With the rapidly developing high-throughput sequencing technologies known as next generation sequencing or NGS, our approach to gene hunting and diagnosis has drastically changed. In <10 years, these technologies have moved from gene panel to whole genome sequencing and from an exclusively research context to clinical practice. Today, the limit is not the sequencing of one, many or all genes but rather the data analysis. Consequently, the challenge is to rapidly and efficiently identify disease-causing mutations within millions of variants. To do so, we developed the VarAFT software to annotate and pinpoint human disease-causing mutations through access to multiple layers of information. VarAFT was designed both for research and clinical contexts and is accessible to all scientists, regardless of bioinformatics training. Data from multiple samples may be combined to address all Mendelian inheritance modes, cancers or population genetics. Optimized filtration parameters can be stored and re-applied to large datasets. In addition to classical annotations from dbNSFP, VarAFT contains unique features at the disease (OMIM), phenotypic (HPO), gene (Gene Ontology, pathways) and variation levels (predictions from UMD-Predictor and Human Splicing Finder) that can be combined to optimally select candidate pathogenic mutations. VarAFT is freely available at: http://varaft.eu.

Deficiency of the dysferlin protein presents as two major clinical phenotypes: limb–girdle muscular dystrophy type 2B and Miyoshi myopathy. Dysferlin is known to participate in membrane repair, providing a potential hypothesis to the underlying pathophysiology of these diseases. The size of the dysferlin cDNA prevents its direct incorporation into an adeno-associated virus (AAV) vector for therapeutic gene transfer into muscle. To bypass this limitation, we split the dysferlin cDNA at the exon 28/29 junction and cloned it into two independent AAV vectors carrying the appropriate splicing sequences. Intramuscular injection of the corresponding vectors into a dysferlin-deficient mouse model led to the expression of full-length dysferlin for at least 1 year. Importantly, systemic injection in the tail vein of the two vectors led to a widespread although weak expression of the full-length protein. Injections were associated with an improvement of the histological aspect of the muscle, a reduction in the number of necrotic fibers, restoration of membrane repair capacity and a global improvement in locomotor activity. Altogether, these data support the use of such a strategy for the treatment of dysferlin deficiency.

Calpain 3 (Capn3) is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. This enigmatic protease has many unique features among the calpain family and, importantly, mutations in Capn3 have been shown to be responsible for limb girdle muscular dystrophy type 2A. Here we demonstrate that the Capn3 activation mechanism is similar to the universal activation of caspases and corresponds to an autolysis within the active site of the protease. We undertook a search for substrates in immature muscle cells, as several lines of evidence suggest that Capn3 is mostly in an inactive state in muscle and needs a signal to be activated. In this model, Capn3 proteolytic activity leads to disruption of the actin cytoskeleton and disorganization of focal adhesions through cleavage of several endogenous proteins. In addition, we show that titin, a previously identified Capn3 partner, and filamin C are further substrates of Capn3. Finally, we report that Capn3 colocalizes in vivo with its substrates at various sites along cytoskeletal structures. We propose that Capn3-mediated cleavage produces an adaptive response of muscle cells to external and/or internal stimuli, establishing Capn3 as a muscle cytoskeleton regulator.

Calpain 3 is a 94‐kDa calcium‐dependent cysteine protease mainly expressed in skeletal muscle. In this tissue, it localizes at several regions of the sarcomere through binding to the giant protein, titin. Loss‐of‐function mutations in the calpain 3 gene have been associated with limb‐girdle muscular dystrophy type 2A (LGMD2A), a common form of muscular dystrophy found world wide. Recently, significant progress has been made in understanding the mode of regulation and the possible function of calpain 3 in muscle. It is now well accepted that it has an unusual zymogenic activation and that cytoskeletal proteins are one class of its substrates. Through the absence of cleavage of these substrates, calpain 3 deficiency leads to abnormal sarcomeres, impairment of muscle contractile capacity, and death of the muscle fibers. These data indicate a role for calpain 3 as a chef d'orchestre in sarcomere remodeling and suggest a new category of LGMD2 pathological mechanisms.