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INTRODUCTION Hereditary inclusion body myopathy (hIBM) refers to a group of clinically and genetically heterogeneous diseases. The overlapping histochemical features of hIBM with other genetic disorders lead to low diagnostic rates with targeted single-gene sequencing. This is true for the most prevalent form of hIBM, GNEpathy. Therefore, we used whole-exome sequencing (WES) to determine whether a cohort of clinically suspected GNEpathy patients undiagnosed by targeted GNE analysis could be genetically characterized. METHODS Twenty patients with hIBM but undiagnosed by targeted GNE sequencing were analyzed by WES before data filtering on 306 genes associated with neuromuscular disorders. RESULTS Seven patients out of 20 were found to have disease-causing mutations in genes associated with hIBM or genes allowing for hIBM in the differential diagnosis or associated with unexpected diagnosis. DISCUSSION Next-generation sequencing is an efficient strategy in the context of hIBM, resulting in a molecular diagnosis for 35% of the patients initially undiagnosed by targeted GNE analysis. Muscle Nerve 56: 993-997, 2017.

Introduction: Autosomal recessive muscular dystrophies are heterogeneous genetic disorders, with 39 genes currently implicated. Genetic diagnosis using targeted single-gene analysis by Sanger sequencing yields negative results in 10−20% of samples, warranting clinical re-evaluation and time-consuming testing of additional genes. This applies to dysferlinopathies caused by mutations in the gene encoding dysferlin (DYSF), which presents mainly as limb-girdle muscular dystrophy (LGMD) or distal myopathy. Methods: We evaluated exome sequencing associated with data filtering for selected genes as a second-tier approach for genetic diagnosis in a cohort of 37 patients with an initial negative result on targeted DYSF analysis. Results: Exome sequencing allowed for establishing (16%) or suggesting (8%) the molecular diagnosis by implicating other known LGMD or distal myopathy genes or by revealing DYSF mutations previously missed using mutation-screening techniques with incomplete detection yields. Conclusions: Exome sequencing associated with data filtering constitutes an efficient second-tier analysis for genes implicated in LGMD or distal myopathies. Muscle Nerve 50: 1007–1010, 2014

Limb-girdle muscular dystrophy type 2A (LGMD2A) due to mutations in the CAPN3 gene is one of the most common of autosomal recessive limb-girdle muscular dystrophies. We describe a patient who had a typical LGMD2A phenotype and posterior compartment involvement on MRI. Different genetic analyses were performed, including microarray analysis. There was an apparently homozygous mutation in exon 24, c.2465G>T, p.(*822Leuext62*), and a lack of correlation in the disease segregation analyses. This suggested the presence of a genomic rearrangement. In fact, a heterozygous deletion of the entire CAPN3 gene was found. This novel deletion comprised the terminal region of the GANC gene and the entire CAPN3 gene. This finding points out the need to reconsider and adapt our current strategy of molecular diagnosis in order to detect these types of genomic rearrangements that escape standard mutation screening procedures. Muscle Nerve 50: 448–453, 2014

Distal myopathies are a heterogeneous group of muscle diseases sharing the clinical pattern of predominant weakness in the feet and/or hands. The classical approach for molecular diagnosis is based on targeted gene-by-gene analysis guided by currently existing combinatorial algorithms.1 Many patients remain undiagnosed. Within the last 5 years, next-generation sequencing (NGS) has emerged as a successful and rapid approach to simultaneously analyse multiple genes in neuromuscular disorders.2 Our objective was to evaluate the efficiency of a targeted NGS approach using a panel of neuromuscular genes on patients with distal myopathies. We first tested its validity in a control group of six index cases (IC) with known molecular diagnosis. Then, we prospectively evaluated this approach by testing a group of 17 IC without molecular diagnosis. ### Patients We prospectively included 54 patients (37 IC and 17 relatives) with a diagnosis of distal myopathy, followed at the Neuromuscular Diseases and ALS Reference Centre of La Timone Hospital, Marseille, France, between 1989 and 2014. Among these 37 IC previously explored by Sanger sequencing, 20 IC had an identified molecular diagnosis: six IC constituted the control group. The remaining 17 undiagnosed IC constituted the test group. A targeted-NGS approach was used to search mutations in 298 neuromuscular genes in both groups. Samples analysed in this study have been prepared and stored by the Center of Biological Resources, Department of Medical Genetics, La Timone Hospital, Marseille, and used following the ethical recommendations of our institution and according to the Declaration of Helsinki. All included patients gave their written consent prior to …

International audience; no abstract

A multiprotein complex encompassing a transcription regulator, cardiac ankyrin repeat protein (CARP), and the calpain 3 protease was identified in the N2A elastic region of the giant sarcomeric protein titin. The present study aimed to investigate the function(s) of this complex in the skeletal muscle. We demonstrate that CARP subcellular localization is controlled by the activity of calpain 3: the higher the calpain 3, the more important the sarcomeric retention of CARP. This regulation would occur through cleavage of the N-terminal end of CARP by the protease. We show that, upon CARP over-expression, the transcription factor nuclear factor NF-κB p65 DNA-binding activity decreases. Taken as a whole, CARP and its regulator calpain 3 appear to occupy a central position in the important cell fate-governing NF-κB pathway. Interestingly, the expression of the atrophying protein MURF1, one of NF-κB main targets, remains unchanged in presence of CARP, suggesting that the pathway encompassing calpain 3/CARP/NF-κB does not play a role in muscle atrophy. With NF-κB also having anti-apoptotic effects, the inability of calpain 3 to lower CARP-driven inhibition of NF-κB could reduce muscle cell survival, hence partly accounting for the dystrophic pattern observed in limb girdle muscular dystrophy 2A, a pathology resulting from the protease deficiency.

In an attempt to identify potential therapeutic targets for the correction of muscle wasting, the gene expression of several pivotal proteins involved in protein metabolism was investigated in experimental atrophy induced by transient or definitive denervation, as well as in four animal models of muscular dystrophies (deficient for calpain 3, dysferlin, alpha-sarcoglycan and dystrophin, respectively). The results showed that: (a) the components of the ubiquitin-proteasome pathway are upregulated during the very early phases of atrophy but do not greatly increase in the muscular dystrophy models; (b) forkhead box protein O1 mRNA expression is augmented in the muscles of a limb girdle muscular dystrophy 2A murine model; and (c) the expression of cardiac ankyrin repeat protein (CARP), a regulator of transcription factors, appears to be persistently upregulated in every condition, suggesting that CARP could be a hub protein participating in common pathological molecular pathway(s). Interestingly, the mRNA level of a cell cycle inhibitor known to be upregulated by CARP in other tissues, p21(WAF1/CIP1), is consistently increased whenever CARP is upregulated. CARP overexpression in muscle fibres fails to affect their calibre, indicating that CARP per se cannot initiate atrophy. However, a switch towards fast-twitch fibres is observed, suggesting that CARP plays a role in skeletal muscle plasticity. The observation that p21(WAF1/CIP1) is upregulated, put in perspective with the effects of CARP on the fibre type, fits well with the idea that the mechanisms at stake might be required to oppose muscle remodelling in skeletal muscle.

International audience; Massively parallel sequencing, otherwise known as high-throughput or next-generation sequencing, is rapidly gaining wide use in clinical practice due to possibility of simultaneous exploration of multiple genomic regions. More than 300 genes have been implicated in neuromuscular disorders, meaning that many genes need to be considered in a differential diagnosis for a patient affected with myopathy. By providing sequencing information for numerous genes at the same time, massively parallel sequencing greatly accelerates the diagnostic processes of myopathies compared to the classical ``gene-after-gene'' approach by Sanger sequencing. In this review, we describe multiple advantages of this powerful sequencing method for applications in myopathy diagnosis. We also outline recent studies that used this approach to discover new myopathy-causing genes and to diagnose cohorts of patients with muscular disorders. Finally, we highlight the key aspects and limitations of massively parallel sequencing that a neurologist considering this test needs to know in order to interpret the results of the test and to deal with other issues concerning the test. (C) 2015 Elsevier Masson SAS. All rights reserved.

International audience; Dysferlinopathies are a family of disabling muscular dystrophies with LGMD2B and Miyoshi myopathy as the main phenotypes. They are associated with molecular defects in DYSF, which encodes dysferlin, a key player in sarcolemmal homeostasis. Previous investigations have suggested that exon skipping may be a promising therapy for a subset of patients with dysferlinopathies. Such an approach aims to rescue functional proteins when targeting modular proteins and specific tissues. We sought to evaluate the dysferlin functional recovery following exon 32 skipping in the cells of affected patients. Exon skipping efficacy was characterized at several levels by use of in vitro myotube formation assays and quantitative membrane repair and recovery tests. Data obtained from these assessments confirmed that dysferlin function is rescued by quasi-dysferlin expression in treated patient cells, supporting the case for a therapeutic antisense-based trial in a subset of dysferlin-deficient patients.

International audience; Background: Facioscapulohumeral muscular dystrophy type 1(FSHD1) is an autosomal dominant disorder associated with the contraction of D4Z4 less than 11 repeat units (RUs) on chromosome 4q35. Penetrance in the range of the largest alleles is poorly known. Our objective was to study the penetrance of FSHD1 in patients carrying alleles ranging between 6 to 10 RUs and to evaluate the influence of sex, age, and several environmental factors on clinical expression of the disease. Methods: A cross-sectional multicenter study was conducted in six French and one Swiss neuromuscular centers. 65 FSHD1 affected patients carrying a 4qA allele of 6-10 RUs were identified as index cases (IC) and their 119 at-risk relatives were included. The age of onset was recorded for IC only. Medical history, neurological examination and manual muscle testing were performed for each subject. Genetic testing determined the allele size (number of RUs) and the 4qA/4qB allelic variant. The clinical status of relatives was established blindly to their genetic testing results. The main outcome was the penetrance defined as the ratio between the number of clinically affected carriers and the total number of carriers. Results: Among the relatives, 59 carried the D4Z4 contraction. At the clinical level, 34 relatives carriers were clinically affected and 25 unaffected. Therefore, the calculated penetrance was 57% in the range of 6-10 RUs. Penetrance was estimated at 62% in the range of 6-8 RUs, and at 47% in the range of 9-10 RUs. Moreover, penetrance was lower in women than men. There was no effect of drugs, anesthesia, surgery or traumatisms on the penetrance. Conclusions: Penetrance of FSHD1 is low for largest alleles in the range of 9-10 RUs, and lower in women than men. This is of crucial importance for genetic counseling and clinical management of patients and families.

International audience; Massively parallel sequencing is rapidly becoming a widely used method in genetic diagnostics. However, there is still no clear consensus as to which approach can most efficiently identify the pathogenic mutations carried by a given patient, while avoiding false negative and false positive results. We developed a targeted exome approach (MyoPanel2) in order to optimize genetic diagnosis of neuromuscular disorders. Using this approach, we were able to analyse 306 genes known to be mutated in myopathies as well as in related disorders, obtaining 98.8% target sequence coverage at 20x. Moreover, MyoPanel2 was able to detect 99.7% of 11,467 known mutations responsible for neuromuscular disorders. We have then used several quality control parameters to compare performance of the targeted exome approach with that of whole exome sequencing. The results of this pilot study of 140 DNA samples suggest that targeted exome sequencing approach is an efficient genetic diagnostic test for most neuromuscular diseases.

International audience; Missense, iso-semantic, and intronic mutations are challenging for interpretation, in particular for their impact in mRNA. Various tools such as the Human Splicing Finder (HSF) system could be used to predict the impact on splicing; however, no diagnosis result could rely on predictions alone, but requires functional testing. Here, we report an in vitro approach to study the impact of DYSF mutations on splicing. It was evaluated on a series of 45 DYSF mutations, both intronic and exonic. We confirmed splicing alterations for all intronic mutations localized in 5 or 3 splice sites. Then, we showed that DYSF missense mutations could also result in splicing defects: mutations c.463G>A and c.2641A>C abolished ESEs and led to exon skipping; mutations c.565C>G and c.1555G>A disrupted Exonic Splicing Enhancer (ESE), while concomitantly creating new 5 or 3 splice site leading to exonic out of frame deletions. We demonstrated that 20% of DYSF missense mutations have a strong impact on splicing. This minigene strategy is an efficient tool for the detection of splicing defects in dysferlinopathies, which could allow for a better comprehension of splicing defects due to mutations and could improve prediction tools evaluating splicing defects.

International audience; Objective: We investigated the link between DNA hypomethylation and clinical penetrance in facioscapulohumeral dystrophy (FSHD) because hypomethylation is moderate and heterogeneous in patients and could not thus far be correlated with disease presence or severity. Methods: To investigate the link between clinical signs of FSHD and DNA methylation, we explored 95 cases (37 FSHD1, 29 asymptomatic individuals carrying a shortened D4Z4 array, 9 patients with FSHD2, and 20 controls) by implementing 2 approaches: methylated DNA immunoprecipitation and sodium bisulfite sequencing. Results: Both methods revealed statistically significant differences between asymptomatic carriers or controls and individuals with clinical FSHD, especially in the proximal region of the repeat. Absence of clinical expression in asymptomatic carriers is associated with a level of methylation similar to controls. Conclusions: We provide a proof of concept that the targeted approaches that we describe could be applied systematically to patient samples in routine diagnosis and suggest that local hypomethylation within D4Z4 might serve as a modifier for clinical expression of FSHD phenotype. Classification of evidence: This study provides Class III evidence that assays for hypomethylation within the D4Z4 region accurately distinguish patients with FSHD from individuals with D4Z4 contraction without FSHD.

Autosomal recessive muscular dystrophies are heterogeneous genetic disorders, with 39 genes currently implicated. Genetic diagnosis using targeted single-gene analysis by Sanger sequencing yields negative results in 10-20% of samples, warranting clinical re-evaluation and time-consuming testing of additional genes. This applies to dysferlinopathies caused by mutations in the gene encoding dysferlin (DYSF), which presents mainly as limb-girdle muscular dystrophy (LGMD) or distal myopathy.We evaluated exome sequencing associated with data filtering for selected genes as a second-tier approach for genetic diagnosis in a cohort of 37 patients with an initial negative result on targeted DYSF analysis.Exome sequencing allowed for establishing (16%) or suggesting (8%) the molecular diagnosis by implicating other known LGMD or distal myopathy genes or by revealing DYSF mutations previously missed using mutation-screening techniques with incomplete detection yields.Exome sequencing associated with data filtering constitutes an efficient second-tier analysis for genes implicated in LGMD or distal myopathies.

International audience; Facioscapulohumeralmuscular dystrophy (FSHD) is linked to copy-number reduction (N < 10) of the 4q D4Z4 subtelomeric array, in association with DUX4-permissive haplo-types. This main form is indicated as FSHD1. FSHD-like phenotypes may also appear in the absence of D4Z4 copy-number reduction. Variants of the SMCHD1 gene have been reported to associate with D4Z4 hypomethylation in DUX4-compatible hap-lotypes, thus defining FSHD2. Recently, mice carrying a muscle-specific knockout of the protocadherin gene Fat1 or its consti-tutive hypomorphic allele were shown to develop muscular and nonmuscular defects mimicking human FSHD. Here, we report FAT1 variants in a group of patients presenting with neuro-muscular symptoms reminiscent of FSHD. The patients do not carry D4Z4 copy-number reduction, 4q hypomethylation, or SM-CHD1 variants. However, abnormal splicing of the FAT1 transcript is predicted for all identified variants. To determine their pathogenicity, we elaborated a minigene approach coupled to an antisense oligonucleotide (AON) assay. In vitro, four out of five selected variants induced partial or complete alteration of splicing by creating new splice sites or modifying splicing regulators. AONs confirmed these effects. Altered transcripts may affect FAT1 protein interactions or stability. Altogether, our data suggest that defective FAT1 is associated with an FSHD-like phenotype. Hum Mutat 36:443–453, 2015. C 2015 Wiley Periodicals, Inc.

Limb-girdle muscular dystrophy type 2A (LGMD2A) due to mutations in the CAPN3 gene is one of the most common of autosomal recessive limb-girdle muscular dystrophies. We describe a patient who had a typical LGMD2A phenotype and posterior compartment involvement on MRI. Different genetic analyses were performed, including microarray analysis. There was an apparently homozygous mutation in exon 24, c.2465GT, p.(*822Leuext62*), and a lack of correlation in the disease segregation analyses. This suggested the presence of a genomic rearrangement. In fact, a heterozygous deletion of the entire CAPN3 gene was found. This novel deletion comprised the terminal region of the GANC gene and the entire CAPN3 gene. This finding points out the need to reconsider and adapt our current strategy of molecular diagnosis in order to detect these types of genomic rearrangements that escape standard mutation screening procedures.

Striatin, an intraneuronal, calmodulin-binding protein addressed to dendrites and spines, is expressed in the motor system, particularly the striatum and motoneurons. Striatin contains a high number of domains mediating protein–protein interactions, suggesting a role within a dendritic Ca2+-signaling pathway. Here, we explored the hypothesis of a direct role of striatin in the motor control of behaving rats, by using an antisense strategy based on oligodeoxynucleotides (ODN). Rats were treated by intracerebroventricular infusion of a striatin antisense ODN (A-ODN) or mismatch ODN (M-ODN) delivered by osmotic pumps over 6 days. A significant decrease in the nocturnal locomotor activity of A-ODN–treated rats was observed after 5 days of treatment. Hypomotricity was correlated with a 60% decrease in striatin content of the striata of A-ODN–treated rats sacrificed on day 6. Striatin thus plays a role in the control of motor function. To approach the cellular mechanisms in which striatin is involved, striatin down-regulation was studied in a comparatively simpler model: purified rat spinal motoneurons which retain their polarity in culture. Treatment of cells by the striatin A-ODN resulted in the impairement of the growth of dendrites but not axon. The decrease in dendritic growth paralleled the loss of striatin. This model allows analysis of the molecular basis of striatin function in the dynamic changes occurring in growing dendrites, and offers clues to unravel its function within spines. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 234–243, 1999

Introduction Les myopathies hereditaires a inclusions (hIBM) representent un groupe heterogene de pathologies musculaires d’origine genetique. Cette heterogeneite a la fois clinique et genetique complique le diagnostic moleculaire. Objectifs Evaluer l’interet du passage de l’approche diagnostique par sequencage gene par gene classique a l’approche par sequencage a haut debit (exome) pour un groupe de pathologies heterogenes (hIBM). Methodes Le departement de genetique medicale a Marseille effectue le diagnostic de routine d’une forme recessive d’hIBM associees au gene GNE (IBM2), en tant que laboratoire de reference national. Ainsi, au cours des 10 dernieres annees, 182 patients ont ete inclus pour une analyse du gene GNE par approche gene par gene. Parmi les patients dont le diagnostic n’a pu etre etabli par cette approche, 19 ont ete selectionnes sur le critere de la presence de vacuoles bordees et etudies par une approche exome. Resultats Le premier groupe constitue des 33 cas index diagnostiques (18 %) par sequencage classique pour une IBM2 a permis de mettre en evidence 13 nouveaux variants associes aux myopathies impliquant le gene GNE representant 8 % de tous les variants pathogenes deja associes a ce type de myopathie. Dans le second groupe, constitue des 19 cas index selectionnes parmi les patients non diagnostiques apres l’analyse par sequencage classique du gene GNE, le diagnostic moleculaire a ete etabli chez 42 % des patients. Discussion Ce projet a permis de caracteriser sur le plan moleculaire la premiere grande cohorte francaise de patients atteints d’IBM2. De plus l’approche de sequencage a haut debit (exome) utilisee en deuxieme intention nous a permis, d’une part, d’eprouver notre pipeline d’analyse et, d’autre part, de determiner le diagnostic moleculaire chez 42 % des patients ouvrant la voie au diagnostic de routine des hIBM par sequencage a haut debit. Conclusion Ce projet permet d’apprecier les limites de l’approche par sequencage gene par gene pour ces pathologies heterogenes, nous conduisant ainsi progressivement vers des strategies diagnostiques par sequencage a haut debit.

Striatin, a 110-kDa protein, is the first member of the tryptophane-aspartate repeat protein family known to bind calmodulin in the presence of Ca2+. We examined the distribution of striatin and its mRNA in the rat central nervous system (CNS) by using immunocytochemistry and in situ hybridization, respectively. Striatin immunostaining and mRNA labeling patterns are generally concordant. Regions showing the most intense staining are the dorsal striatum, nucleus accumbens (anterior and shell parts), olfactory tubercle, red nucleus, subthalamic nucleus, cranial nerve motor nuclei, and layer IX of the spinal cord (motoneurons). Low levels of both striatin and its mRNA are detected in the cerebral cortex, thalamus, septum, amygdala, hippocampus, midbrain and cerebellum. Striatin-immunoreactive neuronal processes are found predominantly in the structures containing striatin-positive neurons, suggesting that these labeled processes represent dendritic arborization rather than axonal processes. Except for the medial forebrain bundle, all axonal fiber tracts examined are devoid of striatin immunolabeling. These data show that the somatodendritic localization of striatin, previously described in the striatum, may be a main feature of the subcellular distribution of this protein throughout the CNS. Although widely distributed in neurons throughout the rat CNS, striatin is expressed prominently in the structures belonging to the motor system, suggesting that this protein may play a preponderant role in motor control. J. Comp. Neurol. 397:41–59, 1998. © 1998 Wiley-Liss, Inc.

Rat striatin, a recently discovered calmodulin-binding protein belonging to the WD repeat family, is expressed in neurons, mostly in the striatum and motor and olfactory systems. Striatin is localized in the somato-dendritic compartment of neurons, mainly in the spines. It may play a role in dendritic Ca2+signaling. Here we report the cloning and sequencing of human striatin cDNA (HGMW-approved symbol STRN), the localization of the gene to chromosome 2p22–p21, and its preferential expression in brain. The human cDNA sequence is predicted to encode a 780-amino-acid protein possessing eight WD repeats. Striatin is highly conserved between rat and human with 96% identity and 98% similarity at the amino acid level. Since theCaenorabditis elegansgenome also contains a closely related striatin coding sequence, the function of striatin is likely to be well conserved.

International audience; Facio-scapulo-humeral dystrophy (FSHD) results from deletions in the subtelomeric macrosatellite D4Z4 array on the 4q35 region. Upregulation of the DUX4 retrogene from the last D4Z4 repeated unit is thought to underlie FSHD pathophysiology. However, no one knows what triggers muscle defect and when alteration arises. To gain further insights into the molecular mechanisms of the disease, we evaluated at the molecular level, the perturbation linked to the FSHD genotype with no a priori on disease onset, severity or penetrance and prior to any infiltration by fibrotic or adipose tissue in biopsies from fetuses carrying a short pathogenic D4Z4 array (n 5 6) compared with fetuses with a non-pathogenic D4Z4 array (n 5 21). By measuring expression of several muscle-specific markers and 4q35 genes including the DUX4 retrogene by an RT-PCR and western blotting, we observed a global dysregulation of genes involved in myogenesis including MYOD1 in samples with

Background— Genetic defects in calpain3 ( CAPN3 ) lead to limb-girdle muscular dystrophy type 2A, a disease of the skeletal muscle that affects predominantly the proximal limb muscles. We previously demonstrated the potential of adeno-associated virus–mediated transfer of the CAPN3 gene to correct the pathological signs in a murine model for limb-girdle muscular dystrophy type 2A after intramuscular and locoregional administrations. Methods and Results— Here, we showed that intravenous injection of calpain3-expressing vector in mice can induce mortality in a dose-dependent manner. An anatomopathological investigation revealed large areas of fibrosis in the heart that we related to unregulated proteolytic activity of calpain3. To circumvent this toxicity, we developed new adeno-associated virus vectors with skeletal muscle–restricted expression by using new muscle-specific promoters that include the CAPN3 promoter itself and by introducing a target sequence of the cardiac-specific microRNA-208a in the cassette. Our results show that CAPN3 transgene expression can be successfully suppressed in the cardiac tissue, preventing the cardiac toxicity, whereas expression of the transgene in skeletal muscle reverts the pathological signs of calpain3 deficiency. Conclusions— The molecular strategies used in this study may be useful for any gene transfer strategy with potential toxicity in the heart.

Background The complexity of the skeletal muscle and the identification of numerous human disease-causing mutations in its constitutive proteins make it an interesting tissue for proteomic studies aimed at understanding functional relationships of interacting proteins in both health and diseases. Method We undertook a large-scale study using two-hybrid screens and a human skeletal-muscle cDNA library to establish a proteome-scale map of protein-protein interactions centered on proteins involved in limb-girdle muscular dystrophies (LGMD). LGMD is a group of more than 20 different neuromuscular disorders that principally affect the proximal pelvic and shoulder girdle muscles. Results and conclusion The interaction network we unraveled incorporates 1018 proteins connected by 1492 direct binary interactions and includes 1420 novel protein-protein interactions. Computational, experimental and literature-based analyses were performed to assess the overall quality of this network. Interestingly, LGMD proteins were shown to be highly interconnected, in particular indirectly through sarcomeric proteins. In-depth mining of the LGMD-centered interactome identified new candidate genes for orphan LGMDs and other neuromuscular disorders. The data also suggest the existence of functional links between LGMD2B/dysferlin and gene regulation, between LGMD2C/γ-sarcoglycan and energy control and between LGMD2G/telethonin and maintenance of genome integrity. This dataset represents a valuable resource for future functional investigations.

International audience; Tubular aggregates are regular arrays of membrane tubules accumulating in muscle with age. They are found as secondary features in several muscle disorders, including alcohol- and drug-induced myopathies, exercise-induced cramps, and inherited myasthenia, but also exist as a pure genetic form characterized by slowly progressive muscle weakness. We identified dominant STIM1 mutations as a genetic cause of tubular-aggregate myopathy (TAM). Stromal interaction molecule 1 (STIM1) is the main Ca2+ sensor in the endoplasmic reticulum, and all mutations were found in the highly conserved intraluminal Ca2+-binding EF hands. Ca2+ stores are refilled through a process called store-operated Ca2+ entry (SOCE). Upon Ca2+-store depletion, wild-type STIM1 oligomerizes and thereby triggers extracellular Ca2+ entry. In contrast, the missense mutations found in our four TAM-affected families induced constitutive STIM1 clustering, indicating that Ca2+ sensing was impaired. By monitoring the calcium response of TAM myoblasts to SOCE, we found a significantly higher basal Ca2+ level in TAM cells and a dysregulation of intracellular Ca2+ homeostasis. Because recessive STIM1 loss-of-function mutations were associated with immunodeficiency, we conclude that the tissue-specific impact of STIM1 loss or constitutive activation is different and that a tight regulation of STIM1-dependent SOCE is fundamental for normal skeletal-muscle structure and function.

International audience; no abstract

International audience; Mutations in the dysferlin gene are the cause of Limb-girdle Muscular Dystrophy type 2B and Miyoshi Myopathy. The dysferlin protein has been implicated in sarcolemmal resealing, leading to the idea that the pathophysiology of dysferlin deficiencies is due to a deficit in membrane repair. Here, we show using two different approaches that fullfiling membrane repair as asseyed by laser wounding assay is not sufficient for alleviating the dysferlin deficient pathology. First, we generated a transgenic mouse overexpressing myoferlin to test the hypothesis that myoferlin, which is homologous to dysferlin, can compensate for the absence of dysferlin. The myoferlin overexpressors show no skeletal muscle abnormalities, and crossing them with a dysferlin-deficient model rescues the membrane fusion defect present in dysferlin-deficient mice in vitro. However, myoferlin overexpression does not correct muscle histology in vivo. Second, we report that AAV-mediated transfer of a minidysferlin, previously shown to correct the membrane repair deficit in vitro, also fails to improve muscle histology. Furthermore, neither myoferlin nor the minidysferlin prevented myofiber degeneration following eccentric exercise. Our data suggest that the pathogenicity of dysferlin deficiency is not solely related to impairment in sarcolemmal repair and highlight the care needed in selecting assays to assess potential therapies for dysferlinopathies.

International audience; Sarcoglycanopathies (SGP) are a group of autosomal recessive muscle disorders caused by primary mutations in one of the four sarcoglycan genes. The sarcoglycans (alpha-, beta-, gamma-, and delta-sarcoglycan) form a tetrameric complex at the muscle membrane that is part of the dystrophin-glycoprotein complex and plays an essential role for membrane integrity during muscle contractions. We previously showed that the most frequent missense mutation in alpha-sarcoglycan (p.R77C) leads to the absence of the protein at the cell membrane due to its blockade by the endoplasmic reticulum (ER) quality control. Moreover, we demonstrated that inhibition of the ER alpha-mannosidase I activity using kifunensine could rescue the mutant protein localization at the cell membrane. Here, we investigate 25 additional disease-causing missense mutations in the sarcoglycan genes with respect to intracellular fate and localization rescue of the mutated proteins by kifunensine. Our studies demonstrate that, similarly to p.R77C, 22 of 25 of the selected mutations lead to defective intracellular trafficking of the SGs proteins. Six of these were saved from ER retention upon kifunensine treatment. The trafficking of SGs mutants rescued by kifunensine was associated with mutations that have moderate structural impact on the protein. Hum Mutat 33:429-439, 2012. (C) 2011 Wiley Periodicals, Inc.

International audience; no abstract

International audience; Mutations in the dysferlin gene (DYSF) lead to a complete or partial absence of the dysferlin protein in skeletal muscles and are at the origin of dysferlinopathies, a heterogeneous group of rare autosomal recessive inherited neuromuscular disorders. As a step towards a better understanding of the DYSF mutational spectrum, and towards possible inclusion of patients in future therapeutic clinical trials, we set up the Universal Mutation Database for Dysferlin (UMD-DYSF), a Locus-Specific Database developed with the UMD (R) software. The main objective of UMD-DYSF is to provide an updated compilation of mutational data and relevant interactive tools for the analysis of DYSF sequence variants, for diagnostic and research purposes. In particular, specific algorithms can facilitate the interpretation of newly identified intronic, missense- or isosemantic-exonic sequence variants, a problem encountered recurrently during genetic diagnosis in dysferlinopathies. UMD-DYSF v1.0 is freely accessible at www.umd.be/DYSF/. It contains a total of 742 mutational entries corresponding to 266 different disease-causing mutations identified in 558 patients worldwide diagnosed with dysferlinopathy. This article presents for the first time a comprehensive analysis of the dysferlin mutational spectrum based on all compiled DYSF disease-causing mutations reported in the literature to date, and using the main bioinformatics tools offered in UMD-DYSF.

Identifying the piece of DNA culprit of a genetic disease has not been as straightforward as expected with the evolution of sequencing capabilities. This difficulty is exemplified by facioscapulohumeral dystrophy (FSHD) study. Unexpectedly, contributions in completing this picture recently came from mice models carrying a muscle specific knock-out of the protocadherin gene Fat1 or its constitutive hypomorphic allele. Both have been shown to develop muscular and non-muscular defects mimicking human FSHD. In humans, this disease is linked to copy number reduction ( n

A multiprotein complex encompassing a transcription regulator, cardiac ankyrin repeat protein (CARP), and the calpain 3 protease was identified in the N2A elastic region of the giant sarcomeric protein titin. The present study aimed to investigate the function(s) of this complex in the skeletal muscle. We demonstrate that CARP subcellular localization is controlled by the activity of calpain 3: the higher the calpain 3, the more important the sarcomeric retention of CARP. This regulation would occur through cleavage of the N-terminal end of CARP by the protease. We show that, upon CARP over-expression, the transcription factor nuclear factor NF-κB p65 DNA-binding activity decreases. Taken as a whole, CARP and its regulator calpain 3 appear to occupy a central position in the important cell fate-governing NF-κB pathway. Interestingly, the expression of the atrophying protein MURF1, one of NF-κB main targets, remains unchanged in presence of CARP, suggesting that the pathway encompassing calpain 3/CARP/NF-κB does not play a role in muscle atrophy. With NF-κB also having anti-apoptotic effects, the inability of calpain 3 to lower CARP-driven inhibition of NF-κB could reduce muscle cell survival, hence partly accounting for the dystrophic pattern observed in limb girdle muscular dystrophy 2A, a pathology resulting from the protease deficiency.

International audience; Dysferlinopathies are autosomal recessive, progressive muscle dystrophies caused by mutations in DYSF, leading to a loss or a severe reduction of dysferlin, a key protein in sarcolemmal repair. Currently, no etiological treatment is available for patients affected with dysferlinopathy. As for other muscular dystrophies, gene therapy approaches based on recombinant adeno-associated virus (rAAV) vectors are promising options. However, because dysferlin messenger RNA is far above the natural packaging size of rAAV, full-length dysferlin gene transfer would be problematic. In a patient presenting with a late-onset moderate dysferlinopathy, we identified a large homozygous deletion, leading to the production of a natural ``minidysferlin'' protein. Using rAAV-mediated gene transfer into muscle, we demonstrated targeting of the minidysferlin to the muscle membrane and efficient repair of sarcolemmal lesions in a mouse model of dysferlinopathy. Thus, as previously demonstrated in the case of dystrophin, a deletion mutant of the dysferlin gene is also functional, suggesting that dysferlin's structure is modular. This minidysferlin protein could be used as part of a therapeutic strategy for patients affected with dysferlinopathies.

International audience; Dysferlinopathies are autosomal recessive muscular dystrophies caused by DYSF mutations, Which lead to a reduced amount or a complete lack of dysferlin. One step in dysferlinopathies diagnosis consists in Western blot analysis of proteins extracted from Muscle biopsy, or blood monocytes. We have taken advantage of dysferlin expression in monocytes to develop a whole blood flow cytometry (WBFC), using antibodies directed against dysferlin. Six patients were submitted to WBFC analysis and immunofluorescence analysis oil monocytes. Results obtained are correlated to Western blot from monocytes and muscle biopsies. The possible usefulness of this flow cytometry analysis in routine diagnosis is presented. (C) 2009 Elsevier B.V. All rights reserved.

We read with interest but also some surprise, the recent ‘Therapeutic exon skipping for Dysferlinopathies?' article by Aartsma-Rus et al,1 published in the Eur J Hum Genet (advance online publication, 10 February 2010; doi:10.1038/ejhg.2010.4). This report contains some inaccuracies and mistakes, and we do not agree with some of its main contents. Therefore, for the sake of the scientific and patients communities, we consider it essential to point out and discuss some of the results and subsequent conclusions appearing in the ‘results and discussion' section, as well as in figures and tables. Antisense-mediated exon skipping constitutes a relevant therapy for several genetic diseases associated with premature termination codons (PTCs) or frameshift mutations lying in dispensable in frame exons. However, knowledge of the specific targeted gene, mRNA and resulting protein, is, among others, an essential pre-requisite toward designing pertinent exon-skipping strategies. In the report by Aartsma-Rus et al,1 it appears that several of these pre-requisites have been overlooked. Here we intend to discuss and contradict some important assertions and results that appear in both the text and the figures of the article by Aartsma-Rus et al,1 and place our comments in the perspective of a recently published report from our group on the same matter.2

International audience; Mutations in DYSF encoding dysferlin cause primary dysferlinopathies, autosomal recessive diseases that mainly present clinically as Limb Girdle Muscular Dystrophy type 213 and Miyoshi myopathy. More than 350 different sequence variants have been reported in DYSE. Like dystrophin, the size of the dysferlin mRNA is above the limited packaging size of AAV vectors. Alternative strategies to AAV gene transfer in muscle cells must then be addressed for patients. A gene therapy approach for Duchenne muscular dystrophy was recently developed, based on exon-skipping strategy. Numerous sequences are recognized by splicing protein complexes and, when specifically blocked by antisense oligoucleotides (AON), the corresponding exon is skipped. We hypothesized that this approach could be useful for patients affected with dysferlinopathics. To confirm this assumption, exon 32 was selected as a prioritary target for exon skipping strategy. This option was initially driven by the report from Sinnreich and colleagues of a patient with a very mild and late-onset phenotype associated to a natural skipping of exon 32. Three different antisense oligonucleotides were tested in myoblasts generated from control and patient MyoD transduced fibroblasts, either as oligonucleotides or after incorporation into lentiviral vectors. These approaches led to a high efficiency of exon 32 skipping. Therefore, these results seem promising, and could be applied to several other exons in the DYSF gene. Patients carrying mutations in exons whose the in-frame suppression has been proven to have no major consequences on the protein function, might benefit of exon-skipping based gene correction.

International audience; Mutations in the gene encoding dysferlin (DYSF; MIM# 603009, 2p13, GenBank NM\₀03494.2) cause primary dysferlinopathies, which are autosomal recessive muscular dystrophies. DYSF has a large mutational spectrum, and genetic diagnosis is complicated by incomplete mutation detection rates. Recently, novel dysferlin transcripts were characterized by identifying alternative exons 1 of DYSF-v1 (GenBank DQ267935), exon 5a (GenBank DQ976379), and exon 40a (GenBank EF015906). To evaluate the frequency of possible mutations in the newly identified DYSF alternative exons, we screened the corresponding genomic regions for mutations in a cohort of 26 patients, carrying only one mutation undoubtedly considered as disease causing in the 55 canonical DYSF exons. No disease-causing mutation was identified in alternative exons 1 of DYSF-v1, exon 5a, and exon 40a, demonstrating a low frequency of disease-causing mutations in these exons.

International audience; Mechanically damaged plasma membrane undergoes rapid calcium-dependent resealing that appears to depend, at least in part, on calpain-mediated cortical cytoskeletal remodeling. Cells null for Capns1, the noncatalytic small subunit present in both m- and mu-calpains, do not undergo calcium-mediated resealing. However, it is not known which of these calpains is needed for repair, or whether other major cytosolic proteinases may participate. Utilizing isozyme-selective siRNAs to decrease expression of Capn1 or Capn2, catalytic subunits of mu- and m-calpains, respectively, in a mouse embryonic fibroblast cell line, we now show that substantial loss of both activities is required to compromise calcium-mediated survival after cell scrape-damage. Using skeletal myotubes derived from Capn3-null mice, we were unable to demonstrate loss of sarcolemma resealing after needle scratch or laser damage. Isolated muscle fibers from Capn3 knockout mice also efficiently repaired laser damage. Employing either a cell line expressing a temperature sensitive El ubiquitin ligase, or lactacystin, a specific proteasome inhibitor, it was not possible to demonstrate an effect of the proteasome on calcium-mediated survival after injury. Moreover, several cell-permeant caspase inhibitors were incapable of significantly decreasing survival or inhibiting membrane repair. Taken together with previous studies, the results show that m- or p-calpain can facilitate repair of damaged plasma membrane. While there was no evidence for the involvement of calpain-3, the proteasome or caspases in early events of plasma membrane repair, our studies do not rule out their participation in downstream events that may link plasma membrane repair to adaptive remodeling after injury. (C) 2009 Elsevier B.V. All rights reserved.

In an attempt to identify potential therapeutic targets for the correction of muscle wasting, the gene expression of several pivotal proteins involved in protein metabolism was investigated in experimental atrophy induced by transient or definitive denervation, as well as in four animal models of muscular dystrophies (deficient for calpain 3, dysferlin, alpha-sarcoglycan and dystrophin, respectively). The results showed that: (a) the components of the ubiquitin-proteasome pathway are upregulated during the very early phases of atrophy but do not greatly increase in the muscular dystrophy models; (b) forkhead box protein O1 mRNA expression is augmented in the muscles of a limb girdle muscular dystrophy 2A murine model; and (c) the expression of cardiac ankyrin repeat protein (CARP), a regulator of transcription factors, appears to be persistently upregulated in every condition, suggesting that CARP could be a hub protein participating in common pathological molecular pathway(s). Interestingly, the mRNA level of a cell cycle inhibitor known to be upregulated by CARP in other tissues, p21(WAF1/CIP1), is consistently increased whenever CARP is upregulated. CARP overexpression in muscle fibres fails to affect their calibre, indicating that CARP per se cannot initiate atrophy. However, a switch towards fast-twitch fibres is observed, suggesting that CARP plays a role in skeletal muscle plasticity. The observation that p21(WAF1/CIP1) is upregulated, put in perspective with the effects of CARP on the fibre type, fits well with the idea that the mechanisms at stake might be required to oppose muscle remodelling in skeletal muscle.

International audience; Limb girdle muscular dystrophy type 2D (LGMD2D, OMIM600119) is a genetic progressive myopathy that is caused by mutations in the human alpha-sarcoglycan gene (SGCA). Here, we have introduced in mice the most prevalent LGMD2D mutation, R77C. It should be noted that the natural murine residue at this position is a histidine. The model is, therefore, referred as Sgca(H77C/H77C). Unexpectedly, we observed an absence of LGMD2D-like phenotype at histological or physiological level. Using a heterologous cellular model of the sarcoglycan complex formation, we showed that the R77C allele encodes a protein that fails to be delivered to its proper cellular localization in the plasma membrane, and consequently to the disappearance of a positively charged residue. Subsequently, we transferred an AAV vector coding for the human R77C protein in the muscle of Sgca-null mice and were able to pharmacologically rescue the R77C protein from endoplasmic reticulum-retention using proteasome or mannosidase I inhibitors. This suggests a therapeutic approach for LGMD2D patients carrying mutations that impair alpha-sarcoglycan trafficking.

International audience; Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in the cysteine protease calpain 3 (CAPN3) that leads to selective muscle wasting. We previously showed that CAPN3 deficiency is associated with a profound perturbation of the NF-kappa B/I kappa B alpha survival pathway. In this study, the consequences of altered NF-kappa B/I kappa B alpha pathway were investigated using biological materials from LGMD2A patients. We first show that die antiapoptotic factor cellular-FUCE inhibitory protein (c-FLIP), which is dependent on the NF-kappa B pathway in normal muscle cells, is down-regulated in LGMD2A biopsies. In muscle cells isolated from LGMD2A patients, NF-kappa B is readily activated on cytokine induction as shown by an increase in its DNA binding activity. However, we observed discrepant transcriptional responses depending on the NF-kappa B target genes. I kappa B alpha is expressed following NF-kappa B activation independent of the CAPN3 status, whereas expression of c-FLIP is obtained only when CAPN3 is present. These data lead us to postulate that CAPN3 intervenes in the regulation of the expression of NF-kappa B-dependent survival genes to prevent apoptosis in skeletal muscle. Deregulations in the NF-kappa B pathway could be part of the mechanism responsible for the muscle wasting resulting from CAPN3 deficiency.

International audience; Myostatin is a negative regulator of muscle mass whose inhibition has been proposed as a therapeutic strategy for muscle-wasting conditions. Indeed, blocking myostatin action through different strategies has proved beneficial for the pathophysiology of the dystrophin-deficient mdx mouse. In this report, we tested the inhibition of myostatin by AAV-mediated expression of a mutated propeptide in animal models of two limb-girdle muscular dystrophies: LGMD2A caused by mutations in the calpain 3 ( CAPN3) gene and LGMD2D caused by mutations in the alpha-sarcoglycan gene (SGCA). In the highly regenerative Sgca-null mice, survival of the alpha-sarcoglycan-deficient muscle fibers did not improve after transfer of the myostatin propeptide. In calpain 3-deficient mice, a boost in muscle mass and an increase in absolute force were obtained, suggesting that myostatin inhibition could constitute a therapeutic strategy in this predominantly atrophic disorder.

International audience; alpha-Sarcoglycanopathy (limb-girdle muscular dystrophy type 2D, LGMD2D) is a recessive muscular disorder caused by deficiency in alpha-sarcoglycan, a transmembrane protein part of the dystrophin-associated complex. To date, no treatment exists for this disease. We constructed recombinant pseudotype-1 adeno-associated virus (rAAV) vectors expressing the human alpha-sarcoglycan cDNA from a ubiquitous or a muscle-specific promoter. Evidence of specific immune response leading to disappearance of the vector was observed with the ubiquitous promoter. In contrast, efficient and sustained transgene expression with correct sarcolemmal localization and without evident toxicity was obtained with the muscle-specific promoter after intra-arterial injection into the limbs of an LGMD2D murine model. Transgene expression resulted in restoration of the sarcoglycan complex, histological improvement, membrane stabilization, and correction of pseudohypertrophy. More importantly, alpha-sarcoglycan transfer produced full rescue of the contractile force deficits and stretch sensibility and led to an increase of the global activity of the animals when both posterior limbs are injected. Our results establish the feasibility for AAV-mediated alpha-sarcoglycan gene transfer as a therapeutic approach.

International audience; Muscular dystrophies are hereditary degenerative muscle diseases that cause life-long disability in patients. They comprise the well-known Duchenne Muscular Dystrophy (DMD) but also the group of Limb Girdle Muscular Dystrophies (LGMD) which account for a third to a fourth of DMD cases. From the clinical point of view, LGMD are characterised by predominant effects on the proximal limb muscles. The LGMD group is still growing today and consists of 19 autosomal dominant and recessive forms (LGMD1A to LGMD1G and LGMD2A to LGMD2M). The proteins involved are very diverse and include sarcomeric, sarcolemmal and enzymatic proteins. With respect to this variability and in line with the intense search for a potent therapeutic approach for DMD, many different strategies have been tested in rodent models. These include replacing the lost function by gene transfer or stem cell transplantation, using a related protein for functional substitution, increasing muscle mass, or blocking the molecular pathological mechanisms by pharmacological means to alleviate the symptoms. The purpose of this review is to summarize current data arising from these preclinical studies and to examine the potential of the tested strategies to lead to clinical applications. (c) 2007 Elsevier Ltd. All rights reserved.