ObjectiveAutosomal dominant centronuclear myopathy (AD-CNM) is a neuromuscular congenital disease caused by mutations in the DNM2 gene that encodes dynamin-2 (DNM2). The main clinical features of AD-CNM are progressive weakness and atrophy of skeletal muscles. However, cognitive defects have also been reported, suggesting that AD-CNM-causing mutations in DNM2 might also affect central nervous system (CNS). We recently demonstrated that defects in excitatory synaptic transmission occur in the brain of transgenic knock-in (KI) mice harboring the DNM2 p.R465W mutation, the most common causing AD-CNM. As DNM2 regulates the trafficking of glutamate-AMPA receptors (AMPARs), major mediators of excitatory synaptic transmission in mammals, it is feasible that the synaptic availability of AMPAR is affected in the context of AD-CNM. The main objective of this work was to evaluate the impact of the p.R465W DNM2 mutation on the GluA1-AMPAR-subunit synaptic availability in the brain of KI mice.MethodsWe addressed an experimental quantitative study. By using subcellular fractionation and western blot we quantified the expression of GluA1 and synaptic proteins in hippocampal total homogenates and postsynaptic densities (PSDs) in the brain of WT and KI mice. By total internal reflection microscopy (TIRFM) we also analyzed the arrival and residence time of GluA1 into the plasma membrane of hippocampal cultured neurons.ResultsAlthough we did not observe significant differences in the GluA1 expression in hippocampal total homogenates, it was significantly reduced in the PSDs of KI compared to wild-type (WT) brains. Moreover, the residence time of GluA1 in the surface membranes of KI hippocampal neurons was significantly reduced compared to WT neurons.ConclusionThese data strongly suggest that the p.R465W mutation in DNM2 perturbs synaptic GluA1-availability in hippocampal neurons, likely leading to defects in excitatory synaptic transmission.

Centronuclear myopathies represent a subset of debilitating genetic disorders, for which no treatment exists. The Unite-CNM trial (NCT04033159) aimed to assess the effect of an antisense oligonucleotide to reduce DNM2 mRNA expression in X-linked myotubular myopathy (XLMTM) and autosomal dominant centronuclear myopathy (ADCNM). The trial was discontinued due to tolerability concerns (hepatic and hematological). This report aims to provide an overview of hepatic involvement in XLMTM and ADCNM adults. The medical history and prospective liver imaging and liver function test results at screening and baseline were assessed. Furthermore, DNM2 protein expression in livers of four other pediatric patients with XLMTM and of healthy children and adults were assessed. Twenty-six patients were screened; 15 with DNM2 mutations (median age 36 years; six females), and 11 with MTM1 mutations (median age 52 years; five females). Overall, six patients had a history of liver disease (6/19;31.6%). One patient with XLMTM had elevated serum alanine transaminase and another XLMTM patient had elevated serum gamma glutamyl transpeptidase. Liver ultrasound showed no features of peliosis hepatis. Liver steatosis was observed in three ADCNM patients and two XLMTM patients. The Fibroscan CAP score was above normal range in three XLMTM patients, and borderline or normal in other patients. The histopathology study showed that DNM2 protein levels in human liver decrease with age and are lower in pediatric individuals with XLMTM compared to controls. This study provides an overview of hepatic involvement in a large group of ADCNM and XLMTM patients. Findings suggest an underlying liver pathology may impact tolerability of therapeutic approaches, and will be important to consider for future trial design and clinical management. The results of DNM2 protein expression warrant further investigations on the role of DNM2 in the liver if it is to be used as a therapeutic target.

CCDC78 was identified as a novel candidate gene for autosomal dominant centronuclear myopathy-4 (CNM4) approximately ten years ago. However, to date, only one family has been described, and the function of CCDC78 remains unclear. Here, we analyze for the first time a family harboring a CCDC78 nonsense mutation to better understand the role of CCDC78 in muscle. We conducted a comprehensive histopathological analysis on muscle biopsies, including immunofluorescent assays to detect multiple sarcoplasmic proteins. We examined CCDC78 transcripts and protein using WB in CCDC78-mutated muscle tissue; these analyses were also performed on muscle, lymphocytes, and fibroblasts from healthy subjects. Subsequently, we conducted RT-qPCR and transcriptome profiling through RNA-seq to evaluate changes in gene expression associated with CCDC78 dysfunction in muscle. Lastly, coimmunoprecipitation (Co-Ip) assays and mass spectrometry (LC-MS/MS) studies were carried out on extracted muscle proteins from both healthy and mutated subjects. The histopathological features in muscle showed novel histological hallmarks, which included areas of dilated and swollen sarcoplasmic reticulum (SR). We provided evidence of nonsense-mediated mRNA decay (NMD), identified the presence of novel CCDC78 transcripts in muscle and lymphocytes, and identified 1035 muscular differentially expressed genes, including several involved in the SR. Through the Co-Ip assays and LC-MS/MS studies, we demonstrated that CCDC78 interacts with two key SR proteins: SERCA1 and CASQ1. We also observed interactions with MYH1, ACTN2, and ACTA1. Our findings provide insight, for the first time, into the interactors and possible role of CCDC78 in skeletal muscle, locating the protein in the SR. Furthermore, our data expand on the phenotype previously associated with CCDC78 mutations, indicating potential histopathological hallmarks of the disease in human muscle. Based on our data, we can consider CCDC78 as the causative gene for CNM4.

Mutations in the LMNA gene-encoding A-type lamins can cause Limb-Girdle muscular dystrophy Type 1B (LGMD1B). This disease presents with weakness and wasting of the proximal skeletal muscles and has a variable age of onset and disease severity. This variability has been attributed to genetic background differences among individuals; however, such variants have not been well characterized. To identify such variants, we investigated a multigeneration family in which affected individuals are diagnosed with LGMD1B. The primary genetic cause of LGMD1B in this family is a dominant mutation that activates a cryptic splice site, leading to a five-nucleotide deletion in the mature mRNA. This results in a frame shift and a premature stop in translation. Skeletal muscle biopsies from the family members showed dystrophic features of variable severity, with the muscle fibers of some family members possessing cores, regions of sarcomeric disruption, and a paucity of mitochondria, not commonly associated with LGMD1B. Using whole genome sequencing (WGS), we identified 21 DNA sequence variants that segregate with the family members possessing more profound dystrophic features and muscle cores. These include a relatively common variant in coiled-coil domain containing protein 78 (CCDC78). This variant was given priority because another mutation in CCDC78 causes autosomal dominant centronuclear myopathy-4, which causes cores in addition to centrally positioned nuclei. Therefore, we analyzed muscle biopsies from family members and discovered that those with both the LMNA mutation and the CCDC78 variant contain muscle cores that accumulated both CCDC78 and RyR1. Muscle cores containing mislocalized CCDC78 and RyR1 were absent in the less profoundly affected family members possessing only the LMNA mutation. Taken together, our findings suggest that a relatively common variant in CCDC78 can impart profound muscle pathology in combination with a LMNA mutation and accounts for variability in skeletal muscle disease phenotypes.

Autosomal dominant centronuclear myopathy (AD-CNM) is a rare congenital myopathy characterized by muscle weakness and centrally located nuclei in muscle fibers in the absence of any regeneration. AD-CNM is due to mutations in the DNM2 gene encoding dynamin 2 (DNM2), a large GTPase involved in intracellular membrane trafficking and a regulator of actin and microtubule cytoskeletons. DNM2 mutations are associated with a broad clinical spectrum ranging from severe neonatal to less severe late-onset forms. The histopathological signature includes nuclear centralization, predominance and atrophy of type 1 myofibers and radiating sarcoplasmic strands. To explain the muscle dysfunction, several pathophysiological mechanisms affecting key mechanisms of muscle homeostasis have been identified. They include defects in excitation-contraction coupling, muscle regeneration, mitochondria or autophagy. Several therapeutic approaches are under development by modulating the expression of DNM2 in a pan-allelic manner or by allele-specific silencing targeting only the mutated allele, which open the era of clinical trials for this pathology. La myopathie centronucléaire liée au gène de la dynamine 2. La myopathie centronucléaire autosomique dominante (AD-CNM) est une myopathie congénitale rare caractérisée par une faiblesse musculaire et par la présence de noyaux centraux dans les fibres musculaires en absence de tout processus de régénération. L’AD-CNM est due à des mutations du gène DNM2 codant la dynamine 2 (DNM2), une volumineuse GTPase impliquée dans le trafic membranaire intracellulaire et un régulateur des cytosquelettes d’actine et de microtubules. Les mutations de la DNM2 sont associées à un large éventail clinique allant de formes sévères néonatales à des formes moins graves à début plus tardif. La signature histopathologique inclut une centralisation nucléaire, une prédominance et une atrophie des fibres lentes, ainsi que des travées sarcoplasmiques en rayons de roue. Pour expliquer la dysfonction musculaire, plusieurs mécanismes physiopathologiques affectant des étapes clés de l’homéostasie musculaire ont été identifiés. Ils incluent des défauts du couplage excitation-contraction, de la régénération musculaire, des mitochondries ou de l’autophagie. Plusieurs approches thérapeutiques sont en développement, en particulier la modulation de l’expression de la DNM2 pan-allélique ou ne ciblant que l’allèle muté, ouvrant ainsi la porte à des essais cliniques dans cette pathologie.