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

AIMS: Limb-girdle congenital myasthenic syndrome (LG-CMS) is a genetically heterogeneous disorder characterized 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, reduced 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.

Clinical, morphological and genetic characterization of Brody disease: an international study of 40 patients.

Brody disease is an autosomal recessive myopathy characterized by exercise-induced muscle stiffness due to mutations in the ATP2A1 gene. Almost 50 years after the initial case presentation, only 18 patients have been reported and many questions regarding the clinical phenotype and results of ancillary investigations remain unanswered, likely leading to incomplete recognition and consequently under-diagnosis. Additionally, little is known about the natural history of the disorder, genotype-phenotype correlations, and the effects of symptomatic treatment. We studied the largest cohort of Brody disease patients to date (n = 40), consisting of 22 new patients (19 novel mutations) and all 18 previously published patients. This observational study shows that the main feature of Brody disease is an exercise-induced muscle stiffness of the limbs, and often of the eyelids. Onset begins in childhood and there was no or only mild progression of symptoms over time. Four patients had episodes resembling malignant hyperthermia. The key finding at physical examination was delayed relaxation after repetitive contractions. Additionally, no atrophy was seen, muscle strength was generally preserved, and some patients had a remarkable athletic build. Symptomatic treatment was mostly ineffective or produced unacceptable side effects. EMG showed silent contractures in approximately half of the patients and no myotonia. Creatine kinase was normal or mildly elevated, and muscle biopsy showed mild myopathic changes with selective type II atrophy. Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) activity was reduced and western blot analysis showed decreased or absent SERCA1 protein. Based on this cohort, we conclude that Brody disease should be considered in cases of exercise-induced muscle stiffness. When physical examination shows delayed relaxation, and there are no myotonic discharges at electromyography, we recommend direct sequencing of the ATP2A1 gene or next generation sequencing with a myopathy panel. Aside from clinical features, SERCA activity measurement and SERCA1 western blot can assist in proving the pathogenicity of novel ATP2A1 mutations. Finally, patients with Brody disease may be at risk for malignant hyperthermia-like episodes, and therefore appropriate perioperative measures are recommended. This study will help improve understanding and recognition of Brody disease as a distinct myopathy in the broader field of calcium-related myopathies.

Atypical nuclear abnormalities in a patient with Brody disease.

Brody disease was first described as a benign pseudo-myotonic disorder with muscular stiffness, which increased with exercise. Biochemical and genetic studies have pointed out its close relationship to a functional defect of the fast-twitch sarcoplasmic reticulum Ca(++) ATPase pump (SERCA1) encoded by the ATP2A1 gene located on chromosome 16. The histopathological features in this form of myopathy were generally described as non-specific, i.e. moderate degree of type 2 fibre atrophy and excess of internal nuclei. We here present the clinical and histopathological features of a patient with Brody disease over a 19-year follow-up period. This patient had two heterozygous ATP2A1 mutations and complained about muscle stiffness immediately after effort. He had suffered from this since early childhood and exhibited clinical symptoms mimicking myotonia. Histological, ultrastructural and cytogenetic analyses revealed morphologically abnormal nuclei with polyploidy. In this report, we discuss the possible links between the consequences of the genetic abnormality and the peculiar aspect of the nuclei.

VCP and PSMF1: Antagonistic regulators of proteasome activity.

Protein turnover and quality control by the proteasome is of paramount importance for cell homeostasis. Dysfunction of the proteasome is associated with aging processes and human diseases such as neurodegeneration, cardiomyopathy, and cancer. The regulation, i.e. activation and inhibition of this fundamentally important protein degradation system, is still widely unexplored. We demonstrate here that the evolutionarily highly conserved type II triple-A ATPase VCP and the proteasome inhibitor PSMF1/PI31 interact directly, and antagonistically regulate proteasomal activity. Our data provide novel insights into the regulation of proteasomal activity.

Clinical, histological and genetic characterisation of patients with tubular aggregate myopathy caused by mutations in STIM1.

BACKGROUND: Tubular aggregate myopathies (TAMs) are muscle disorders characterised by abnormal accumulations of densely packed single-walled or double-walled membrane tubules in muscle fibres. Recently, STIM1, encoding a major calcium sensor of the endoplasmic reticulum, was identified as a TAM gene. METHODS: The present study aims to define the clinical, histological and ultrastructural phenotype of tubular aggregate myopathy and to assess the STIM1 mutation spectrum. RESULTS: We describe six new TAM families harbouring one known and four novel STIM1 mutations. All identified mutations are heterozygous missense mutations affecting highly conserved amino acids in the calcium-binding EF-hand domains, demonstrating the presence of a mutation hot spot for TAM. We show that the mutations induce constitutive STIM1 clustering, strongly suggesting that calcium sensing and consequently calcium homoeostasis is impaired. Histological and ultrastructural analyses define a common picture with tubular aggregates labelled with Gomori trichrome and Nicotinamide adenine dinucleotide (NADH) tetrazolium reductase, substantiating their endoplasmic reticulum origin. The aggregates were observed in both fibre types and were often accompanied by nuclear internalisation and fibre size variability. The phenotypical spectrum ranged from childhood onset progressive muscle weakness and elevated creatine kinase levels to adult-onset myalgia without muscle weakness and normal CK levels. CONCLUSIONS: The present study expands the phenotypical spectrum of STIM1-related tubular aggregate myopathy. STIM1 should therefore be considered for patients with tubular aggregate myopathies involving either muscle weakness or myalgia as the first and predominant clinical sign.

Advances in the understanding of skeletal muscle weakness in murine models of diseases affecting nerve-evoked muscle activity, motor neurons, synapses and myofibers.

Disease processes and trauma affecting nerve-evoked muscle activity, motor neurons, synapses and myofibers cause different levels of muscle weakness, i.e., reduced maximal force production in response to voluntary activation or nerve stimulation. However, the mechanisms of muscle weakness are not well known. Using murine models of amyotrophic lateral sclerosis (SOD1(G93A) transgenic mice), congenital myasthenic syndrome (AChE knockout mice and Musk(V789M/-) mutant mice), Schwartz-Jampel syndrome (Hspg2(C1532YNEO/C1532YNEO) mutant mice) and traumatic nerve injury (Neurotomized wild-type mice), we show that the reduced maximal activation capacity (the ability of the nerve to maximally activate the muscle) explains 52%, 58% and 100% of severe weakness in respectively SOD1(G93A), Neurotomized and Musk mice, whereas muscle atrophy only explains 37%, 27% and 0%. We also demonstrate that the impaired maximal activation capacity observed in SOD1, Neurotomized, and Musk mice is not highly related to Hdac4 gene upregulation. Moreover, in SOD1 and Neurotomized mice our results suggest LC3, Fn14, Bcl3 and Gadd45a as candidate genes involved in the maintenance of the severe atrophic state. In conclusion, our study indicates that muscle weakness can result from the triggering of different signaling pathways. This knowledge may be helpful in designing therapeutic strategies and finding new drug targets for amyotrophic lateral sclerosis, congenital myasthenic syndrome, Schwartz-Jampel syndrome and nerve injury.

Hereditary myopathy with early respiratory failure: occurrence in various populations.

OBJECTIVE: Several families with characteristic features of hereditary myopathy with early respiratory failure (HMERF) have remained without genetic cause. This international study was initiated to clarify epidemiology and the genetic underlying cause in these families, and to characterise the phenotype in our large cohort. METHODS: DNA samples of all currently known families with HMERF without molecular genetic cause were obtained from 12 families in seven different countries. Clinical, histopathological and muscle imaging data were collected and five biopsy samples made available for further immunohistochemical studies. Genotyping, exome sequencing and Sanger sequencing were used to identify and confirm sequence variations. RESULTS: All patients with clinical diagnosis of HMERF were genetically solved by five different titin mutations identified. One mutation has been reported while four are novel, all located exclusively in the FN3 119 domain (A150) of A-band titin. One of the new mutations showed semirecessive inheritance pattern with subclinical myopathy in the heterozygous parents. Typical clinical features were respiratory failure at mid-adulthood in an ambulant patient with very variable degree of muscle weakness. Cytoplasmic bodies were retrospectively observed in all muscle biopsy samples and these were reactive for myofibrillar proteins but not for titin. CONCLUSIONS: We report an extensive collection of families with HMERF with five different mutations in exon 343 of TTN, which establishes this exon as the primary target for molecular diagnosis of HMERF. Our relatively large number of new families and mutations directly implies that HMERF is not extremely rare, not restricted to Northern Europe and should be considered in undetermined myogenic respiratory failure.

Constitutive activation of the calcium sensor STIM1 causes tubular-aggregate myopathy.

Tubular aggregates are regular arrays of membrane tubules accumulating in muscle with age. They are found as secondary features in several muscle disorders, including alcohol- and drug-induced myopathies, exercise-induced cramps, and inherited myasthenia, but also exist as a pure genetic form characterized by slowly progressive muscle weakness. We identified dominant STIM1 mutations as a genetic cause of tubular-aggregate myopathy (TAM). Stromal interaction molecule 1 (STIM1) is the main Ca(2+) sensor in the endoplasmic reticulum, and all mutations were found in the highly conserved intraluminal Ca(2+)-binding EF hands. Ca(2+) stores are refilled through a process called store-operated Ca(2+) entry (SOCE). Upon Ca(2+)-store depletion, wild-type STIM1 oligomerizes and thereby triggers extracellular Ca(2+) entry. In contrast, the missense mutations found in our four TAM-affected families induced constitutive STIM1 clustering, indicating that Ca(2+) sensing was impaired. By monitoring the calcium response of TAM myoblasts to SOCE, we found a significantly higher basal Ca(2+) level in TAM cells and a dysregulation of intracellular Ca(2+) homeostasis. Because recessive STIM1 loss-of-function mutations were associated with immunodeficiency, we conclude that the tissue-specific impact of STIM1 loss or constitutive activation is different and that a tight regulation of STIM1-dependent SOCE is fundamental for normal skeletal-muscle structure and function.

Identification of an agrin mutation that causes congenital myasthenia and affects synapse function.

We report the case of a congenital myasthenic syndrome due to a mutation in AGRN, the gene encoding agrin, an extracellular matrix molecule released by the nerve and critical for formation of the neuromuscular junction. Gene analysis identified a homozygous missense mutation, c.5125G>C, leading to the p.Gly1709Arg variant. The muscle-biopsy specimen showed a major disorganization of the neuromuscular junction, including changes in the nerve-terminal cytoskeleton and fragmentation of the synaptic gutters. Experiments performed in nonmuscle cells or in cultured C2C12 myotubes and using recombinant mini-agrin for the mutated and the wild-type forms showed that the mutated form did not impair the activation of MuSK or change the total number of induced acetylcholine receptor aggregates. A solid-phase assay using the dystrophin glycoprotein complex showed that the mutation did not affect the binding of agrin to alpha-dystroglycan. Injection of wild-type or mutated agrin into rat soleus muscle induced the formation of nonsynaptic acetylcholine receptor clusters, but the mutant protein specifically destabilized the endogenous neuromuscular junctions. Importantly, the changes observed in rat muscle injected with mutant agrin recapitulated the pre- and post-synaptic modifications observed in the patient. These results indicate that the mutation does not interfere with the ability of agrin to induce postsynaptic structures but that it dramatically perturbs the maintenance of the neuromuscular junction.

The origin of tubular aggregates in human myopathies.

Tubular aggregates are morphological abnormalities characterized by the accumulation of densely packed tubules in skeletal muscle fibres. To improve knowledge of tubular aggregates, the formation and role of which are still unclear, the present study reports the electron microscopic analysis and protein characterization of tubular aggregates in six patients with 'tubular aggregate myopathy'. Three of the six patients also presented with myasthenic features. A large panel of immunochemical markers located in the sarcoplasmic reticulum, T-tubules, mitochondria, and nucleus was used. Despite differences in clinical phenotype, the composition of tubular aggregates, which contained proteins normally segregated differently along the sarcoplasmic reticulum architecture, was similar in all patients. All of these proteins, calsequestrin, RyR, triadin, SERCAs, and sarcalumenin, are involved in calcium uptake, storage, and release. The dihydropyridine receptor, DHPR, specifically located in the T-tubule, was also present in tubular aggregates in all patients. COX-2 and COX-7 mitochondrial proteins were not found in tubular aggregates, despite being observed close to them in the muscle fibre. The nuclear membrane protein emerin was found in only one case. Electron microscopy revealed vesicular budding from nuclei, and the presence of SAR-1 GTPase protein in tubular aggregates shown by immunochemistry, in all patients, suggests that tubular aggregates could arise from endoplasmic reticulum exit sites. Taken together, these results cast new light on the composition and significance of tubular aggregates.

[Pathophysiological characterization of congenital myasthenic syndromes: the example of mutations in the MUSK gene]. / Caractérisation physiopathologique des syndromes myasthéniques congénitaux: l'exemple de mutations dans le gène MUSK.

Congenital myasthenic syndromes (CMS) are rare genetic diseases affecting the neuromuscular junction (NMJ) and are characterized by a dysfunction of the neurotransmission. They are heterogeneous at their pathophysiological level and can be classified in three categories according to their presynaptic, synaptic and postsynaptic origins. We report here the first case of a human neuromuscular transmission dysfunction due to mutations in the gene encoding a postsynaptic molecule, the muscle-specific receptor tyrosine kinase (MuSK). Gene analysis identified two heteroallelic mutations, a frameshift mutation (c.220insC) and a missense mutation (V790M). The muscle biopsy showed dramatic pre- and postsynaptic structural abnormalities of the neuromuscular junction and severe decrease in acetylcholine receptor (AChR) epsilon-subunit and MuSK expression. In vitro and in vivo expression experiments were performed using mutant MuSK reproducing the human mutations. The frameshift mutation led to the absence of MuSK expression. The missense mutation did not affect MuSK catalytic kinase activity but diminished expression and stability of MuSK leading to decreased agrin-dependent AChR aggregation, a critical step in the formation of the neuromuscular junction. In electroporated mouse muscle, overexpression of the missense mutation induced, within a week, a phenotype similar to the patient muscle biopsy: a severe decrease in synaptic AChR and an aberrant axonal outgrowth. These results strongly suggest that the missense mutation, in the presence of a null mutation on the other allele, is responsible for the dramatic synaptic changes observed in the patient.

MUSK, a new target for mutations causing congenital myasthenic syndrome.

We report the first case of a human neuromuscular transmission dysfunction due to mutations in the gene encoding the muscle-specific receptor tyrosine kinase (MuSK). Gene analysis identified two heteroallelic mutations, a frameshift mutation (c.220insC) and a missense mutation (V790M). The muscle biopsy showed dramatic pre- and postsynaptic structural abnormalities of the neuromuscular junction and severe decrease in acetylcholine receptor (AChR) epsilon-subunit and MuSK expression. In vitro and in vivo expression experiments were performed using mutant MuSK reproducing the human mutations. The frameshift mutation led to the absence of MuSK expression. The missense mutation did not affect MuSK catalytic kinase activity but diminished expression and stability of MuSK leading to decreased agrin-dependent AChR aggregation, a critical step in the formation of the neuromuscular junction. In electroporated mouse muscle, overexpression of the missense mutation induced, within a week, a phenotype similar to the patient muscle biopsy: a severe decrease in synaptic AChR and an aberrant axonal outgrowth. These results strongly suggest that the missense mutation, in the presence of a null mutation on the other allele, is responsible for the dramatic synaptic changes observed in the patient.