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The term neuromuscular disorder (NMD) includes many genetic and acquired diseases and differential diagnosis can be challenging. Next-generation sequencing (NGS) is especially useful in this setting given the large number of possible candidate genes, the clinical, pathological, and genetic heterogeneity, the absence of an established genotype-phenotype correlation, and the exceptionally large size of some causative genes such as TTN , NEB and RYR1. We evaluated the diagnostic value of a custom targeted next-generation sequencing gene panel to study the mutational spectrum of a subset of NMD patients in Spain. In an NMD cohort of 207 patients with congenital myopathies, distal myopathies, congenital and adult-onset muscular dystrophies, and congenital myasthenic syndromes, we detected causative mutations in 102 patients (49.3%), involving 42 NMD-related genes. The most common causative genes, TTN and RYR1, accounted for almost 30% of cases. Thirty-two of the 207 patients (15.4%) carried variants of uncertain significance or had an unidentified second mutation to explain the genetic cause of the disease. In the remaining 73 patients (35.3%), no candidate variant was identified. In combination with patients' clinical and myopathological data, the custom gene panel designed in our lab proved to be a powerful tool to diagnose patients with myopathies, muscular dystrophies and congenital myasthenic syndromes. Targeted NGS approaches enable a rapid and cost-effective analysis of NMD- related genes, offering reliable results in a short time and relegating invasive techniques to a second tier.

Mutations in the SGCA gene cause limb girdle muscular dystrophy type 2D (LGMD2D). We report a family with three affected siblings with a mild phenotype consisting of late onset glutei and axial muscle weakness produced by a new mutation in the SGCA gene leading to a partial expression of the alpha-sarcoglycan protein. The MRI showed muscle atrophy involving paraspinal, pelvic and thigh muscles and a dystrophic pattern was observed in the muscle biopsy. Exome sequencing revealed a homozygous intronic deletion of SGCA and mRNA analysis showed the presence of three different transcripts. The presence, though in a lower proportion, of wild type transcript leads to a milder presentation of the disease. Although clinical symptoms did not entirely correspond with a sarcoglycanopathy, a compatible muscle MRI drove us to look for changes in the sarcoglycan genes. These cases are an example of how clinical, radiological and pathological data enriches the interpretation of exome analysis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Recent advances in molecular therapies for Duchenne muscular dystrophy (DMD) require precise genetic diagnosis because most therapeutic strategies are mutation-specific. To understand more about the genotype-phenotype correlations of the DMD gene we performed a comprehensive analysis of the DMD mutational spectrum in a large series of families. Here we provide the clinical, pathological and genetic features of 576 dystrophinopathy patients. DMD gene analysis was performed using the MLPA technique and whole gene sequencing in blood DNA and muscle cDNA. The impact of the DNA variants on mRNA splicing and protein functionality was evaluated by in silico analysis using computational algorithms. DMD mutations were detected in 576 unrelated dystrophinopathy families by combining the analysis of exonic copies and the analysis of small mutations. We found that 471 of these mutations were large intragenic rearrangements. Of these, 406 (70.5%) were exonic deletions, 64 (11.1%) were exonic duplications, and one was a deletion/duplication complex rearrangement (0.2%). Small mutations were identified in 105 cases (18.2%), most being nonsense/frameshift types (75.2%). Mutations in splice sites, however, were relatively frequent (20%). In total, 276 mutations were identified, 85 of which have not been previously described. The diagnostic algorithm used proved to be accurate for the molecular diagnosis of dystrophinopathies. The reading frame rule was fulfilled in 90.4% of DMD patients and in 82.4% of Becker muscular dystrophy patients (BMD), with significant differences between the mutation types. We found that 58% of DMD patients would be included in single exon-exon skipping trials, 63% from strategies directed against multiexon-skipping exons 45 to 55, and 14% from PTC therapy. A detailed analysis of missense mutations provided valuable information about their impact on the protein structure.

Background: Mutations in the dysferlin (DYSF) gene cause 3 different phenotypes of muscular dystrophies: Miyoshi myopathy, limb-girdle muscular dystrophy type 2B, and distal anterior compartment myopathy., Objective: To present the results of clinical and molecular analysis of 8 patients with dysferlinopathy from 5 unrelated families., Design: Clinical assessment was performed with a standardized protocol. A muscle biopsy specimen was obtained and studied by immunohistochemistry. Genetic analysis was performed using single-stranded conformation polymorphism and direct sequencing of genomic DNA., Results: All the patients presented the R1905X mutation in the DYSF gene in homozygosity, and the haplotype analysis at the DYSF locus revealed that it was a novel and founder mutation. A C-to-T transition at nucleotide position 6086 changes an arginine into a stop codon, leading to premature termination of translation. This mutation was expressed as 3 different clinical phenotypes (limb-girdle muscular dystrophy type 2B, Miyoshi distal myopathy, and distal anterior dysferlinopathy), but only 1 phenotype was found in the same family., Conclusions: The new R1905X DYSF founder mutation produced the 3 possible dysferlinopathy phenotypes without intrafamilial heterogeneity. This homogeneous population in Sueca, Spain, should be helpful in studying the modifying factors responsible for the phenotypic variability.