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Inherited neuromuscular diseases comprise a highly heterogeneous group of disorders characterized by the impairment of the neural structures or motor unit components responsible for the generation of movement. While as single gene-associated disorder the majority of them are rare, taken together their estimated prevalence reaches 1 – 3 cases / 1000 individuals. Due to their elevated morbidity and mortality, they represent a significant health burden for the affected individuals, their families, and the healthcare systems. Moreover, their clinical and genetic heterogeneity makes their diagnosis a long and complex process, which often requires specialized diagnostic procedures and poses a challenge in about half of the cases. However, thanks to decreasing costs and increased availability of next-generation sequencing technologies, the last years had witnessed a rise in the number of novel genes associated to neuromuscular disorders. In this study, we identified three novel neuromuscular disease-causing genes: PIEZO2, whose biallelic loss-of-function mutations cause distal arthrogryposis with impaired proprioception and touch; VAMP1, whose biallelic loss-of-function mutations cause a novel presynaptic congenital myasthenic syndrome; CAPRIN1, whose specific p.Pro512Leu mutation causes a neurodegenerative disorder characterized by ataxia and muscle weakness. For PIEZO2, we identified biallelic loss-of-function mutations using exome sequencing, SNPchip-based linkage analysis, DNA microarray, and Sanger sequencing in ten affected individuals of four independent families showing arthrogryposis, hypotonia, respiratory insufficiency at birth, scoliosis, and delayed motor development. This phenotype is clearly distinct from distal arthrogryposis with ocular anomalies which characterize the autosomal dominant distal arthrogryposis 3 (DA3), distal arthrogryposis 5 (DA5), and Marden-Walker syndrome (MWKS). While these disorders are caused by heterozygous gain-of-function mutations in PIEZO2, the novel reported mutations result in the loss of PIEZO2, since they lead to nonsense-mediated mRNA decay in patient-derived fibroblast cell lines. PIEZO2 is a mechanosensitive ion channel playing a major role in light-touch sensation and proprioception. Mice ubiquitously depleted of PIEZO2 die postnatally because of respiratory distress, while individuals lacking PIEZO2 develop a neuromuscular disorder, likely due to the loss of proprioception inputs in muscles. For VAMP1, we identified biallelic loss-of-function mutations using exome or genome sequencing in two pairs of siblings from two independent families affected by a novel congenital myasthenic syndrome. Electrodiagnostic examination showed severely low compound muscle action potentials and presynaptic impairment. The two described homozygous mutations are a frameshift and a missense mutation of a highly conserved residue, therefore are likely to result in the loss of VAMP1 function. Indeed, the phenotype is resembled by VAMP1lew/lew mice, which carry a homozygous VAMP1 truncating mutation and show neurophysiological features of presynaptic impairment. For CAPRIN1, we identified the identical de novo c.1535C>T (p.Pro512Leu) missense variant using trio exome sequencing in two unrelated individuals displaying early-onset ataxia, dysarthria, cognitive decline and muscle weakness. This mutation causes the substitution of a highly conserved residue and in silico tools predict an increase in the protein aggregation propensity. Overexpression of CAPRIN1-P512L caused the formation of insoluble ubiquitinated aggregates, sequestrating proteins associated with neurodegenerative disorders, such as ATXN2, GEMIN5, SNRNP200, and SNCA. Upon differentiation in cortical neurons of induced pluripotent stem cell (iPSC) lines where the CAPRIN1-P512L was introduced via CRISPR/Cas9, reduced neuronal activity and altered stress granules dynamics were observed in the lines harboring the mutation. Moreover, nano-differential scanning fluorimetry revealed that CAPRIN1-P512L adopts an extended conformation, and fluorescence microscopy demonstrated that RNA greatly enhances its aggregation in vitro. Taken together, this study associates: (1) biallelic loss-of-function mutations in PIEZO2 with the autosomal recessive distal arthrogryposis with impaired proprioception and touch; (2) biallelic loss-of-function mutations in VAMP1 with an autosomal recessive presynaptic congenital myasthenic syndrome; (3) a recurrent de novo p.Pro512Leu mutation of CAPRIN1 with a neurodegenerative disorder characterized by ataxia and muscle weakness. |
