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Objective: Anoctamin 5 (ANO5) is a putative intracellular calcium-activated chloride channel. Recessive mutations in ANO5 may present from asymptomatic hyperCKemia and exercise-induced myalgia to proximal and/or distal muscle weakness. Here we describe the clinical, pathological, and molecular findings of three unrelated patients with ANO5-related muscular dystrophy. Methods: In this retrospective study, we analyzed our database which includes 1700 muscle biopsies performed for diagnostic purposes from October 2004 to February 2019. Patients were attended by two myology experts, who performed and analyzed the muscle biopsies. Muscle biopsies were frozen in cooled isopenthane, cryostat sectioned, and routinely stained and reacted (minimum 16 stainings). A custom panel, including 115 genes (Nextera Rapid Capture, Illumina) and whole-exome sequencing analysis, was used for next-generation sequencing in cases without a definite pathological diagnosis. Results: Three patients were diagnosed with ANO5-related muscular dystrophy, with all presenting the common exon 5 mutation c.191dup plus a compound heterozygous missense mutation. They showed three different phenotypes (distal myopathy, LGMD2L, and asymptomatic hyperCKemia). Curiously, all three muscle biopsies showed different patterns, but numerous ragged-red fibers with little endomysial inflammation and partial invasion cell by T lymphocytes were observed in one. Conclusion: ANO5-related muscular dystrophy is a heterogeneous disease with different clinical phenotypes as well as different histological patterns, which may even mimic a mitochondrial myopathy. The results of this study provide further knowledge of the clinical, histological, and pathological features related to ANO5 mutations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. [ABSTRACT FROM AUTHOR]

TRAPPC11 was identified as a component of the TRAPP III complex that functions in membrane trafficking and autophagy. Variants in TRAPPC11 have been reported to be associated with a broad spectrum of phenotypes but all affected individuals display muscular pathology. Identifying additional variants will further our understanding of the clinical spectrum of phenotypes and will reveal regions of the protein critical for its functions. Here we report three individuals from unrelated families that have bi-allellic TRAPPC11 variants. Subject 1 harbors a compound heterozygous variant (c.1287 + 5G > A and c.3379_3380insT). The former variant results in a partial deletion of the foie gras domain (p.Ala372_Ser429del), while the latter variant results in a frame-shift and extension at the carboxy terminus (p.Asp1127Valfs*47). Subjects 2 and 3 both harbour a homozygous missense variant (c.2938G > A; p.Gly980Arg). Fibroblasts from all three subjects displayed membrane trafficking defects manifested as delayed endoplasmic reticulum (ER)-to-Golgi transport and/or a delay in protein exit from the Golgi. All three individuals also show a defect in glycosylation of an ER-resident glycoprotein. However, only the compound heterozygous subject displayed an autophagic flux defect. Collectively, our characterization of these individuals with bi-allelic TRAPPC11 variants highlights the functional importance of the carboxy-terminal portion of the protein. [ABSTRACT FROM AUTHOR]

• P3A is an alternatively-spliced evolutionary exon in CHRNA1. • c.257G>A results in P3A inclusion and expression of a non-functional AChR α-subunit. • Patients harbouring c.257G>A show an unusual pattern of facial and distal weakness. • We stress the importance of including alternative exons in mutation analysis and functional studies. Primary acetylcholine receptor deficiency is the most common subtype of congenital myasthenic syndrome, resulting in reduced amount of acetylcholine receptors expressed at the muscle endplate and impaired neuromuscular transmission. AChR deficiency is caused mainly by pathogenic variants in the ε-subunit of the acetylcholine receptor encoded by CHRNE , although pathogenic variants in other subunits are also seen. We report the clinical and molecular features of 13 patients from nine unrelated kinships with acetylcholine receptor deficiency harbouring the CHRNA1 variant NM_001039523.3:c.257G>A (p.Arg86His) in homozygosity or compound heterozygosity. This variant results in the inclusion of an alternatively-spliced evolutionary exon (P3A) that causes expression of a non-functional acetylcholine receptor α-subunit. We compare the clinical findings of this group to the other cases of acetylcholine receptor deficiency within our cohort. We report differences in phenotype, highlighting a predominant pattern of facial and distal weakness in adulthood, predominantly in the upper limbs, which is unusual for acetylcholine receptor deficiency syndromes, and more in keeping with slow-channel syndrome or distal myopathy. Finally, we stress the importance of including alternative exons in variant analysis to increase the probability of achieving a molecular diagnosis. [ABSTRACT FROM AUTHOR]

Background: Congenital myopathies (CMs) are a clinically and genetically heterogeneous group of hereditary muscular disorders. The distribution of genetic and histologic subtypes has been addressed in only a few cohorts, and the relationship between phenotypes and genotypes is only partially understood.Methods: This is a retrospective cross-sectional data collection study conducted at a single center. The clinical, histopathological, and molecular characterization of 104 patients with CM is reported.Results: The most common histopathological subtype was core myopathy (42%). Patients with severe endomysial fibrosis were more commonly unable to walk than patients with only a mild-grade endomysial fibrosis (56% vs 16%). Inability to walk was also more prevalent in patients with severe fatty replacement (44% vs 19%). The genetic etiology was more frequently identified among those patients with "specific" histologic findings (74% vs 62%). A definite molecular diagnosis was reached in 65 of 104 patients (62%), with RYR1 (24/104) and TTN (8/104) being the most frequent causative genes. Neonatal onset occurred in 56%. Independent ambulation was achieved by 74%. Patients who walked late were more likely to become wheelchair-dependent. Respiratory support was needed in one of three patients. Gastrostomy placement was required in 15%. Cardiac involvement was observed in 3%, scoliosis in 43%, and intellectual disability in 6%.Conclusions: This study provides an updated picture of the clinical, histopathological, and molecular landscape of CMs. Independently of the causative gene, fibrosis and fatty replacement in muscle biopsy specimens are associated with clinical severity. Mutations in TTN are responsible for a higher proportion of cases than previously thought. [ABSTRACT FROM AUTHOR]