Sex differences in the involvement of skeletal and cardiac muscles in myopathic Ano5-/- mice.

Limb-girdle muscular dystrophy R12 (LGMD-R12) is caused by recessive mutations in the Anoctamin-5 gene (ANO5, TMEM16E). Although ANO5 myopathy is not X-chromosome linked, we performed a meta-analysis of the research literature and found that three-quarters of patients with LGMD-R12 are males. Females are less likely to present with moderate to severe skeletal muscle and/or cardiac pathology. Because these sex differences could be explained in several ways, we compared males and females in a mouse model of LGMD-R12. This model recapitulates the sex differences in human LGMD-R12. Only male Ano5-/- mice had elevated serum creatine kinase after exercise and exhibited defective membrane repair after laser injury. In contrast, by these measures, female Ano5-/- mice were indistinguishable from wild type. Despite these differences, both male and female Ano5-/- mice exhibited exercise intolerance. Although exercise intolerance of male mice can be explained by skeletal muscle dysfunction, echocardiography revealed that Ano5-/- female mice had features of cardiomyopathy that may be responsible for their exercise intolerance. These findings heighten concerns that mutations of ANO5 in humans may be linked to cardiac disease.

A phase Ib/IIa, open-label, multiple ascending-dose trial of domagrozumab in fukutin-related protein limb-girdle muscular dystrophy.

INTRODUCTION/AIMS: In this study we report the results of a phase Ib/IIa, open-label, multiple ascending-dose trial of domagrozumab, a myostatin inhibitor, in patients with fukutin-related protein (FKRP)-associated limb-girdle muscular dystrophy. METHODS: Nineteen patients were enrolled and assigned to one of three dosing arms (5, 20, or 40 mg/kg every 4 weeks). After 32 weeks of treatment, participants receiving the lowest dose were switched to the highest dose (40 mg/kg) for an additional 32 weeks. An extension study was also conducted. The primary endpoints were safety and tolerability. Secondary endpoints included muscle strength, timed function testing, pulmonary function, lean body mass, pharmacokinetics, and pharmacodynamics. As an exploratory outcome, muscle fat fractions were derived from whole-body magnetic resonance images. RESULTS: Serum concentrations of domagrozumab increased in a dose-dependent manner and modest levels of myostatin inhibition were observed in both serum and muscle tissue. The most frequently occurring adverse events were injuries secondary to falls. There were no significant between-group differences in the strength, functional, or imaging outcomes studied. DISCUSSION: We conclude that, although domagrozumab was safe in patients in limb-girdle muscular dystrophy type 2I/R9, there was no clear evidence supporting its efficacy in improving muscle strength or function.

Over three decades of natural history of limb girdle muscular dystrophy type R1/2A and R2/2B: Mathematical modelling of a multifactorial study.

We aimed to describe the natural history of Limb Girdle Muscular Dystrophy type 2A and 2B over more than three decades by considering muscular strength, motor, cardiac and respiratory function. 428 visits of nineteen 2A and twenty 2B patients were retrospectively analysed through a regression model to create the curves of evolution with disease duration of muscle strength (through Medical Research Council grading), motor function measure scale (D1, D2 and D3 domains) and cardio-pulmonary function tests. Clinically relevant muscular and motor function alterations occurred after the first decade of disease, while mild respiratory function alterations started after the second, with preserved cardiac function. Although type 2A showed relatively stronger distal lower limb muscles, while type 2B started with relatively stronger upper limb muscles, the corresponding motor functions were similar, becoming severely compromised after 25 years of disease. This was the longest retrospective study in types 2A and 2B. It defined curves of disease evolution not only from a neuromuscular, but also from functional, cardiac, and respiratory points of view, to be used to evaluate how the natural progression is changed by therapies. Due to slow disease progression, it was not possible to identify time sensitive endpoints.

Assessing Dysferlinopathy Patients Over Three Years With a New Motor Scale.

OBJECTIVE: Dysferlinopathy is a muscular dystrophy with a highly variable clinical presentation and currently unpredictable progression. This variability and unpredictability presents difficulties for prognostication and clinical trial design. The Jain Clinical Outcomes Study of Dysferlinopathy aims to establish the validity of the North Star Assessment for Limb Girdle Type Muscular Dystrophies (NSAD) scale and identify factors that influence the rate of disease progression using NSAD. METHODS: We collected a longitudinal series of functional assessments from 187 patients with dysferlinopathy over 3 years. Rasch analysis was used to develop the NSAD, a motor performance scale suitable for ambulant and nonambulant patients. Generalized estimating equations were used to evaluate the impact of patient factors on outcome trajectories. RESULTS: The NSAD detected significant change in clinical progression over 1 year. The steepest functional decline occurred during the first 10 years after symptom onset, with more rapid decline noted in patients who developed symptoms at a younger age (p = 0.04). The most rapidly deteriorating group over the study was patients 3 to 8 years post symptom onset at baseline. INTERPRETATION: The NSAD is the first validated limb girdle specific scale of motor performance, suitable for use in clinical practice and clinical trials. Longitudinal analysis showed it may be possible to identify patient factors associated with greater functional decline both across the disease course and in the short-term for clinical trial preparation. Through further work and validation in this cohort, we anticipate that a disease model incorporating functional performance will allow for more accurate prognosis for patients with dysferlinopathy. ANN NEUROL 2021;89:967-978.

Mutational Spectrum of CAPN3 with Genotype-Phenotype Correlations in Limb Girdle Muscular Dystrophy Type 2A/R1 (LGMD2A/LGMDR1) Patients in India.

BACKGROUND: Limb girdle muscular dystrophy recessive type 1 (LGMDR1, Previously LGMD2A) is characterized by inactivating mutations in CAPN3. Despite the significant burden of muscular dystrophy in India, and particularly of LGMDR1, its genetic characterization and possible phenotypic manifestations are yet unidentified. MATERIAL AND METHODS: We performed bidirectional CAPN3 sequencing in 95 LGMDR1 patient samples characterized by calpain-3 protein analysis, and these findings were correlated with clinical, biochemical and histopathological features. RESULTS: We identified 84 (88.4%) cases of LGMDR1 harboring 103 CAPN3 mutations (71 novel and 32 known). At least two mutant alleles were identified in 79 (94.2%) of patients. Notably, 76% exonic variations were enriched in nine CAPN3 exons and overall, 41 variations (40%) correspond to only eight exonic and intronic mutations. Patients with two nonsense/out of frame/splice-site mutations showed significant loss of calpain-3 protein as compared to those with two missense/inframe mutations (P = 0.04). We observed a slow progression of disease and less severity in our patients compared to European population. Rarely, presenting clinical features were atypical, and mimicked other muscle diseases like FSHMD, distal myopathy and metabolic myopathies. CONCLUSION: This is first systematic study to characterize the genetic framework of LGMDR1 in the Indian population. Preliminary calpain-3 immunoblot screening serves well to direct genetic testing. Our findings prioritized nine CAPN3 exons for LGMDR1 diagnosis in our population; therefore, a targeted-sequencing panel of nine exons could serve well for genetic diagnosis, carrier testing, counseling and clinical trial feasibility study in LGMDR1 patients in India.

Costameric integrin and sarcoglycan protein levels are altered in a Drosophila model for Limb-girdle muscular dystrophy type 2H.

Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult, as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. We previously established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. Here we show that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N, and 520fs) induce myofibril abnormalities, altered nuclear morphology, and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, we also report for the first time that the protein levels of ßPS integrin and sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant aberrantly accumulate α- and ß-dystroglycan and α-sarcoglycan. We speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration.

Myopathies with finger flexor weakness: Not only inclusion-body myositis.

Muscle disorders are characterized by differential involvement of various muscle groups. Among these, weakness predominantly affecting finger flexors is an uncommon pattern, most frequently found in sporadic inclusion-body myositis. This finding is particularly significant when the full range of histopathological findings of inclusion-body myositis is not found on muscle biopsy. Prominent finger flexor weakness, however, is also observed in other myopathies. It occurs commonly in myotonic dystrophy types 1 and 2. In addition, individual reports and small case series have documented finger flexor weakness in sarcoid and amyloid myopathy, and in inherited myopathies caused by ACTA1, CRYAB, DMD, DYSF, FLNC, GAA, GNE, HNRNPDL, LAMA2, MYH7, and VCP mutations. Therefore, the finding of finger flexor weakness requires consideration of clinical, myopathological, genetic, electrodiagnostic, and sometimes muscle imaging findings to establish a diagnosis.

N-Acetylcysteine Reduces Skeletal Muscles Oxidative Stress and Improves Grip Strength in Dysferlin-Deficient Bla/J Mice.

Dysferlinopathy is an autosomal recessive muscular dystrophy resulting from mutations in the dysferlin gene. Absence of dysferlin in the sarcolemma and progressive muscle wasting are hallmarks of this disease. Signs of oxidative stress have been observed in skeletal muscles of dysferlinopathy patients, as well as in dysferlin-deficient mice. However, the contribution of the redox imbalance to this pathology and the efficacy of antioxidant therapy remain unclear. Here, we evaluated the effect of 10 weeks diet supplementation with the antioxidant agent N-acetylcysteine (NAC, 1%) on measurements of oxidative damage, antioxidant enzymes, grip strength and body mass in 6 months-old dysferlin-deficient Bla/J mice and wild-type (WT) C57 BL/6 mice. We found that quadriceps and gastrocnemius muscles of Bla/J mice exhibit high levels of lipid peroxidation, protein carbonyls and superoxide dismutase and catalase activities, which were significantly reduced by NAC supplementation. By using the Kondziela's inverted screen test, we further demonstrated that NAC improved grip strength in dysferlin deficient animals, as compared with non-treated Bla/J mice, without affecting body mass. Together, these results indicate that this antioxidant agent improves skeletal muscle oxidative balance, as well as muscle strength and/or resistance to fatigue in dysferlin-deficient animals.

Early pathological signs in young dysf-/- mice are improved by halofuginone.

Dysferlinopathies are a non-lethal group of late-onset muscular dystrophies. Here, we evaluated the fusion ability of primary myoblasts from young dysf-/- mice and the muscle histopathology prior to, and during early stages of disease onset. The ability of primary myoblasts of 5-week-old dysf-/- mice to form large myotubes was delayed compared to their wild-type counterparts, as evaluated by scanning electron microscopy. However, their fusion activity, as reflected by the presence of actin filaments connecting several cells, was enhanced by the antifibrotic drug halofuginone. Early dystrophic signs were already apparent in 4-week-old dysf-/- mice; their collagen level was double that in wild-type mice and continued to rise until 5 months of age. Continuous treatment with halofuginone from 4 weeks to 5 months of age reduced muscle fibrosis in a phosphorylated-Smad3 inhibition-related manner. Halofuginone also enhanced myofiber hypertrophy, reduced the percentage of centrally nucleated myofibers, and increased muscle performance. Together, the data suggest an inhibitory effect of halofuginone on the muscle histopathology at very early stages of dysferlinopathy, and enhancement of muscle performance. These results offer new opportunities for early pharmaceutical treatment in dysferlinopathies with favorable outcomes at later stages of life.

The effects of concentric and eccentric training in murine models of dysferlin-associated muscular dystrophy.

INTRODUCTION: Dysferlin-deficient murine muscle sustains severe damage after repeated eccentric contractions. METHODS: With a robotic dynamometer, we studied the response of dysferlin-sufficient and dysferlin-deficient mice to 12 weeks of concentrically or eccentrically biased contractions. We also studied whether concentric contractions before or after eccentric contractions reduced muscle damage in dysferlin-deficient mice. RESULTS: After 12 weeks of concentric training, there was no net gain in contractile force in dysferlin-sufficient or dysferlin-deficient mice, whereas eccentric training produced a net gain in force in both mouse strains. However, eccentric training induced more muscle damage in dysferlin-deficient vs dysferlin-sufficient mice. Although concentric training produced minimal muscle damage in dysferlin-deficient mice, it still led to a prominent increase in centrally nucleated fibers. Previous exposure to concentric contractions conferred slight protection on dysferlin-deficient muscle against damage from subsequent injurious eccentric contractions. DISCUSSION: Concentric contractions may help dysferlin-deficient muscle derive the benefits of exercise without inducing damage.

Longitudinal functional and imaging outcome measures in FKRP limb-girdle muscular dystrophy.

BACKGROUND: Pathogenic variants in the FKRP gene cause impaired glycosylation of α-dystroglycan in muscle, producing a limb-girdle muscular dystrophy with cardiomyopathy. Despite advances in understanding the pathophysiology of FKRP-associated myopathies, clinical research in the limb-girdle muscular dystrophies has been limited by the lack of normative biomarker data to gauge disease progression. METHODS: Participants in a phase 2 clinical trial were evaluated over a 4-month, untreated lead-in period to evaluate repeatability and to obtain normative data for timed function tests, strength tests, pulmonary function, and body composition using DEXA and whole-body MRI. Novel deep learning algorithms were used to analyze MRI scans and quantify muscle, fat, and intramuscular fat infiltration in the thighs. T-tests and signed rank tests were used to assess changes in these outcome measures. RESULTS: Nineteen participants were observed during the lead-in period for this trial. No significant changes were noted in the strength, pulmonary function, or body composition outcome measures over the 4-month observation period. One timed function measure, the 4-stair climb, showed a statistically significant difference over the observation period. Quantitative estimates of muscle, fat, and intramuscular fat infiltration from whole-body MRI corresponded significantly with DEXA estimates of body composition, strength, and timed function measures. CONCLUSIONS: We describe normative data and repeatability performance for multiple physical function measures in an adult FKRP muscular dystrophy population. Our analysis indicates that deep learning algorithms can be used to quantify healthy and dystrophic muscle seen on whole-body imaging. TRIAL REGISTRATION: This study was retrospectively registered in clinicaltrials.gov (NCT02841267) on July 22, 2016 and data supporting this study has been submitted to this registry.

Self-reported physical activity in people with limb-girdle muscular dystrophy and Charcot-Marie-Tooth disease in Norway.

BACKGROUND: Physical activity is associated with positive health effects, but individuals with neuromuscular disease (NMD) may experience constraints being physically active. There is a gap in the literature on the activity level of people with NMDs, and therefore we did a study to determine the physical activity level in people with Limb-Girdle muscular dystrophy (LGMD) and Charcot-Marie-Tooth disease (CMT). METHODS: This study used a cross-sectional design to obtain self-reported physical activity and sitting time among individuals with LGMD and CMT who were recruited from the Norwegian registry for hereditary and congenital neuromuscular diseases. RESULTS: A total of 127 respondents who filled out questionnaires about either physical activity or sitting time were included in the analysis. Seventy (55.1%) had a diagnosis of CMT and 57 (44.9%) had a diagnosis of LGMD. Seventy-three (57.5%) respondents were female and 54 (42.5%) were male. Among the 108 respondents with available physical activity data, 44.4% reported being physically inactive. Among the 109 respondents with available sitting time data, the average sitting time was 8.6 h. Longer sitting time was associated with higher physical inactivity. CONCLUSION: Among people with LGMD and CMT in our study, 55.6% reported being physically active. Respondents with LGMD and CMT reported longer sitting time and less physical activity compared with healthy respondents in other studies. Further research should explore variables and measures that can promote physical activity among people with neuromuscular conditions.

Global FKRP Registry: observations in more than 300 patients with Limb Girdle Muscular Dystrophy R9.

OBJECTIVE: The Global FKRP Registry is a database for individuals with conditions caused by mutations in the Fukutin-Related Protein (FKRP) gene: limb girdle muscular dystrophy R9 (LGMDR9, formerly LGMD2I) and congenital muscular dystrophies MDC1C, Muscle-Eye-Brain Disease and Walker-Warburg Syndrome. The registry seeks to further understand the natural history and prevalence of FKRP-related conditions; aid the rapid identification of eligible patients for clinical studies; and provide a source of information to clinical and academic communities. METHODS: Registration is patient-initiated through a secure online portal. Data, reported by both patients and their clinicians, include: age of onset, presenting symptoms, family history, motor function and muscle strength, respiratory and cardiac function, medication, quality of life and pain. RESULTS: Of 663 registered participants, 305 were genetically confirmed LGMDR9 patients from 23 countries. A majority of LGMDR9 patients carried the common mutation c.826C > A on one or both alleles; 67.9% were homozygous and 28.5% were compound heterozygous for this mutation. The mean ages of symptom onset and disease diagnosis were higher in individuals homozygous for c.826C > A compared with individuals heterozygous for c.826C > A. This divergence was replicated in ages of loss of running ability, wheelchair-dependence and ventilation assistance; consistent with the milder phenotype associated with individuals homozygous for c.826C > A. In LGMDR9 patients, 75.1% were currently ambulant and 24.6%, nonambulant (unreported in 0.3%). Cardiac impairment was reported in 23.2% (30/129). INTERPRETATION: The Global FKRP Registry enables the collection of patient natural history data, which informs academics, healthcare professionals and industry. It represents a trial-ready cohort of individuals and is centrally placed to facilitate recruitment to clinical studies.

A novel heterozygous mutation in the C-terminal region of HSPB8 leads to limb-girdle rimmed vacuolar myopathy.

Mutations in heat shock protein B8 were initially identified in inherited neuropathies and were more recently found to cause a predominantly distal myopathy with myofibrillar pathology and rimmed vacuoles. Rare patients also had proximal weakness. Only very few pathogenic variants have been identified in HSPB8. Disruption of the chaperone activity of heat shock protein B8 impairs chaperone-assisted selective autophagy and results in protein aggregation. We report a 23-year-old patient who presented with a 4-year history of predominantly proximal lower limb weakness due to a novel variant in HSPB8. The creatine kinase level was mildly elevated. Electrodiagnostic studies demonstrated a proximal-predominant myopathy without evidence of neuropathy, and muscle histopathology revealed rimmed vacuoles and myofibrillar protein aggregates. Whole exome sequencing identified a de novo frameshift variant in the C-terminal region of HSPB8 (c.577_580dupGTCA, p.Thr194Serfs*23). This case demonstrates that HSPB8-related disorders can present with early onset limb-girdle myopathy without associated neuropathy.

Clinical and genetic characterization of limb girdle muscular dystrophy R7 telethonin-related patients from three unrelated Chinese families.

Limb girdle muscular dystrophy LGMD R7 telethonin-related is a rare autosomal recessive muscle disorder characterized by proximal muscle weakness of pelvic and shoulder girdles. Mutation in TCAP is responsible for LGMD R7, and the disease has a wide geographic distribution in diverse populations, but genotype-phenotype relationships remain unclear. We collected 5 LGMD R7 patients from three unrelated Chinese families. The average onset age was 16 ± 1.41; the initial symptoms included progressive proximal muscle weakness in limbs, difficulty in fast running, and asymmetric muscle atrophy in calves. Muscle MR imaging showed varying severity of fatty infiltration in the pelvic girdle, thigh, and calf muscles, and the severity of muscle infiltration was related to the length of the disease course. Muscle histopathology revealed aberrantly sized muscle fibers, internal nuclei, split fibers, rimmed vacuoles, monocyte invasion, and necrotic fibers. Sequencing identified one novel and one previously reported TCAP mutation. Our study extends the known distribution of this rare muscular dystrophy and presents the first detailed clinical and genetic characterizations of LGMD R7 cases from the Chinese population. Our work expands the mutation spectrum known for LGMD R7 and emphasizes the need for clinicians to consider TCAP mutations when evaluating patients with symptoms of limb girdle muscular dystrophy.

Cardiac Intervention Improves Heart Disease and Clinical Outcomes in Patients With Muscular Dystrophy in a Multidisciplinary Care Setting.

Background Patients with muscular dystrophy (MD) represent a vulnerable patient population with no clearly defined care model in modern-day clinical practice to manage a high burden of heart disease and comorbidities. We demonstrate the effectiveness of cardiac interventions, namely the initiation and optimization of medical and device therapies, as part of a multidisciplinary care approach to improve clinical outcomes in patients with MD. Methods and Results We conducted a prospective cohort study at the Neuromuscular Multidisciplinary clinic following patients with dystrophinopathies, limb-girdle MD, type 1 myotonic dystrophy, and facioscapulohumeral MD. A negative control group classified as non-MD myopathies without heart disease, was also tracked. Our cohort of 185 patients (median age: 42 years; 79 [42.7%] women), included 145 patients with MD. Cardiomyopathy was present in 65.6% of the patients with dystrophinopathies (21 of 32) and 27.3% of the patients with limb-girdle MD (9 of 33). Conduction abnormalities were common in type 1 myotonic dystrophy (33.3% [20/60] patients). Cardiac intervention reversed systolic dysfunction, with left ventricular ejection fraction improving from 43% to 50.0% over a 3-year period. A sustained reduction in healthcare utilization was also observed. The number of outpatient clinic visits decreased from 3.0 to 1.5 visits per year, the duration of hospitalizations was reduced from 14.2 to 0.9 days per year, and the number of cardiac-related hospitalizations decreased from 0.4 to 0.1 hospitalizations per year associated with low mortality. Conclusions Our study demonstrates that cardiac intervention as part of a comprehensive multidisciplinary care approach to treating patients with MD leads to a sustained improvement in clinical outcomes.

European muscle MRI study in limb girdle muscular dystrophy type R1/2A (LGMDR1/LGMD2A).

BACKGROUND: Limb girdle muscular dystrophy type R1/2A (LGMDR1/LGMD2A) is a progressive myopathy caused by deficiency of calpain 3, a calcium-dependent cysteine protease of skeletal muscle, and it represents the most frequent type of LGMD worldwide. In the last few years, muscle magnetic resonance imaging (MRI) has been proposed as a tool for identifying patterns of muscular involvement in genetic disorders and as a biomarker of disease progression in muscle diseases. In this study, 57 molecularly confirmed LGMDR1 patients from a European cohort (age range 7-78 years) underwent muscle MRI and a global evaluation of functional status (Gardner-Medwin and Walton score and ability to raise the arms). RESULTS: We confirmed a specific pattern of fatty substitution involving predominantly the hip adductors and hamstrings in lower limbs. Spine extensors were more severely affected than spine rotators, in agreement with higher incidence of lordosis than scoliosis in LGMDR1. Hierarchical clustering of lower limb MRI scores showed that involvement of anterior thigh muscles discriminates between classes of disease progression. Severity of muscle fatty substitution was significantly correlated with CAPN3 mutations: in particular, patients with no or one "null" alleles showed a milder involvement, compared to patients with two null alleles (i.e., predicting absence of calpain-3 protein). Expectedly, fat infiltration scores strongly correlated with functional measures. The "pseudocollagen" sign (central areas of sparing in some muscle) was associated with longer and more severe disease course. CONCLUSIONS: We conclude that skeletal muscle MRI represents a useful tool in the diagnostic workup and clinical management of LGMDR1.

Phenotypic and genetic spectrum of patients with limb-girdle muscular dystrophy type 2A from Serbia.

Limb-girdle muscular dystrophy (LGMD) type 2A (calpainopathy) is an autosomal recessive disease caused by mutation in the CAPN3 gene. The aim of this study was to examine genetic and phenotypic features of Serbian patients with calpainopathy. The study comprised 19 patients with genetically confirmed calpainopathy diagnosed at the Neurology Clinic, Clinical Center of Serbia and the Clinic for Neurology and Psychiatry for Children and Youth in Belgrade, Serbia during a ten-year period. Eighteen patients in this cohort had c.550delA mutation, with nine of them being homozygous. In majority of the patients, disease started in childhood or early adulthood. The disease affected shoulder girdle - upper arm and pelvic girdle - thigh muscles with similar frequency, with muscles of lower extremities being more severely impaired. Facial and bulbar muscles were spared. All patients in this cohort, except two, remained ambulant. None of the patients had cardiomyopathy, while 21% showed mild conduction defects. Respiratory function was mildly impaired in 21% of patients. Standard muscle histopathology showed myopathic and dystrophic pattern. In conclusion, the majority of Serbian LGMD2A patients have the same mutation and similar phenotype.