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Background: Mutations in the LMNA gene, encoding human lamin A/C, have been associated with an increasing number of disorders often involving skeletal and cardiac muscle, but no clear genotype/phenotype correlation could be established to date., Methods: We analyzed the LMNA gene in a large cohort of patients mainly affected by neuromuscular or cardiac disease and clustered mutated patients in two groups to unravel possible correlations., Results: We identified 28 variants, 9 of which reported for the first time. The two groups of patients were characterized by clinical and genetic differences: 1) patients with childhood onset displayed skeletal muscle involvement with predominant scapuloperoneal and facial weakness associated with missense mutations; 2) patients with adult onset mainly showed cardiac disorders or myopathy with limb girdle distribution, often associated with frameshift mutations presumably leading to a truncated protein., Conclusions: Our findings, supported by meta-analysis of previous literature, suggest the presence of two different pathogenetic mechanisms: late onset phenotypes may arise through loss of function secondary to haploinsufficiency, while dominant negative or toxic gain of function mechanisms may explain the severity of early phenotypes. This model of patient stratification may help patient management and facilitate future studies aimed at deciphering lamin A/C pathogenesis.

Over the past 30 years, more than 50 repeat expansion disorders have been identified, thanks to the exponential development of genetic tools and next-generation sequencing technologies. Long-read sequencing has emerged as a new milestone in the diagnosis of these disorders. Despite these major achievements, the comprehensive characterization of short tandem repeats in a pathological context remains challenging, primarily due to their inherent characteristics such as motif complexity, high GC content, and variable length. In this study, we implemented CRISPR/Cas9-based enrichment strategy combined to Oxford Nanopore (ONT) long-read sequencing. Our aim was to thoroughly characterize trinucleotide repeat expansion in two neuromuscular diseases: oculopharyngodistal myopathy (OPDM) and myotonic dystrophy type 1 (DM1). We conducted precise analyses of the OPDM and DM1 loci, determining repeat size, repeat length distribution, and DNA methylation. We achieved this by distinguishing normal and expanded alleles, using three different basecallers (Guppy, Bonito and Dorado). Our observations revealed miscalling of CGG and CTG repeats, with errors highly dependent on library preparation protocols (ONT R.9.4 vs R10.4 chemistries) and the chosen basecalling strategy. Our findings highlight the importance of the choice of basecalling strategies for the true characterization of repeated regions. Given our results, we propose guidelines for the CRISPR/Cas9-enrichment long-read sequencing strategy for repeat expansion diseases, which could be readily applicable in research but also in diagnostic settings. [ABSTRACT FROM AUTHOR]

Background and purpose: The aim was to determine the genetic background of unknown muscular dystrophy in five French families. Methods: Twelve patients with limb girdle muscular dystrophy or distal myopathy were clinically evaluated. Gene mutations were identified using targeted exon sequencing and mutated DNAJB6 was tested in vitro. Results: Five patients presented with distal lower limb weakness whilst others had proximal presentation with a variable rate of progression starting at the mean age of 38.5 years. Two novel mutations (c.284A>T, p.Asn95Ile, two families; and c.293_295delATG, p.Asp98del, one family) as well as the previously reported c.279C>G (p.Phe93Leu, two families) mutation in DNAJB6 were identified. All showed a reduced capacity to prevent protein aggregation. Conclusions: The mutational and phenotypical spectrum of DNAJB6-caused muscle disease is larger than previously reported, including also dysphagia. The originally reported c.279C>G (p.Phe93Leu) mutation is now identified in four different populations and appears to be a mutational hotspot. Our report confirms that some DNAJB6 mutations cause distal-onset myopathy and hence DNAJB6 defects should be considered broadly in dominant muscular dystrophy families. [ABSTRACT FROM AUTHOR]

Background: Skeletal muscle disorders associated with mutations of lamin A/C gene include autosomal Emery­Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B. The pathogenic mechanism underlying these diseases is unknown. Recent data suggest an impairment of signalling mechanisms as a possible cause of muscle malfunction. A molecular complex in muscle cells formed by lamin A/C, emerin, and nuclear actin has been identified. The stability of this protein complex appears to be related to phosphorylation mechanisms. Objective: To analyse lamin A/C phosphorylation in control and laminopathic muscle cells. Methods: Lamin A/C N-terminal phosphorylation was determined in cultured mouse myoblasts using a specific antibody. Insulin treatment of serum starved myoblast cultures was carried out to evaluate involvement of insulin signalling in the phosphorylation pathway. Screening of four Emery­Dreifuss and one limb girdle muscular dystrophy 1B cases was undertaken to investigate lamin A/C phosphorylation in both cultured myoblasts and mature muscle fibres. Results: Phosphorylation of lamin A was observed during myoblast differentiation or proliferation, along with reduced lamin A/C phosphorylation in quiescent myoblasts. Lamin A N-terminus phosphorylation was induced by an insulin stimulus, which conversely did not affect lamin C phosphorylation. Lamin A/C was also hyperphosphorylated in mature muscle, mostly in regenerating fibres. Lamin A/C phosphorylation was strikingly reduced in laminopathic myoblasts and muscle fibres, while it was preserved in interstitial fibroblasts. Conclusions: Altered lamin A/C interplay with a muscle specific phosphorylation partner might be involved in the pathogenic mechanism of Emery­Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B. [ABSTRACT FROM AUTHOR]

Abstract: Dropped head syndrome is characterized by severe weakness of neck extensor muscles with sparing of the flexors. It is a prominent sign in several neuromuscular conditions, but it may also be an isolated feature with uncertain aetiology. We report two children in whom prominent weakness of neck extensor muscles is associated with mutations in lamin A/C (LMNA) and selenoprotein N1 (SEPN1) genes, respectively. This report expands the underlying causes of the dropped head syndrome which may be the presenting feature of a congenital muscular dystrophy. [Copyright &y& Elsevier]

Abstract: Collagen VI-myopathies regroup several forms of neuromuscular disorders caused by the deficiency of collagen type VI (COLVI), a protein that contributes to the architectural structure of muscle, its maintenance and survival. COLVI-myopathies constitute a spectrum of clinical presentations ranging from severe, early-onset phenotypes, to intermediate and milder phenotypes, with Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy at each extremity. To investigate in details the pathogenesis and to evaluate therapeutic strategies, we developed a novel mouse model carrying a nonsense mutation in the Col6a2 gene, based on the mutation identified in a patient with classical UCMD. Overall, homozygous KI-Col6a2 animals (KI) are viable and fertile, but males fail to thrive and display a significantly reduced body weight compared to wild type and heterozygous littermates, detectable as early as 4weeks of age. We examined in detail the skeletal muscle phenotype of the KI-Col6a2 mice in terms of histology, morphology and function. The mutation introduced leads to a premature termination codon which triggers the specific degradation of the Col6a2 transcript through the nonsense-mediated RNA decay and the absence of COLVI in skeletal muscle from KI animals. These mice develop a myopathic phenotype with pronounced fiber size variability, increased internal nuclei and fibrosis. In addition, Tibialis anterior (TA) muscles of KI animals display significant hypoplasia, associated with an increase in type IIA fibers and a decrease in type IIB fibers. Accordingly, SDH and COX assays revealed more abundant oxidative fibers. At the functional level, the maximal specific force of TA muscle is significantly diminished in 9week-old KI males. Taken together, these data validate the KI-Col6a2 mouse as a model of COLVI-myopathy and identify quantifiable markers of muscle dysfunction that will be extremely useful to assess the potential benefit of therapeutic approaches. [Copyright &y& Elsevier]

Abstract: Currently in most laboratories, molecular investigations of neuromuscular disorders (NMDs) are based on a differential molecular diagnosis by a complex and time-consuming gene by gene approach guided by a clinical approach. As a consequence, it is estimated that 35–50% of patients remain devoid of a precise molecular diagnosis. On one hand, many causal genes for NMDs are still to be identified but, on the other hand, it becomes evident that the current approaches are not sufficiently powerful and accurate to perform exhaustive screenings even in known genes, and that around 50% of patients without a definite molecular diagnosis carry unidentified mutations in known genes. In the recent years, technological tools have been developed towards performing massive molecular analyses using DNA sequence capture (SC) and next generation sequencing (NGS). In the context of the European project “NMD-Chip” (FP7 Health call), we have taken advantage of these technologies to develop specific custom SC DNA arrays to explore via NGS, known and candidate genes for NMDs. A custom in-solution DNA SC library has been designed. It includes 820 genes: 50 are known to be involved in common muscular dystrophies or myopathies (Duchenne/Becker, limb girdle, congenital), the others are candidate genes for those disorders, selected from experimental and published data. To validate this DNA chip, we have selected 20 patient DNAs: five with one known mutation, including point mutations and large rearrangements (1 COL6A2, 1 COL6A1, 1 DOK7, 1 DES, 1 TNXB) and 15 with no molecular diagnosis. We also explored the DNA of three of these patients via Exome sequencing to further compare and validate our custom SC design. The analysis of our data will help to demonstrate the proof of concept of NGS as a robust and accurate approach towards exploring multiple genes in NMD patients. [Copyright &y& Elsevier]