Neuromuscular disorders (NMD) with neonatal or early infantile onset are usually severe and differ in symptoms, complications, and treatment options. The establishment of a diagnosis relies on the combination of clinical examination, morphological analyses of muscle biopsies, and genetic investigations. Here, we re-evaluated and classified a unique collection of 535 muscle biopsies from NMD infants aged 0-6 months examined over a period of 52 years. We aimed to assess the importance and contribution of morphological muscle biopsy analyses for the establishment of a precise and accurate molecular diagnosis. Altogether, 82% of the biopsies showed typical structural myofiber anomalies highly suggestive of specific NMD classes (congenital myopathies, metabolic myopathies, lower motor neuron (LMN) and neuromuscular junction (NMJ) disorders, muscular dystrophies, inflammatory myopathies), while the remaining 18% showed no or only non-specific histological abnormalities. The diagnostic success rate differed among the NMD classes and was particularly high for congenital myopathies as illustrated by the identification of causative genes in 61% of cases. This is essentially due to the presence of characteristic histopathological hallmarks on biopsies visible by light or electron microscopy often pointing to specific genes. In contrast, metabolic myopathies commonly displayed non-specific features on muscle sections, led to the identification of causative genes in only 19% of the patients, and typically required additional enzymatic tests to establish a more precise diagnosis. The evolution of sequencing technologies fundamentally improved molecular diagnosis and also shifted the relevance of muscle biopsies within the diagnostic process. Depending on the clinical presentation of the patients, direct gene or panel sequencing may be the preferred method nowadays. However, histological and ultrastructural examinations of muscle sections are still frequently useful and can constitute an elemental step in the diagnostic process-either by directing purposeful gene sequencing or pointing to genes and pathogenic variants identified by next-generation sequencing (NGS), or by complementing clinical findings and biochemical analysis methods.

In a neuropathological series of 20 COVID-19 cases, we analyzed six cases (three biopsies and three autopsies) with multiple foci predominantly affecting the white matter as shown by MRI. The cases presented with microhemorrhages evocative of small artery diseases. This COVID-19 associated cerebral microangiopathy (CCM) was characterized by perivascular changes: arterioles were surrounded by vacuolized tissue, clustered macrophages, large axonal swellings and a crown arrangement of aquaporin-4 immunoreactivity. There was evidence of blood-brain-barrier leakage. Fibrinoid necrosis, vascular occlusion, perivascular cuffing and demyelination were absent. While no viral particle or viral RNA was found in the brain, the SARS-CoV-2 spike protein was detected in the Golgi apparatus of brain endothelial cells where it closely associated with furin, a host protease known to play a key role in virus replication. Endothelial cells in culture were not permissive to SARS-CoV-2 replication. The distribution of the spike protein in brain endothelial cells differed from that observed in pneumocytes. In the latter, the diffuse cytoplasmic labeling suggested a complete replication cycle with viral release, notably through the lysosomal pathway. In contrast, in cerebral endothelial cells the excretion cycle was blocked in the Golgi apparatus. Interruption of the excretion cycle could explain the difficulty of SARS-CoV-2 to infect endothelial cells in vitro and to produce viral RNA in the brain. Specific metabolism of the virus in brain endothelial cells could weaken the cell walls and eventually lead to the characteristic lesions of COVID-19 associated cerebral microangiopathy. Furin as a modulator of vascular permeability could provide some clues for the control of late effects of microangiopathy.

Nemaline myopathy (NM) is a muscle disorder with broad clinical and genetic heterogeneity. The clinical presentation of affected individuals ranges from severe perinatal muscle weakness to milder childhood-onset forms, and the disease course and prognosis depends on the gene and mutation type. To date, 14 causative genes have been identified, and ACTA1 accounts for more than half of the severe NM cases. ACTA1 encodes α-actin, one of the principal components of the contractile units in skeletal muscle. We established a homogenous cohort of ten unreported families with severe NM, and we provide clinical, genetic, histological, and ultrastructural data. The patients manifested antenatal or neonatal muscle weakness requiring permanent respiratory assistance, and most deceased within the first months of life. DNA sequencing identified known or novel ACTA1 mutations in all. Morphological analyses of the muscle biopsy specimens showed characteristic features of NM histopathology including cytoplasmic and intranuclear rods, cytoplasmic bodies, and major myofibrillar disorganization. We also detected structural anomalies of the perinuclear space, emphasizing a physiological contribution of skeletal muscle α-actin to nuclear shape. In-depth investigations of the nuclei confirmed an abnormal localization of lamin A/C, Nesprin-1, and Nesprin-2, forming the main constituents of the nuclear lamina and the LINC complex and ensuring nuclear envelope integrity. To validate the relevance of our findings, we examined muscle samples from three previously reported ACTA1 cases, and we identified the same set of structural aberrations. Moreover, we measured an increased expression of cardiac α-actin in the muscle samples from the patients with longer lifespan, indicating a potential compensatory effect. Overall, this study expands the genetic and morphological spectrum of severe ACTA1 -related nemaline myopathy, improves molecular diagnosis, highlights the enlargement of the perinuclear space as an ultrastructural hallmark, and indicates a potential genotype/phenotype correlation.

Tropomyosin 3 (TPM3) gene mutations associate with autosomal dominant and recessive nemaline myopathy 1 (NEM1), congenital fiber type disproportion myopathy (CFTD) and cap myopathy (CAPM1), and a combination of caps and nemaline bodies. We report on a 47-year-old man with polyglobulia, restricted vital capacity and mild apnea hypopnea syndrome, requiring noninvasive ventilation. Physical assessment revealed bilateral ptosis and facial paresis, with high arched palate and retrognathia; global hypotonia and diffuse axial weakness, including neck and upper and lower limb girdle and foot dorsiflexion weakness. Whole body MRI showed a diffuse fatty replacement with an unspecific pattern. A 122 gene NGS neuromuscular disorders panel revealed the heterozygous VUS c.709G>A (p.Glu237Lys) on exon 8 of TMP3. A deltoid muscle biopsy showed a novel histological pattern combining fiber type disproportion and caps. Our findings support the pathogenicity of the novel TPM3 variant and widen the phenotypic gamut of TMP3-related congenital myopathy.

Nemaline myopathy type 6 (NEM6), KBTBD13-related congenital myopathy is caused by mutated KBTBD13 protein that interacts improperly with thin filaments/actin, provoking impaired muscle-relaxation kinetics. We describe muscle morphology in 18 Dutch NEM6 patients and correlate it with clinical phenotype and pathophysiological mechanisms. Rods were found in in 85% of biopsies by light microscopy, and 89% by electron microscopy. A peculiar ring disposition of rods resulting in ring-rods fiber was observed. Cores were found in 79% of NEM6 biopsies by light microscopy, and 83% by electron microscopy. Electron microscopy also disclosed granulofilamentous protein material in 9 biopsies. Fiber type 1 predominance and prominent nuclear internalization were found. Rods were immunoreactive for α-actinin and myotilin. Areas surrounding the rods showed titin overexpression suggesting derangement of the surrounding sarcomeres. NEM6 myopathology hallmarks are prominent cores, rods including ring-rods fibers, nuclear clumps, and granulofilamentous protein material. This material might represent the histopathologic epiphenomenon of altered interaction between mutated KBTBD13 protein and thin filaments. We claim to classify KBTBD13-related congenital myopathy as rod-core myopathy.

BACKGROUND:Diaphragm pacing allows certain ventilator-dependent patients to achieve weaning from mechanical ventilation. The reference method consists in implanting intrathoracic contact electrodes around the phrenic nerve during video-assisted thoracic surgery, which involves time-consuming phrenic nerve dissection with a risk of nerve damage. Identifying a phrenic segment suitable for dissection-free implantation of electrodes would constitute progress. STUDY DESIGN:This study characterizes a free terminal phrenic segment never fully described before. We conducted a cadaver study (n = 14) and a clinical observational study during thoracic procedures (n = 54). RESULTS:A free terminal phrenic segment was observed on both sides in 100% of cases, "jumping" from the pericardium to the diaphragm and measuring 60 mm [95% confidence interval; 48-63] and 72.5 mm [65-82] (right left, respectively; p = 0.0038; cadaver study). This segment rolled up on itself at end-expiration and became unravelled and elongated with diaphragm descent (clinical study). Three categories of fat pads were defined (type 1: pericardiophrenic bundle free of surrounding fat; type 2: single fatty fringe leaving the phrenic nerve visible until diaphragmatic entry; type 3: multiple fatty fringes masking the site of penetration of the phrenic nerve) that depended on body mass index (p = 0.001, clinical study). Hematoxylin-eosin and toluidine blue staining (cadaver study) showed that all of the phrenic fibers in the distal, pre-branching part of the terminal segment were contained within a single epineurium containing a variable number of fascicles (right: 1 [95%CI 0.65-4.01]; left 5 [3.37-7.63]; p = 0.03). CONCLUSION:Diaphragm pacing through periphrenic electrodes positioned on the terminal phrenic segment should be tested.

Objective Glycogen storage diseases (GSDs) are severe human disorders resulting from abnormal glucose metabolism, and all previously described GSDs segregate as autosomal recessive or X‐linked traits. In this study, we aimed to molecularly characterize the first family with a dominant GSD. Methods We describe a dominant GSD family with 13 affected members presenting with adult‐onset muscle weakness, and we provide clinical, metabolic, histological, and ultrastructural data. We performed exome sequencing to uncover the causative gene, and functional experiments in the cell model and on recombinant proteins to investigate the pathogenic effect of the identified mutation. Results We identified a heterozygous missense mutation in PYGM segregating with the disease in the family. PYGM codes for myophosphorylase, the enzyme catalyzing the initial step of glycogen breakdown. Enzymatic tests revealed that the PYGM mutation impairs the AMP‐independent myophosphorylase activity, whereas the AMP‐dependent activity was preserved. Further functional investigations demonstrated an altered conformation and aggregation of mutant myophosphorylase, and the concurrent accumulation of the intermediate filament desmin in the myofibers of the patients. Interpretation Overall, this study describes the first example of a dominant glycogen storage disease in humans, and elucidates the underlying pathomechanisms by deciphering the sequence of events from the PYGM mutation to the accumulation of glycogen in the muscle fibers. ANN NEUROL 2020;88:274–282.

Autosomal dominant pathogenic variants in the filamin C gene (FLNC) have been associated with myofibrillar myopathies, distal myopathies, and isolated cardiomyopathies. Mutations in different functional domains of FLNC can cause various clinical phenotypes. A novel heterozygous missense variant c.608G>A, p.(Cys203Tyr) in the actin binding domain of FLCN was found to cause an upper limb distal myopathy (MIM #614065). The muscle MRI findings are similar to those observed in FLNC-myofibrillar myopathy (MIM #609524). However, the muscle biopsy revealed >20% of muscle fibers with nemaline bodies, in addition to numerous ring fibers and a predominance of type 1 fibers. Overall, this case shows some unique and rare aspects of FLNC-myopathy constituting a new morphologic phenotype of FLNC-related myopathies.

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

Several morphological phenotypes have been associated to RYR1-recessive myopathies. We recharacterized the RYR1-recessive morphological spectrum by a large monocentric study performed on 54 muscle biopsies from a large cohort of 48 genetically confirmed patients, using histoenzymology, immunohistochemistry, and ultrastructural studies. We also analysed the level of RyR1 expression in patients' muscle biopsies. We defined "dusty cores" the irregular areas of myofibrillar disorganisation characterised by a reddish-purple granular material deposition with uneven oxidative stain and devoid of ATPase activity, which represent the characteristic lesion in muscle biopsy in 54% of patients. We named Dusty Core Disease (DuCD) the corresponding entity of congenital myopathy. Dusty cores had peculiar histological and ultrastructural characteristics compared to the other core diseases. DuCD muscle biopsies also showed nuclear centralization and type1 fibre predominance. Dusty cores were not observed in other core myopathies and centronuclear myopathies. The other morphological groups in our cohort of patients were: Central Core (CCD: 21%), Core-Rod (C&R:15%) and Type1 predominance "plus" (T1P+:10%). DuCD group was associated to an earlier disease onset, a more severe clinical phenotype and a lowest level of RyR1 expression in muscle, compared to the other groups. Variants located in the bridge solenoid and the pore domains were more frequent in DuCD patients. In conclusion, DuCD is the most frequent histopathological presentation of RYR1-recessive myopathies. Dusty cores represent the unifying morphological lesion among the DuCD pathology spectrum and are the morphological hallmark for the recessive form of disease.

Autosomal dominant limb girdle muscular dystrophy D3 HNRNPDL-related is a rare dominant myopathy caused by mutations in HNRNPDL. Only three unrelated families have been described worldwide, a Brazilian and a Chinese carrying the mutation c.1132G>A p.(Asp378Asn), and one Uruguayan with the mutation c.1132G>C p. (Asp378His), both mutations occurring in the same codon. The present study enlarges the clinical, morphological and muscle MRI spectrum of AD-HNRNPDL-related myopathies demonstrating the significant particularities of the disease. We describe two new unrelated Argentinean families, carrying the previously reported c.1132G>C p.(Asp378His) HNRNPDL mutation. There was a wide phenotypic spectrum including oligo-symptomatic cases, pure limb girdle muscle involvement or distal lower limb muscle weakness. Scapular winging was the most common finding, observed in all patients. Muscle MRIs of the thigh, at different stages of the disease, showed particular involvement of adductor magnus and vastus besides a constant preservation of the rectus femoris and the adductor longus muscles, defining a novel MRI pattern. Muscle biopsy findings were characterized by the presence of numerous rimmed vacuoles, cytoplasmic bodies, and abundant autophagic material at the histochemistry and ultrastructural levels. HNRNPDL-related LGMD D3 results in a wide range of clinical phenotypes from the classic proximal form of LGMD to a more distal phenotype. Thigh MRI suggests a specific pattern. Codon 378 of HNRNPDL gene can be considered a mutation hotspot for HNRNPDL-related myopathy. Pathologically, the disease can be classified among the autophagic rimmed vacuolar myopathies as with the other multisystem proteinopathies.

Clathrin plaques are stable features of the plasma membrane observed in several cell types. They are abundant in muscle, where they localize at costameres that link the contractile apparatus to the sarcolemma and connect the sarcolemma to the basal lamina. Here, we show that clathrin plaques and surrounding branched actin filaments form microdomains that anchor a three-dimensional desmin intermediate filament (IF) web. Depletion of clathrin plaque and branched actin components causes accumulation of desmin tangles in the cytoplasm. We show that dynamin 2, whose mutations cause centronuclear myopathy (CNM), regulates both clathrin plaques and surrounding branched actin filaments, while CNM-causing mutations lead to desmin disorganization in a CNM mouse model and patient biopsies. Our results suggest a novel paradigm in cell biology, wherein clathrin plaques act as platforms capable of recruiting branched cortical actin, which in turn anchors IFs, both essential for striated muscle formation and function.

Background: Protein aggregate myopathies (PAM) represent a group of familial or sporadic neuromuscular conditions with marked clinical and genetic heterogeneity that occur in children and adults. Familial PAM includes myofibrillar myopathies defined by the presence of desmin-positive protein aggregates and degenerative intermyofibrillar network changes. PAM is often caused by dysfunctional genes, such as DES, PLEC 1, CRYAB, FLNC, MYOT, ZASP, BAG3, FHL1, and DNAJB6. Objective: To retrospectively analyze genetic mutations and demographic, clinical, and morphological aspects of PAM in a French population. Methods: Forty-eight PAM patients (29 men, 19 women) were divided into two groups, those with genetically (GIM) and nongenetically identified (NGIM) mutations associated with myofibrillar myopathy. Results: Age of myopathy onset ranged from 13 to 68 years. GIM group mutations (81.25%) included DES (14), ZASP (8), FLNC (4), MYOT (4), BAG3 (1), CRYAB (2), and DNAJB6 (6). The MYOT subgroup displayed a significantly older onset age (p=0.029). Autosomal dominant inheritance was found in 74.3% of GIM and 44.4% of NGIM subjects. Overall, 22.9% had Maghrebian heritage, 72.9% Caucasian, and 4.2% Asian. The most common clinical sign was distal muscle weakness (66%) followed by simultaneous distal and proximal weakness in 49%. Eleven patients had contractures, one had a rigid spine, and five had respiratory dysfunction. GIM subjects had greater cardiac involvement (51.7%) versus the NGIM group (33.3%). The average serum creatine kinase level was 393U/L in GIM and 382U/L in NGIM subjects. Morphological analysis showed significant differences among GIM subgroups, including the number of vacuoles and regenerated fibers (ZASP), group atrophy (ZASP), and rubbed out fibers (MYOT). Ultrastructural findings showed significant differences in intranuclear rods, Z-disc thickness, and intranuclear inclusions between gene mutation subgroups. Paracrystalline inclusions were present in three patients (DNAJB6). The most frequent mutation in was in DES, followed by ZASP.Conclusions: GIM and NGIM PAM subjects showed similar results, suggesting that any unknown genes, which cause this disease have characteristics similar to those already described. Considering the complexity of clinical, morphological, and genetic data related to PAM, particularly myofibrillar myopathies, physicians should be careful when diagnosing patients with sporadic PAM.

Background The activating signal cointegrator 1 (ASC-1) complex acts as a transcriptional coactivator for a variety of transcription factors and consists of four subunits: ASCC1, ASCC2, ASCC3 and TRIP4. A single homozygous mutation in ASCC1 has recently been reported in two families with a severe muscle and bone disorder. Objective We aim to contribute to a better understanding of the ASCC1-related disorder. Methods Here, we provide a clinical, histological and genetic description of three additional ASCC1 families. Results All patients presented with severe prenatal-onset muscle weakness, neonatal hypotonia and arthrogryposis, and congenital bone fractures. The muscle biopsies from the affected infants revealed intense oxidative rims beneath the sarcolemma and scattered remnants of sarcomeres with enlarged Z-bands, potentially representing a histopathological hallmark of the disorder. Sequencing identified recessive nonsense or frameshift mutations in ASCC1, including two novel mutations. Conclusion Overall, this work expands the ASCC1 mutation spectrum, sheds light on the muscle histology of the disorder and emphasises the physiological importance of the ASC-1 complex in fetal muscle and bone development.

Large flat clathrin plaques are stable features of the plasma membrane associated with sites of strong adhesion suggesting that they could also play a role in force transduction. Here, we analyzed how clathrin plaques interact with the cytoskeleton and how they respond to mechanical cues in skeletal muscle myotubes. We show that branched actin networks surrounding clathrin plaques are directly regulated by dynamin 2, anchor intermediate filaments and sequester YAP at the plasma membrane. Dynamin 2, clathrin and desmin intermediate filaments are all required for basal YAP nucleocytoplasmic distribution and efficient nuclear translocation in response to mechanical stimuli. Dynamin 2 mutations that are responsible for centronuclear myopathy in humans disorganize the desmin network and deregulate YAP signaling both in vitro and in vivo . Thus, clathrin plaques and associated dynamin 2 are defined here as a new sensor conveying mechanical cues and integrate cell signaling with cytoskeletal regulation.

Few genes (RYR1, NEB, ACTA1, CFL2, KBTBD13) have been associated with core-rod congenital myopathies [7]. KBTBD13 belongs to the Kelch-repeat super-family of proteins and is implicated in the ubiquitination pathway. Dominant mutations in KBTBD13 have been associated with a peculiar form of core-rod myopathy (NEM6) so far [10]. Childhood onset, slowly progressive proximal muscle weakness with characteristic slowness of movements and combination of nemaline rods, irregular shaped cores and unusual type2 fibres hypotrophy at muscle biopsy, were the main characteristics shared in all the affected members of the four KBTBD13 families reported in the literature [12]. We report on a 65 years old patient, of Sardinian origin, with atypical clinical and morphological presentation of NEM6 due to a novel mutation in KBTBD13 gene.

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.

Mutations in GFPT1 (glutamine-fructose-6-phosphate transaminase 1), a gene encoding an enzyme involved in glycosylation of ubiquitous proteins, cause a limb-girdle congenital myasthenic syndrome (LG-CMS) with tubular aggregates (TAs) characterized predominantly by affection of the proximal skeletal muscles and presence of highly organized and remodeled sarcoplasmic tubules in patients' muscle biopsies. We report here the first long-term clinical follow-up of 11 French individuals suffering from LG-CMS with TAs due to GFPT1 mutations, of which nine are new. Our retrospective clinical evaluation stresses an evolution toward a myopathic weakness that occurs concomitantly to ineffectiveness of usual CMS treatments. Analysis of neuromuscular biopsies from three unrelated individuals demonstrates that the maintenance of neuromuscular junctions (NMJs) is dramatically impaired with loss of post-synaptic junctional folds and evidence of denervation–reinnervation processes affecting the three main NMJ components. Moreover, molecular analyses of the human muscle biopsies confirm glycosylation defects of proteins with reduced O-glycosylation and show reduced sialylation of transmembrane proteins in extra-junctional area. Altogether, these results pave the way for understanding the etiology of this rare neuromuscular disorder that may be considered as a "tubular aggregates myopathy with synaptopathy".