myopathy, predominantly limb-girdle muscular dystrophies and myotonic dystrophies. The main reasons for ED visits in the KD group were respiratory issues, worsening of muscle weakness, and gastrointestinal problems. Heart complications were less prevalent. In the UD group, 27 patients received a new diagnosis of a specific primary muscle disorder after the ED access, mostly an inflammatory myopathy. Death during hospitalization was recorded in 26 patients, with a higher rate in the KD group and in patients affected by mitochondrial and inflammatory myopathies. Sepsis and dyspnea were associated with increased death risk. CONCLUSIONS: Respiratory complications are the most common reason for myopathic patients accessing the ED, followed by gastrointestinal issues. Infections are severe threats and, once hospitalized, these patients have relatively high mortality.

Title-molecular diagnostics of dystrophinopathies in Sri Lanka towards phenotype predictions: an insight from a South Asian resource limited setting.

BACKGROUND: The phenotype of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) patients is determined by the type of DMD gene variation, its location, effect on reading frame, and its size. The primary objective of this investigation was to determine the frequency and distribution of DMD gene variants (deletions/duplications) in Sri Lanka through the utilization of a combined approach involving multiplex polymerase chain reaction (mPCR) followed by Multiplex Ligation Dependent Probe Amplification (MLPA) and compare to the international literature. The current consensus is that MLPA is a labor efficient yet expensive technique for identifying deletions and duplications in the DMD gene. METHODOLOGY: Genetic analysis was performed in a cohort of 236 clinically suspected pediatric and adult myopathy patients in Sri Lanka, using mPCR and MLPA. A comparative analysis was conducted between our findings and literature data. RESULTS: In the entire patient cohort (n = 236), mPCR solely was able to identify deletions in the DMD gene in 131/236 patients (DMD-120, BMD-11). In the same cohort, MLPA confirmed deletions in 149/236 patients [DMD-138, BMD -11]. These findings suggest that mPCR has a detection rate of 95% (131/138) among all patients who received a diagnosis. The distal and proximal deletion hotspots for DMD were exons 45-55 and 6-15. Exon 45-60 identified as a novel in-frame variation hotspot. Exon 45-59 was a hotspot for BMD deletions. Comparisons with the international literature show significant variations observed in deletion and duplication frequencies in DMD gene across different populations. CONCLUSION: DMD gene deletions and duplications are concentrated in exons 45-55 and 2-20 respectively, which match global variation hotspots. Disparities in deletion and duplication frequencies were observed when comparing our data to other Asian and Western populations. Identified a 95% deletion detection rate for mPCR, making it a viable initial molecular diagnostic approach for low-resource countries where MLPA could be used to evaluate negative mPCR cases and cases with ambiguous mutation borders. Our findings may have important implications in the early identification of DMD with limited resources in Sri Lanka and to develop tailored molecular diagnostic algorithms that are regional and population specific and easily implemented in resource limited settings.

Findings from the Longitudinal CINRG Becker Natural History Study.

BACKGROUND: Becker muscular dystrophy is an X-linked, genetic disorder causing progressive degeneration of skeletal and cardiac muscle, with a widely variable phenotype. OBJECTIVE: A 3-year, longitudinal, prospective dataset contributed by patients with confirmed Becker muscular dystrophy was analyzed to characterize the natural history of this disorder. A better understanding of the natural history is crucial to rigorous therapeutic trials. METHODS: A cohort of 83 patients with Becker muscular dystrophy (5-75 years at baseline) were followed for up to 3 years with annual assessments. Muscle and pulmonary function outcomes were analyzed herein. Age-stratified statistical analysis and modeling were conducted to analyze cross-sectional data, time-to-event data, and longitudinal data to characterize these clinical outcomes. RESULTS: Deletion mutations of dystrophin exons 45-47 or 45-48 were most common. Subgroup analysis showed greater pairwise association between motor outcomes at baseline than association between these outcomes and age. Stronger correlations between outcomes for adults than for those under 18 years were also observed. Using cross-sectional binning analysis, a ceiling effect was seen for North Star Ambulatory Assessment but not for other functional outcomes. Longitudinal analysis showed a decline in percentage predicted forced vital capacity over the life span. There was relative stability or improved median function for motor functional outcomes through childhood and adolescence and decreasing function with age thereafter. CONCLUSIONS: There is variable progression of outcomes resulting in significant heterogeneity of the clinical phenotype of Becker muscular dystrophy. Disease progression is largely manifest in adulthood. There are implications for clinical trial design revealed by this longitudinal analysis of a Becker natural history dataset.

Magnetic resonance imaging-based criteria to differentiate dysferlinopathy from other genetic muscle diseases.

The identification of disease-characteristic patterns of muscle fatty replacement in magnetic resonance imaging (MRI) is helpful for diagnosing neuromuscular diseases. In the Clinical Outcome Study of Dysferlinopathy, eight diagnostic rules were described based on MRI findings. Our aim is to confirm that they are useful to differentiate dysferlinopathy (DYSF) from other genetic muscle diseases (GMD). The rules were applied to 182 MRIs of dysferlinopathy patients and 1000 MRIs of patients with 10 other GMD. We calculated sensitivity (S), specificity (Sp), positive and negative predictive values (PPV/NPV) and accuracy (Ac) for each rule. Five of the rules were more frequently met by the DYSF group. Patterns observed in patients with FKRP, ANO5 and CAPN3 myopathies were similar to the DYSF pattern, whereas patterns observed in patients with OPMD, laminopathy and dystrophinopathy were clearly different. We built a model using the five criteria more frequently met by DYSF patients that obtained a S 95.9%, Sp 46.1%, Ac 66.8%, PPV 56% and NPV 94% to distinguish dysferlinopathies from other diseases. Our findings support the use of MRI in the diagnosis of dysferlinopathy, but also identify the need to externally validate "disease-specific" MRI-based diagnostic criteria using MRIs of other GMD patients.

Health-Related Quality of Life in FKRP-Related Limb-Girdle Muscular Dystrophy R9.

BACKGROUND: Limb-girdle muscular dystrophy R9 (LGMDR9) is a chronic progressive hereditary muscle disease, related to the Fukutin Related Protein (FKRP) gene, that may cause major disabilities, cardiomyopathy, and ventilatory failure. Knowledge of how LGMDR9 affects health-related quality of life (HRQoL) is relevant in treatment and care. OBJECTIVE: To investigate HRQoL in the Norwegian LGMDR9 population over 14 months and relation to fatigue and sleep quality. METHODS: Participants (16+ years) of the Norwegian LGMDR9 cohort study completed two HRQoL measures, i.e., Individualized Neuromuscular Quality of Life questionnaire (INQoL) and the 36-item Short Form (SF-36) at baseline, 8, and 14 months and measures of fatigue and sleep quality at 9 months. RESULTS: HRQoL response rate was 84/90 (75 c.826 C > A homozygotes and nine c.826 C > A compound heterozygotes). Compared to Norwegian normative data, all SF-36 domain scores were impaired (p≤0.006) except mental health in males (p = 0.05) and pain scores. During 14 months, perceived muscle weakness and the INQoL index (disease burden) worsened in c.826 C > A homozygotes. Compound heterozygotes reported more dysphagia and physical difficulties than homozygotes and showed a tendency towards worsening in weakness over time but some improvement on the INQoL index. Homozygous females reported generally poorer HRQoL and a higher burden than males. The INQoL index was related to perceived muscle weakness and fatigue, and fatigue to myalgia and mental distress. The prevalence of fatigue and poor sleep was 40% and 49%, respectively. CONCLUSIONS: The 14-month follow-up period shows a worsening of perceived weakness and burden in c.826 C > A homozygotes, which can then be expected. The prevalence and impact of fatigue indicate a need for awareness and treatment of fatigue. Myalgia and mental distress are potential targets in the treatment of fatigue, which future studies need to establish. Sleep issues and gender-specific care needs also require attention in LGMDR9.

Insomnia and sleep-disordered breathing in FKRP-related limb-girdle muscular dystrophy R9. The Norwegian LGMDR9 cohort study (2020).

Limb-girdle muscular dystrophy R9 (LGMDR9) is a progressive and disabling genetic muscle disease. Sleep is relevant in the patient care as it impacts on health, functioning, and well-being. LGMDR9 may potentially affect sleep by physical or emotional symptoms, myalgia, or sleep-disordered breathing (SDB) through cardiorespiratory involvement. The objective was to investigate the occurrence of insomnia and unrecognized or untreated SDB in LGMDR9, associated factors, and relationships with fatigue and health-related quality of life (HRQoL). All 90 adults in a Norwegian LGMDR9 cohort received questionnaires on sleep, fatigue, and HRQoL. Forty-nine of them underwent clinical assessments and 26 without mask-based therapy for respiration disorders additionally underwent polysomnography (PSG) and capnometry. Among 77 questionnaire respondents, 31% received mask-based therapy. The prevalence of insomnia was 32% of both those with and without such therapy but was significantly increased in fatigued respondents (54% vs 21%). Insomnia levels correlated inversely with mental HRQoL. Among 26 PSG candidates, an apnea-hypopnea index (AHI) ≥ 5/h was observed in 16/26 subjects (≥ 15/h in 8/26) with median 6.8 obstructive apneas and 0.2 central apneas per hour of sleep. The AHI was related to advancing age and an ejection fraction < 50%. Sleep-related hypoventilation was detected in one subject. Fatigue severity did not correlate with motor function or nocturnal metrics of respiration or sleep but with Maximal Inspiratory Pressure (r = - 0.46). The results indicate that insomnia and SDB are underrecognized comorbidities in LGMDR9 and associated with HRQoL impairment and heart failure, respectively. We propose an increased attention to insomnia and SDB in the interdisciplinary care of LGMDR9. Insomnia and pulmonary function should be examined in fatigued patients.

Our journey with François Gros.

Gillian Butler-Browne began working on muscle at the Institut Pasteur in the laboratory of François Gros in 1978. She characterized the expression profile of different myosin isoforms during both human and rodent development. Vincent Mouly joined this laboratory for his PhD in 1982, and defined the different populations of myoblasts appearing during development in birds and then in humans. Together, they demonstrated the impact of the limit in proliferation of the precursor cells on the regenerative capacity of human skeletal muscle, and their group developed models to evaluate the regenerative potential of skeletal muscle in vitro, measuring the telomeric erosion, and identified the involvement of a stress pathway in the proliferative arrest of muscle progenitors. A platform to produce human immortalized muscle cell lines was the successful result of this research, initiated with François Gros and W. E. Wright. The in vivo regenerative potential of human muscle cells was evaluated by injection into muscles of immunodeficient mice. Their group in collaboration with the clinical team of Professor Jean Lacau St-Guily and Professor Sophie Perié completed a successful autologous myoblast transplantation clinical trial for Oculo-pharyngeal muscular dystrophy. This common scientific career was made possible thanks to the precious and always benevolent support of François Gros.

Gillian Butler-Browne a commencé à travailler sur le muscle à l'Institut Pasteur dans le laboratoire de François Gros en 1978. Elle a caractérisé le profil d'expression des différentes isoformes de myosine au cours du développement de l'homme et du rongeur. Vincent Mouly a rejoint ce laboratoire pour son doctorat en 1982, et a défini les différentes populations de myoblastes apparaissant au cours du développement chez les oiseaux puis chez l'homme. Ensemble, ils ont démontré l'impact de la limite de prolifération des cellules précurseurs sur la capacité de régénération du muscle squelettique humain, et leur groupe a développé des modèles pour évaluer le potentiel de régénération du muscle squelettique in vitro, en mesurant l'érosion télomérique, et a identifié l'implication d'une voie de stress dans l'arrêt de la prolifération des progéniteurs musculaires. Une plateforme de production de lignées de cellules musculaires humaines immortalisées a été le résultat fructueux de cette recherche, initiée avec François Gros et W. E. Wright. Le potentiel de régénération in vivo des cellules musculaires humaines a été évalué par injection dans des muscles de souris immunodéficientes. Leur groupe, en collaboration avec l'équipe clinique des professeurs Jean-Lacau-St Guily et Sophie Perié, a réalisé avec succès un essai clinique de transplantation autologue de myoblastes visant à traiter la dystrophie musculaire oculo-pharyngée. Cette carrière scientifique commune a été rendue possible par le soutien précieux et toujours bienveillant de François Gros.

Health checkups during the pandemic and clinical impact of COVID-19 in children with neuromuscular disease. / Controles de salud durante la pandemia e impacto clínico del COVID-19 en niños con enfermedad neuromuscular.

Introduction. Children with neuromuscular disease (NMD) require chronic health care (CHC) and may develop severe COVID-19. Objectives. To describe CHC for children with NMD during the pandemic and the course of COVID-19 in this group. Population and methods. Prospective, single-center cohort. Patients aged 2 to 18 years with ≥ 1 year of follow-up prior to the pandemic were included. Demographic variables in relation to CHC and COVID-19 were collected from medical records and via telephone surveys. Results. A total of 226 patients with a median age of 11.3 years were included; 71% were males. They had muscular dystrophy (55.7%) and spinal muscular atrophy (23%). When comparing the first year of the pandemic with the previous year, 30% did not have a health checkup and 25% did not receive kinesiotherapy. Others did, but with a lower frequency. A total of 52 COVID-19 cases were reported; 82% were symptomatic: 88.4% were mild/moderate and 11.6%, severe. No patient died. Conclusions. The pandemic had a negative impact on CHC, and COVID-19 cases were mostly mild.

Introducción. Los niños con enfermedad neuromuscular (ENM) requieren cuidados crónicos de salud (CCS) y podrían presentar COVID-19 grave. Objetivos. Describir CCS para niños con ENM durante la pandemia y evolución del COVID-19 en este grupo. Población y métodos. Cohorte prospectiva unicéntrica. Se incluyeron pacientes de 2-18 años, con ≥ 1 año de seguimiento previo a la pandemia. Se recolectaron variables demográficas, relativas a los CCS y al COVID-19 mediante historias clínicas y encuestas telefónicas. Resultados. Se incluyeron 226 pacientes; el 71 % varones, mediana de edad 11,3 años. Presentaban distrofias musculares (55,7 %) y atrofia muscular espinal (23 %). Comparando el primer año de pandemia con el previo, el 30 % no realizó controles médicos y el 25 % no realizó kinesioterapia. Otros disminuyeron la frecuencia. Hubo 52 casos de COVID-19. Fueron sintomáticos el 82 %: el 88,4 % leves/moderados y el 11,6 % graves. No hubo fallecidos. Conclusiones. La pandemia impactó negativamente en los CCS y los casos de COVID-19 fueron mayormente leves.

MicroRNA as potential biomarker for severity, progression, and therapeutic monitoring in animal models of limb-girdle muscular dystrophy: a systematic review.

Limb-girdle muscular dystrophies (LGMD) constitute a heterogeneous group of neuromuscular disorders in which there are alterations in proteins responsible for the preservation of muscle architecture and function, leading to proximal and progressive muscle weakness. There is, however, significant phenotypic and genotypic variation, as well as difficulty in establishing biomarkers that help to define pathogenic mechanisms and assess disease severity and progression. In this field, there is special attention to microRNAs, small non-coding RNA molecules related to the regulation of gene expression and, consequently, the production of proteins. Thus, this research aimed to verify the correlation between the expression of microRNAs and the severity, progression, and therapeutic response of LGMD animal models. A search was carried out in the PubMed, Embase, Scopus, ScienceDirect, Cochrane, and SciELO databases, with articles in English and without a time limit. The PRISMA 2020 checklist was used, and the protocol of this review was submitted to PROSPERO. The bibliographic survey of the 434 records found that 5 original articles met the inclusion criteria. The studies explored myomicroRNAs or miRNA panels with gene expression analysis. The analysis demonstrates that miR-1, 133a, and 206 are differentially expressed in serum and muscle. They change according to the degree of inflammation, fibrosis, muscle regeneration, and progression of the dystrophic process. MicroRNAs are up-regulated in dystrophic muscles, which are reversed after treatment in a dose-dependent manner. The present study inferred that miRs are essential in severity, progression, and therapeutic response in LGMD models and may be a useful biomarker in clinical research and prognosis. However, the practical application of these findings should be further explored.

Clinical features, imaging findings and molecular data of limb-girdle muscular dystrophies in a cohort of Chinese patients.

BACKGROUND: Limb-girdle muscular dystrophies (LGMDs) are a group of heterogeneous inherited diseases predominantly characterized by limb-girdle muscle weakness and dystrophic changes on histological analysis. The frequency of LGMD subtypes varies among regions in China and ethnic populations worldwide. Here, we analyzed the prevalence of LGMD subtypes, their corresponding clinical manifestations, and molecular data in a cohort of LGMD patients in Southeast China. METHODS: A total of 81 consecutive patients with clinically suspected LGMDs from 62 unrelated families across Southeast China were recruited for targeted next-generation sequencing and whole-exome sequencing from July 2017 to February 2020. RESULTS: Among 50 patients (41 families) with LGMDs, the most common subtypes were LGMD-R2/LGMD2B (36.6%) and LGMD-R1/LGMD2A (29.3%). Dystroglycanopathies (including LGMD-R9/LGMD2I, LGMD-R11/LGMD2K, LGMD-R14/LGMD2N and LGMD-R20/LGMD2U) were the most common childhood-onset subtypes and were found in 12.2% of the families. A total of 14.6% of the families had the LGMD-R7/LGMD2G subtype, and the mutation c.26_33dupAGGTGTCG in TCAP was the most frequent (83.3%). The only patient with the rare subtype LGMD-R18/LGMD2S had TRAPPC11 mutations; had a later onset than those previously reported, and presented with proximal‒distal muscle weakness, walking aid dependency, fatty liver disease and diabetes at 33 years of age. A total of 22.0% of the patients had cardiac abnormalities, and one patient with LMNA-related muscular dystrophy/LGMD1B experienced sudden cardiac death at 37 years of age. A total of 15.4% of the patients had restrictive respiratory insufficiency. Muscle imaging in patients with LGMD-R1/LGMD2A and LGMD-R2/LGMD2B showed subtle differences, including more severe fatty infiltration of the posterior thigh muscles in those with LGMD-R1/LGMD2A and edema in the lower leg muscles in those with LGMD-R2/LGMD2B. CONCLUSION: We determined the prevalence of different LGMD subtypes in Southeast China, described the detailed clinical manifestations and distinct muscle MRI patterns of these LGMD subtypes and reported the frequent mutations and the cardiorespiratory involvement frequency in our cohort, all of which might facilitate the differential diagnosis of LGMDs, allowing more timely treatment and guiding future clinical trials.

Higher Prevalence of Nonsense Pathogenic DMD Variants in a Single-Center Cohort from Brazil: A Genetic Profile Study That May Guide the Choice of Disease-Modifying Treatments.

Dystrophinopathies are muscle diseases caused by pathogenic variants in DMD, the largest gene described in humans, representing a spectrum of diseases ranging from asymptomatic creatine phosphokinase elevation to severe Duchenne muscular dystrophy (DMD). Several therapeutic strategies are currently in use or under development, each targeting different pathogenic variants. However, little is known about the genetic profiles of northeast Brazilian patients with dystrophinopathies. We describe the spectrum of pathogenic DMD variants in a single center in northeast Brazil. This is an observational, cross-sectional study carried out through molecular-genetic analysis of male patients diagnosed with dystrophinopathies using Multiplex Ligation-dependent Probe Amplification (MLPA) followed by Next-Generation Sequencing (NGS)-based strategies. A total of 94 male patients were evaluated. Deletions (43.6%) and duplications (10.6%) were the most recurring patterns of pathogenic variants. However, small variants were present in 47.1% of patients, most of them nonsense variants (27.6%). This is the largest South American single-center case series of dystrophinopathies to date. We found a higher frequency of treatment-amenable nonsense single-nucleotide variants than most previous studies. These findings may have implications for diagnostic strategies in less-known populations, as a higher frequency of nonsense variants may mean a higher possibility of treating patients with disease-modifying drugs.

Considering the Promise of Vamorolone for Treating Duchenne Muscular Dystrophy.

This commentary provides an independent consideration of data related to the drug vamorolone (VBP15) as an alternative steroid proposed for treatment of Duchenne muscular dystrophy (DMD). Glucocorticoids such as prednisone and deflazacort have powerful anti-inflammatory benefits and are the standard of care for DMD, but their long-term use can result in severe adverse side effects; thus, vamorolone was designed as a unique dissociative steroidal anti-inflammatory drug, to retain efficacy and minimise these adverse effects. Extensive clinical trials (ongoing) have investigated the use of vamorolone for DMD, with two trials also for limb-girdle muscular dystrophies including dysferlinopathy (current), plus a variety of pre-clinical trials published. Vamorolone looks very promising, with similar efficacy and some reduced adverse effects (e.g., related to height) compared with other glucocorticoids, specifically prednisone/prednisolone, although it has not yet been directly compared with deflazacort. Of particular interest to clarify is the optimal clinical dose and other aspects of vamorolone that are proposed to provide additional benefits for membranes of dystrophic muscle: to stabilise and protect the sarcolemma from damage and enhance repair. The use of vamorolone (and other glucocorticoids) needs to be evaluated in terms of overall long-term efficacy and cost, and also in comparison with many candidate non-steroidal drugs with anti-inflammatory and other benefits for DMD.

Novel mutations in the SGCA gene in unrelated Vietnamese patients with limb-girdle muscular dystrophies disease.

Background: Limb-girdle muscular dystrophy (LGMD) is a group of inherited neuromuscular disorders characterized by atrophy and weakness in the shoulders and hips. Over 30 subtypes have been described in five dominant (LGMD type 1 or LGMDD) and 27 recessive (LGMD type 2 or LGMDR). Each subtype involves a mutation in a single gene and has high heterogeneity in age of onset, expression, progression, and prognosis. In addition, the lack of understanding of the disease and the vague, nonspecific symptoms of LGMD subtypes make diagnosis difficult. Even as next-generation sequencing (NGS) genetic testing has become commonplace, some patients remain undiagnosed for many years. Methods: To identify LGMD-associated mutations, Targeted sequencing was performed in the patients and Sanger sequencing was performed in patients and family members. The in silico analysis tools such as Fathmm, M-CAP, Mutation Taster, PolyPhen 2, PROVEAN, REVEL, SIFT, MaxEntScan, Spliceailookup, Human Splicing Finder, NetGene2, and Fruitfly were used to predict the influence of the novel mutations. The pathogenicity of the mutation was interpreted according to the ACMG guidelines. Results: In this study, six patients from four different Vietnamese families were collected for genetic analysis at The Center for Gene and Protein Research and The Department of Molecular Pathology Faculty of Medical Technology, Hanoi Medical University, Hanoi, Vietnam. Based on clinical symptoms and serum creatine kinase (CK) levels, the patients were diagnosed with limb-girdle muscular dystrophies. Five mutations, including four (c.229C>T, p.Arg77Cys; exon one to three deletion; c.983 + 5G>C; and c.257_258insTGGCT, p.Phe88Leufs*125) in the SGCA gene and one (c.946-4_946-1delACAG) in the CAPN3 gene, were detected in six LGMD patients from four unrelated Vietnamese families. Two homozygous mutations (c.983 + 5G>C and c.257_258insTGGCT) in the SGCA gene were novel. These mutations were identified as the cause of the disease in the patients. Conclusion: Our results contribute to the general understanding of the etiology of the disease and provide the basis for definitive diagnosis and support genetic counseling and prenatal screening.

Modeling Sarcoglycanopathy in Danio rerio.

Sarcoglycanopathies, also known as limb girdle muscular dystrophy 3-6, are rare muscular dystrophies characterized, although heterogeneous, by high disability, with patients often wheelchair-bound by late adolescence and frequently developing respiratory and cardiac problems. These diseases are currently incurable, emphasizing the importance of effective treatment strategies and the necessity of animal models for drug screening and therapeutic verification. Using the CRISPR/Cas9 genome editing technique, we generated and characterized δ-sarcoglycan and ß-sarcoglycan knockout zebrafish lines, which presented a progressive disease phenotype that worsened from a mild larval stage to distinct myopathic features in adulthood. By subjecting the knockout larvae to a viscous swimming medium, we were able to anticipate disease onset. The δ-SG knockout line was further exploited to demonstrate that a δ-SG missense mutant is a substrate for endoplasmic reticulum-associated degradation (ERAD), indicating premature degradation due to protein folding defects. In conclusion, our study underscores the utility of zebrafish in modeling sarcoglycanopathies through either gene knockout or future knock-in techniques. These novel zebrafish lines will not only enhance our understanding of the disease's pathogenic mechanisms, but will also serve as powerful tools for phenotype-based drug screening, ultimately contributing to the development of a cure for sarcoglycanopathies.

Gpr18 agonist dampens inflammation, enhances myogenesis, and restores muscle function in models of Duchenne muscular dystrophy.

Introduction: Muscle wasting in Duchenne Muscular Dystrophy is caused by myofiber fragility and poor regeneration that lead to chronic inflammation and muscle replacement by fibrofatty tissue. Our recent findings demonstrated that Resolvin-D2, a bioactive lipid derived from omega-3 fatty acids, has the capacity to dampen inflammation and stimulate muscle regeneration to alleviate disease progression. This therapeutic avenue has many advantages compared to glucocorticoids, the current gold-standard treatment for Duchenne Muscular Dystrophy. However, the use of bioactive lipids as therapeutic drugs also faces many technical challenges such as their instability and poor oral bioavailability. Methods: Here, we explored the potential of PSB-KD107, a synthetic agonist of the resolvin-D2 receptor Gpr18, as a therapeutic alternative for Duchenne Muscular Dystrophy. Results and discussion: We showed that PSB-KD107 can stimulate the myogenic capacity of patient iPSC-derived myoblasts in vitro. RNAseq analysis revealed an enrichment in biological processes related to fatty acid metabolism, lipid biosynthesis, small molecule biosynthesis, and steroid-related processes in PSB-KD107-treated mdx myoblasts, as well as signaling pathways such as Peroxisome proliferator-activated receptors, AMP-activated protein kinase, mammalian target of rapamycin, and sphingolipid signaling pathways. In vivo, the treatment of dystrophic mdx mice with PSB-KD107 resulted in reduced inflammation, enhanced myogenesis, and improved muscle function. The positive impact of PSB-KD107 on muscle function is similar to the one of Resolvin-D2. Overall, our findings provide a proof-of concept that synthetic analogs of bioactive lipid receptors hold therapeutic potential for the treatment of Duchenne Muscular Dystrophy.