Severe ACTA1-related nemaline myopathy: intranuclear rods, cytoplasmic bodies, and enlarged perinuclear space as characteristic pathological features on muscle biopsies.

peer reviewedNemaline 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

Caveolae and Bin1 form ring-shaped platforms for T-tubule initiation

Excitation-contraction coupling requires a highly specialized membrane structure, the triad, composed of a plasma membrane invagination, the T-tubule, surrounded by two sarcoplasmic reticulum terminal cisternae. Although the precise mechanisms governing T-tubule biogenesis and triad formation remain largely unknown, studies have shown that caveolae participate in T-tubule formation and mutations of several of their constituents induce muscle weakness and myopathies. Here, we demonstrate that, at the plasma membrane, Bin1 and caveolae composed of caveolin-3 assemble into ring-like structures from which emerge tubes enriched in the dihydropyridine receptor. Bin1 expression lead to the formation of both rings and tubes and we show that Bin1 forms scaffolds on which caveolae accumulate to form the initial T-tubule. Cav3 deficiency caused by either gene silencing or pathogenic mutations results in defective ring formation and perturbed Bin1-mediated tubulation that may explain defective T-tubule organization in mature muscles. Our results uncover new pathophysiological mechanisms that may prove relevant to myopathies caused by Cav3 or Bin1 dysfunction

Pathogenic DPAGT1 variants in limb‐girdle congenital myasthenic syndrome (LG‐CMS) associated with tubular aggregates and ORAI1 hypoglycosylation

International audienceAbstract Aims Limb‐girdle congenital myasthenic syndrome (LG‐CMS) is a genetically heterogeneous disorder characterised by muscle weakness and fatigability. The LG‐CMS gene DPAGT1 codes for an essential enzyme of the glycosylation pathway, a posttranslational modification mechanism shaping the structure and function of proteins. In DPAGT1 ‐related LG‐CMS, decreased glycosylation of the acetylcholine receptor (AChR) reduces its localization at the neuromuscular junction (NMJ) and results in diminished neuromuscular transmission. LG‐CMS patients also show tubular aggregates on muscle biopsy, but the origin and potential contribution of the aggregates to disease development are not understood. Here, we describe two LG‐CMS patients with the aim of providing a molecular diagnosis and to shed light on the pathways implicated in tubular aggregate formation. Methods Following clinical examination of the patients, we performed next‐generation sequencing (NGS) to identify the genetic causes, analysed the biopsies at the histological and ultrastructural levels, investigated the composition of the tubular aggregates and performed experiments on protein glycosylation. Results We identified novel pathogenic DPAGT1 variants in both patients and pyridostigmine treatment quantitatively improved muscle force and function. The tubular aggregates contained proteins of the sarcoplasmic reticulum (SR) and structurally conformed to the aggregates observed in tubular aggregate myopathy (TAM). TAM arises from overactivation of the plasma membrane calcium channel ORAI1, and functional studies on muscle extracts from our LG‐CMS patients evidenced abnormal ORAI1 glycosylation. Conclusions We expand the genetic variant spectrum of LG‐CMS and provide a genotype/phenotype correlation for pathogenic DPAGT1 variants. The discovery of ORAI1 hypoglycosylation in our patients highlights a physiopathological link between LG‐CMS and TAM

‘Dusty core disease’ (DuCD): expanding morphological spectrum of RYR1 recessive myopathies

International audienceSeveral 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

NEM6, KBTBD13-Related Congenital Myopathy: Myopathological Analysis in 18 Dutch Patients Reveals Ring Rods Fibers, Cores, Nuclear Clumps, and Granulo-Filamentous Protein Material

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

ACTN2 mutations cause "Multiple structured Core Disease" (MsCD)

The identification of genes implicated in myopathies is essential for diagnosis and for revealing novel therapeutic targets. Here we characterize a novel subclass of congenital myopathy at the morphological, molecular, and functional level. Through exome sequencing, we identified de novo ACTN2 mutations, a missense and a deletion, in two unrelated patients presenting with progressive early-onset muscle weakness and respiratory involvement. Morphological and ultrastructural analyses of muscle biopsies revealed a distinctive pattern with the presence of muscle fibers containing small structured cores and jagged Z-lines. Deeper analysis of the missense mutation revealed mutant alpha-actinin-2 properly localized to the Z-line in differentiating myotubes and its level was not altered in muscle biopsy. Modelling of the disease in zebrafish and mice by exogenous expression of mutated alpha-actinin-2 recapitulated the abnormal muscle function and structure seen in the patients. Motor deficits were noted in zebrafish, and muscle force was impaired in isolated muscles from AAV-transduced mice. In both models, sarcomeric disorganization was evident, while expression of wild-type alpha-actinin-2 did not result in muscle anomalies. The murine muscles injected with mutant ACTN2 displayed cores and Z-line defects. Dominant ACTN2 mutations were previously associated with cardiomyopathies, and our data demonstrate that specific mutations in the well-known Z-line regulator alpha-actinin-2 can cause a skeletal muscle disorder

QIL1 mutation causes MICOS disassembly and early onset fatal mitochondrial encephalopathy with liver disease

International audiencePreviously, we identified QIL1 as a subunit of mitochondrial contact site (MICOS) complex and demonstrated a role for QIL1 in MICOS assembly, mitochondrial respiration, and cristae formation critical for mitochondrial architecture (Guarani et al., 2015). Here, we identify QIL1 null alleles in two siblings displaying multiple clinical symptoms of early-onset fatal mitochondrial encephalopathy with liver disease, including defects in respiratory chain function in patient muscle. QIL1 absence in patients' fibroblasts was associated with MICOS disassembly, abnormal cristae, mild cytochrome c oxidase defect, and sensitivity to glucose withdrawal. QIL1 expression rescued cristae defects, and promoted re-accumulation of MICOS subunits to facilitate MICOS assembly. MICOS assembly and cristae morphology were not efficiently rescued by over-expression of other MICOS subunits in patient fibroblasts. Taken together, these data provide the first evidence of altered MICOS assembly linked with a human mitochondrial disease and confirm a central role for QIL1 in stable MICOS complex formation

Deep morphological analysis of muscle biopsies from type III glycogenesis (GSDIII), debranching enzyme deficiency, revealed stereotyped vacuolar myopathy and autophagy impairment

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Loss of Sarcomeric Scaffolding as a Common Baseline Histopathologic Lesion in Titin-Related Myopathies

International audienceTitin-related myopathies are heterogeneous clinical conditions associated with mutations in TTN. To define their histopathologic boundaries and try to overcome the difficulty in assessing the pathogenic role of TTN variants, we performed a thorough morphological skeletal muscle analysis including light and electron microscopy in 23 patients with different clinical phenotypes presenting pathogenic autosomal dominant or autosomal recessive (AR) mutations located in different TTN domains. We identified a consistent pattern characterized by diverse defects in oxidative staining with prominent nuclear internalization in congenital phenotypes (AR-CM) (n = 10), ± necrotic/regenerative fibers, associated with endomysial fibrosis and rimmed vacuoles (RVs) in AR early-onset Emery-Dreifuss-like (AR-ED) (n = 4) and AR adult-onset distal myopathies (n = 4), and cytoplasmic bodies (CBs) as predominant finding in hereditary myopathy with early respiratory failure (HMERF) patients (n = 5). Ultrastructurally, the most significant abnormalities, particularly in AR-CM, were multiple narrow core lesions and/or clear small areas of disorganizations affecting one or a few sarcomeres with M-band and sometimes A-band disruption and loss of thick filaments. CBs were noted in some AR-CM and associated with RVs in HMERF and some AR-ED cases. As a whole, we described recognizable histopathological patterns and structural alterations that could point toward considering the pathogenicity of TTN mutations

Severe ACTA1-related nemaline myopathy: intranuclear rods, cytoplasmic bodies, and enlarged perinuclear space as characteristic pathological features on muscle biopsies

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.SCOPUS: ar.jinfo:eu-repo/semantics/publishe