Altered splicing of the BIN1 muscle-specific exon in humans and dogs with highly progressive centronuclear myopathy

Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM), a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM). However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD). Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies

To retain or remove the syndesmotic screw: a review of literature

Introduction: Syndesmotic positioning screws are frequently placed in unstable ankle fractures. Many facets of adequate placement techniques have been the subject of various studies. Whether or not the syndesmosis screw should be removed prior to weight-bearing is still debated. In this study, the recent literature is reviewed concerning the need for removal of the syndesmotic screw. Materials and methods: A comprehensive literature search was conducted in the electronic databases of the Cochrane Library, Pubmed Medline and EMbase from January 2000 to October 2010. Results: A total of seven studies were identified in the literature. Most studies found no difference in outcome between retained or removed screws. Patients with screws that were broken, or showed loosening, had similar or improved outcome compared to patients with removed screws. Removal of the syndesmotic screws, when deemed necessary, is usually not performed before 8-12 weeks. Conclusion: There is paucity in randomized controlled trials on the absolute need for removal of the syndesmotic screw. However, current literature suggests that it might be reserved for intact screws that cause hardware irritation or reduced range of motion after 4-6 months

Unusual multisystemic involvement and a novel BAG3 mutation revealed by NGS screening in a large cohort of myofibrillar myopathies

Background: Myofibrillar myopathies (MFM) are a group of phenotypically and genetically heterogeneous neuromuscular disorders, which are characterized by protein aggregations in muscle fibres and can be associated with multisystemic involvement. Methods: We screened a large cohort of 38 index patients with MFM for mutations in the nine thus far known causative genes using Sanger and next generation sequencing (NGS). We studied the clinical and histopathological characteristics in 38 index patients and five additional relatives (n = 43) and particularly focused on the associated multisystemic symptoms. Results: We identified 14 heterozygous mutations (diagnostic yield of 37%), among them the novel p.Pro209Gln mutation in the BAG3 gene, which was associated with onset in adulthood, a mild phenotype and an axonal sensorimotor polyneuropathy, in the absence of giant axons at the nerve biopsy. We revealed several novel clinical phenotypes and unusual multisystemic presentations with previously described mutations: hearing impairment with a FLNC mutation, dysphonia with a mutation in DES and the first patient with a FLNC mutation presenting respiratory insufficiency as the initial symptom. Moreover, we described for the first time respiratory insufficiency occurring in a patient with the p.Gly154Ser mutation in CRYAB. Interestingly, we detected a polyneuropathy in 28% of the MFM patients, including a BAG3 and a MYOT case, and hearing impairment in 13%, including one patient with a FLNC mutation and two with mutations in the DES gene. In four index patients with a mutation in one of the MFM genes, typical histological findings were only identified at the ultrastructural level (29%). Conclusions: We conclude that extraskeletal symptoms frequently occur in MFM, particularly cardiac and respiratory involvement, polyneuropathy and/or deafness. BAG3 mutations should be considered even in cases with a mild phenotype or an adult onset. We identified a genetic defect in one of the known genes in less than half of the MFM patients, indicating that more causative genes are still to be found. Next generation sequencing techniques should be helpful in achieving this aim

An integrated diagnosis strategy for congenital myopathies

Congenital myopathies are severe muscle disorders affecting adults as well as children in all populations. The diagnosis of congenital myopathies is constrained by strong clinical and genetic heterogeneity. Moreover, the majority of patients present with unspecific histological features, precluding purposive molecular diagnosis and demonstrating the need for an alternative and more efficient diagnostic approach. We used exome sequencing complemented by histological and ultrastructural analysis of muscle biopsies to identify the causative mutations in eight patients with clinically different skeletal muscle pathologies, ranging from a fatal neonatal myopathy to a mild and slowly progressive myopathy with adult onset. We identified RYR1 (ryanodine receptor) mutations in six patients and NEB (nebulin) mutations in two patients. We found novel missense and nonsense mutations, unraveled small insertions/deletions and confirmed their impact on splicing and mRNA/protein stability. Histological and ultrastructural findings of the muscle biopsies of the patients validated the exome sequencing results. We provide the evidence that an integrated strategy combining exome sequencing with clinical and histopathological investigations overcomes the limitations of the individual approaches to allow a fast and efficient diagnosis, accelerating the patient's access to a better healthcare and disease management. This is of particular interest for the diagnosis of congenital myopathies, which involve very large genes like RYR1 and NEB as well as genetic and phenotypic heterogeneity

A comparison of extended spectrum β-lactamase producing Escherichia coli from clinical, recreational water and wastewater samples associated in time and location

Extended spectrum β-lactamase producing Escherichia coli (ESBL-EC) are excreted via effluents and sewage into the environment where they can re-contaminate humans and animals. The aim of this observational study was to detect and quantify ESBL-EC in recreational water and wastewater, and perform a genetic and phenotypic comparative analysis of the environmental strains with geographically associated human urinary ESBL-EC. Recreational fresh- and saltwater samples from four different beaches and wastewater samples from a nearby sewage plant were filtered and cultured on differential and ESBL-selective media. After antimicrobial susceptibility testing and multi-locus variable number of tandem repeats assay (MLVA), selected ESBL-EC strains from recreational water were characterized by whole genome sequencing (WGS) and compared to wastewater and human urine isolates from people living in the same area. We detected ESBL-EC in recreational water samples on 8/20 occasions (40%), representing all sites. The ratio of ESBL-EC to total number of E. coli colony forming units varied from 0 to 3.8%. ESBL-EC were present in all wastewater samples in ratios of 0.56–0.75%. ST131 was most prevalent in urine and wastewater samples, while ST10 dominated in water samples. Eight STs and identical ESBL-EC MLVA-types were detected in all compartments. Clinical ESBL-EC isolates were more likely to be multidrug-resistant (p<0.001). This study confirms that ESBL-EC, including those that are capable of causing human infection, are present in recreational waters where there is a potential for human exposure and subsequent gut colonisation and infection in bathers. Multidrug-resistant E. coli strains are present in urban aquatic environments even in countries where antibiotic consumption in both humans and animals is highly restricted

Comparative structural analysis of 20S proteasome ortholog protein complexes by native mass spectrometry

Ortholog protein complexes are responsible for equivalent functions in different organisms. However, during evolution, each organism adapts to meet its physiological needs and the environmental challenges imposed by its niche. This selection pressure leads to structural diversity in protein complexes, which are often difficult to specify, especially in the absence of high-resolution structures. Here, we describe a multi-level experimental approach based on native mass spectrometry (MS) tools for elucidating the structural preservation and variations among highly related protein complexes. The 20S proteasome, an essential protein degradation machinery, served as our model system, wherein we examined five complexes isolated from different organisms. We show that throughout evolution, from the T. acidophilum archaeal prokaryotic complex to the eukaryotic 20S proteasomes in yeast (S. cerevisiae) and mammals (rat - R. norvegicus, rabbit - O. cuniculus and human - HEK293 cells), the proteasome increased both in size and stability. Native Ms structural signatures of the rat and rabbit 20S proteasomes, which heretofore lacked high-resolution three-dimensional structures, highly resembled that of the human complex. Using cryo-electron microscopy single-particle analysis we were able to obtain a high-resolution structure of the rat 20S proteasome, allowing us to validate the MS-based results. Our study also revealed that the yeast complex, and not those in mammals, was the largest in size, and displayed the greatest degree of kinetic stability. Moreover, we also identified a new proteoform of the PSMA7 subunit that resides within the rat and rabbit complexes, which to our knowledge have not been previously described. Altogether, our strategy enables elucidation of the unique structural properties of protein complexes that are highly similar to one another, a framework that is valid not only to ortholog protein complexes, but also for other highly related protein assemblies. </p