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The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70-90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.

Mutations in the small heat shock protein B8 gene ( HSPB8/HSP22 ) have been associated with distal hereditary motor neuropathy, Charcot–Marie–Tooth disease, and recently distal myopathy. It is so far not clear how mutant HSPB8 induces the neuronal and muscular phenotypes and if a common pathogenesis lies behind these diseases. Growing evidence points towards a role of HSPB8 in chaperone-associated autophagy, which has been shown to be a determinant for the clearance of poly-glutamine aggregates in neurodegenerative diseases but also for the maintenance of skeletal muscle myofibrils. To test this hypothesis and better dissect the pathomechanism of mutant HSPB8, we generated a new transgenic mouse model leading to the expression of the mutant protein (knock-in lines) or the loss-of-function (functional knock-out lines) of the endogenous protein Hspb8. While the homozygous knock-in mice developed motor deficits associated with degeneration of peripheral nerves and severe muscle atrophy corroborating patient data, homozygous knock-out mice had locomotor performances equivalent to those of wild-type animals. The distal skeletal muscles of the post-symptomatic homozygous knock-in displayed Z-disk disorganisation, granulofilamentous material accumulation along with Hspb8, αB-crystallin (HSPB5/CRYAB), and desmin aggregates. The presence of the aggregates correlated with reduced markers of effective autophagy. The sciatic nerve of the homozygous knock-in mice was characterized by low autophagy potential in pre-symptomatic and Hspb8 aggregates in post-symptomatic animals. On the other hand, the sciatic nerve of the homozygous knock-out mice presented a normal morphology and their distal muscle displayed accumulation of abnormal mitochondria but intact myofiber and Z-line organisation. Our data, therefore, suggest that toxic gain-of-function of mutant Hspb8 aggregates is a major contributor to the peripheral neuropathy and the myopathy. In addition, mutant Hspb8 induces impairments in autophagy that may aggravate the phenotype.

Background Distinguishing between distal myopathies (DMs) and distal hereditary motor neuropathies (dHMNs) can be challenging because clinical, EMG and biopsy findings sometimes overlap. This study aims to identify distinctive muscle MRI features that can guide the diagnosis. Methods We collected clinical, genetic and muscle MRI data from patients with a confirmed diagnosis of DM and dHMN. We analyzed potential MRI characteristics to distinguish these conditions and to guide molecular diagnosis, such as the texture and pattern of infiltration. Results Seventy‐eight (71.5%) patients diagnosed with DMs and thirty‐one (28.4%) with dHMNs were included. A length‐dependent pattern of muscle involvement, a distal to proximal gradient of fat replacement along the length of the muscles and severe and widespread involvement of foot muscles were more common in patients with dHMNs. Muscle hypertrophy and asymmetry were more frequently observed in the DMs. A reticular pattern of fat infiltration was exclusive to patients with dHMNs, while the moth‐eaten pattern predominated in DMs. Muscle islands were more commonly identified in dHMNs (54.8%) but were also observed in 32% of patients with DMs. Conclusions Analysis of MRI features can help distinguish between DMs and dHMNs. A reticular pattern is an early feature of dHMNs while muscle islands are identified in advanced stages and in some forms of DMs, though not specific to neurogenic conditions. We recommend including foot muscles in the MRI protocol as they show extensive involvement in most dHMNs, while in DMs their involvement correlates with greater fatty infiltration of lower leg muscles. This study assesses muscle MRI features for the differential diagnosis of patients with distal myopathies and distal hereditary motor neuropathies (dHMNs). A reticular pattern of fat infiltration, together with diffuse and marked involvement of intrinsic foot muscles, emerged as characteristic of dHMNs. Muscle islands, previously described as a distinguishing feature of neurogenic conditions, were predominantly observed in dHMNs but were also present in up to one‐third of patients with distal myopathies. Additional features suggestive of neurogenic disorders included a distal‐to‐proximal gradient or a length‐dependent pattern, whereas asymmetric involvement and hypertrophy were more frequently associated with distal myopathies. The severity of muscle fat replacement correlated with both functional disability and disease duration. These findings support the use of muscle MRI as a valuable tool for distinguishing distal myopathies from dHMNs and for clinical monitoring of patients affected by these conditions.

Introduction GNE myopathy is a rare autosomal recessive hereditary myopathy resulting in impaired sialic acid biosynthesis. The features of this condition include distal muscle weakness with relatively preserved quadriceps femoris strength and the presence of rimmed vacuoles in muscle fibers. Methods We performed a retrospective clinical analysis of patients diagnosed with GNE myopathy at our hospital from 2017 to 2024. Result All six patients exhibited weakness in the distal lower limbs. Rimmed vacuoles were observed in the muscle fibers of five patients. Additionally, several relatively uncommon clinical manifestations were identified in this study, including Beevor’s sign, respiratory dysfunction, ragged red fibers, and elevated acid phosphatase enzyme activity. Genetic analysis revealed five novel variants in the GNE gene: c.1691G > T (p.G564V), c.1877 C > G (p.A626G), c.935_936insAA (p.H312fs), c.1247 A > T (p.K416M), and c.974 C > T (p.A325V). Furthermore, three patients carried the hotspot variant p.D207V. Conclusion The clinical and histopathological findings underscore the distinctive characteristics and heterogeneity of the disease. Significantly, this study broadens the mutational spectrum of GNE myopathy by identifying five novel variants.

Proline substitutions within the coiled-coil rod region of the β-myosin gene (MYH7) are the predominant mutations causing Laing distal myopathy (MPD1), an autosomal dominant disorder characterized by progressive weakness of distal/proximal muscles. We report that the MDP1 mutation R1500P, studied in what we believe to be the first mouse model for the disease, adversely affected myosin motor activity despite being in the structural rod domain that directs thick filament assembly. Contractility experiments carried out on isolated mutant muscles, myofibrils, and myofibers identified muscle fatigue and weakness phenotypes, an increased rate of actin-myosin detachment, and a conformational shift of the myosin heads toward the more reactive disordered relaxed (DRX) state, causing hypercontractility and greater ATP consumption. Similarly, molecular analysis of muscle biopsies from patients with MPD1 revealed a significant increase in sarcomeric DRX content, as observed in a subset of myosin motor domain mutations causing hypertrophic cardiomyopathy. Finally, oral administration of MYK-581, a small molecule that decreases the population of heads in the DRX configuration, significantly improved the limited running capacity of the R1500P-transgenic mice and corrected the increased DRX state of the myofibrils from patients. These studies provide evidence of the molecular pathogenesis of proline rod mutations and lay the groundwork for the therapeutic advancement of myosin modulators.

The myosin inhibitor mavacamten has transformed the management of obstructive hypertrophic cardiomyopathy (HCM) by targeting myosin ATPase activity to mitigate cardiac hypercontractility. This therapeutic mechanism has proven effective for patients with HCM independent of having a primary gene mutation in myosin. In this issue of the JCI, Buvoli et al. report that muscle hypercontractility is a mechanism of pathogenesis underlying muscle dysfunction in Laing distal myopathy, a disorder characterized by mutations altering the rod domain of β myosin heavy chain. The authors performed detailed physiological, molecular, and biomechanical analyses and demonstrated that myosin ATPase inhibition can correct a large extent of muscle abnormalities. The findings offer a therapeutic avenue for Laing distal myopathy and potentially other myopathies. This Commentary underscores the importance of reevaluating myosin activity's role across myopathies in general for the potential development of targeted myosin inhibitors to treat skeletal muscle disorders.

Using deep phenotyping and high-throughput sequencing, we have identified a novel type of distal myopathy caused by mutations in the Small muscle protein X-linked (SMPX) gene. Four different missense mutations were identified in ten patients from nine families in five different countries, suggesting that this disease could be prevalent in other populations as well. Haplotype analysis of patients with similar ancestry revealed two different founder mutations in Southern Europe and France, indicating that the prevalence in these populations may be higher. In our study all patients presented with highly similar clinical features: adult-onset, usually distal more than proximal limb muscle weakness, slowly progressing over decades with preserved walking. Lower limb muscle imaging showed a characteristic pattern of muscle involvement and fatty degeneration. Histopathological and electron microscopic analysis of patient muscle biopsies revealed myopathic findings with rimmed vacuoles and the presence of sarcoplasmic inclusions, some with amyloid-like characteristics. In silico predictions and subsequent cell culture studies showed that the missense mutations increase aggregation propensity of the SMPX protein. In cell culture studies, overexpressed SMPX localized to stress granules and slowed down their clearance.

GNE myopathy is an ultra-rare autosomal recessive disease, which starts as a distal muscle weakness and ultimately leads to a wheelchair bound state. Molecular research and animal modelling significantly moved forward understanding of GNE myopathy mechanisms and suggested therapeutic interventions to alleviate the symptoms. Multiple therapeutic attempts are being made to supplement sialic acid depleted in GNE myopathy muscle cells. Translational research field provided valuable knowledge through natural history studies, patient registries and clinical trial, which significantly contributed to bringing forward an era of GNE myopathy treatment. In this review, we are summarising current GNE myopathy, scientific trends and open questions, which would be of significant interest for a wide neuromuscular diseases community.

BackgroundInherited skeletal muscle diseases are genetically heterogeneous diseases caused by mutations in more than 150 genes. This has made it challenging to establish a high-throughput screening method for identifying causative gene mutations in clinical practice.AimIn the present study, we developed a useful method for screening gene mutations associated with the pathogenesis of skeletal muscle diseases.MethodsWe established four target gene panels, each covering all exonic and flanking regions of genes involved in the pathogenesis of the following muscle diseases: (1) muscular dystrophy (MD), (2) congenital myopathy/congenital myasthenic syndrome, (3) metabolic myopathy and (4) myopathy with protein aggregations/rimmed vacuoles. We assigned one panel to each patient based on the results of clinical and histological analyses of biopsied muscle samples and performed high-throughput sequencing by using Ion PGM next-generation sequencer. We also performed protein analysis to confirm defective proteins in patients with major muscular dystrophies. Further, we performed muscle-derived cDNA analysis to identify splice-site mutations.ResultsWe identified possible causative gene mutations in 33% of patients (62/188) included in this study. Our results showed that the MD panel was the most useful, with a diagnostic rate of 46.2%.ConclusionsThus, we developed a high-throughput sequencing technique for diagnosing inherited muscle diseases. The use of this technique along with histological and protein analyses may be useful and cost-effective for screening mutations in patients with inherited skeletal muscle diseases.

A novel FLNC c.5161delG (p.Gly1722ValfsTer61) mutation was identified in two members of a French family affected by distal myopathy and in one healthy relative. This FLNC c.5161delG mutation is one nucleotide away from a previously reported FLNC mutation (c.5160delC) that was identified in patients and in asymptomatic carriers of three Bulgarian families with distal muscular dystrophy, indicating a low penetrance of the FLNC frameshift mutations. Given these similarities, we believe that the two FLNC mutations alone can be causative of distal myopathy without full penetrance. Moreover, comparative analysis of the clinical manifestations indicates that patients of the French family show an earlier onset and a complete segregation of the disease. As a possible explanation of this, the two French patients also carry a OBSCN c.13330C>T (p.Arg4444Trp) mutation. The p.Arg4444Trp variant is localized within the OBSCN Ig59 domain that, together with Ig58, binds to the ZIg9/ZIg10 domains of titin at Z-disks. Structural and functional studies indicate that this OBSCN p.Arg4444Trp mutation decreases titin binding by ~15-fold. On this line, we suggest that the combination of the OBSCN p.Arg4444Trp variant and of the FLNC c.5161delG mutation, can cooperatively affect myofibril stability and increase the penetrance of muscular dystrophy in the French family.