No description available.

No description available.

Facioscapulohumeral muscular dystrophy (FSHD) is a progressive muscle-wasting disease that affects about 7/100,000 people. The first symptoms usually appear in the muscles of the shoulder, the face and the foot. The molecular cause of FSHD has been traced to the macrosatellite D4Z4, located on the chromosome 4 subtelomere. D4Z4 is usually composed of 11- 120 copies of 3.3 kb repeats with identical DNA sequences. The repeats are oriented head-to-tail in a tandem array, and there is a copy of the intronless gene DUX4 embedded within each. Healthy individuals have large D4Z4 macrosatellites with more than 11 repeats; the high repeat number is linked to the epigenetic silencing of D4Z4. Most FSHD patients, however, only have 1- 10 D4Z4 repeats (,FSHD 1 '). This causes the D4Z4 chromatin to become 'de-repressed ' and epigenetically active, resulting in the aberrant production of DUX4 transcripts. These transcripts are thought to cause the muscle wasting symptoms in FSHD. Interestingly, a small number of 'FSHD2' patients share the chromatin de-repression and DUX4 expression with FSHDl patients, without having a reduction in D4Z4 repeat number. In this thesis, I discuss two different aspects of this unusual disease. The function of DUX genes is still unknown, and a better understanding of their evolutionary history would aid the interpretation of functional studies of related homologues. Using a combination of bioinformatics and laboratory experiments, I dissect the evolutionary origins of DUX4 and other DUX genes in mammals. I also show that the unusual high-copy tandem array arrangement of DUX4 is shared by the related gene DUXC. My data suggest a model for the origin of DUX4, its intron-loss and its high copy number. It is unknown what triggers the epigenetic D4Z4 changes in FSHD2. Here, I report the molecular characterisation of patients with FSHD2, who may participate in a future exome sequencing mutation screen. In a proof-of-principle study, I demonstrate how exome sequencing can be used to correct a clinical misdiagnosis. i

Duchenne muscular dystrophy (DMD) is an X-linked muscle wasting disorder caused by mutations in the gene for dystrophin, a 427kDa cytoskeletal protein important for maintaining the integrity of muscle fibres. A number of DMD mutations result in an absence of functional protein due to disruption of the translational reading frame. It has been shown previously that antisense oligonucleotide (AON) reagents can modulate dystrophin pre-mRNA splicing to specifically exclude an out-of-frame exon from the mRNA. This restores the open reading frame resulting in production of a semi-functional internally-truncated dystrophin protein, mimicking what occurs in the milder allelic Becker muscular dystrophy (BMD). Previous work in this laboratory demonstrated successful exclusion of nonsense mutation carrying exon 23 in the mdx mouse model of DMD. This thesis is concerned with extension of the investigation by examining the bioactivity of alternative AON backbone chemistries 2' -O-methyl phosphorothioate (20Me), peptide nucleic acid (PNA) and phosphorodiamidate morpholino oligomer (PMO) targeting the 5'splice site of exon 23 in vitro and in vivo. In cultured murine muscle cells both the 20Me and PMO-based AON reagents induced detectable exon 23 exclusion. In the mdx mouse model intramuscular delivery of the 20Me-based AON reagent resulted in de novo dystrophin expression correctly localised at the sarcolemma that persisted for up to 4 weeks after a single dose. The PMO-based reagent resulted in de novo dystrophin expression that persisted for up to 10 weeks after a single intramuscular dose and for up to 8 weeks after a single intravenous dose. To broaden the investigation nine additional murine dystrophin exons were selected and AON regents designed targeting the 5'splice site and putative exonic splicing enhancer (ESE) sequences. Overall the results demonstrate that the AONs employed here induce detectable, reproducible exclusion of exon 23. The PMO-based reagent is currently superior for modulation of dystrophin pre-mRNA splicing.

Mutations in C0L6A], COL6A2 and COL6A3, the genes which encode the extracellular matrix component collagen VI, lead to Bethlem myopathy BM Ullrich Congenital Muscular Dystrophy (UCMD) and myosclerosis myopathy. Unlike UCMD, BM is difficult to diagnose due to its clinical overlap with other contractural phenotypes and the lack of sensitivity of standard immunohistochemica! diagnostic techniques in muscle biopsy samples. This thesis investigates the potential of two prospective diagnostic techniques, and proposes an algorithm for better BM diagnosis based on immunofluorescent labelling of collagen VI in skin biopsy-derived fibroblast cells.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder caused by absence of functional dystrophin protein. This thesis describes investigations into the role of small non-coding RNAs in both DMD pathology, and as potential therapeutic molecules. MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression and are implicated in wide-ranging cellular processes and pathological conditions. This study has compared differential miRNA expression in proximal and distal limb muscles, diaphragm, heart and serum in the mdx dystrophic mouse model relative to wild-type controls. Global transcriptome analysis revealed muscle-specific patterns of differential miRNA expression as well as commonalities between tissues, including previously identified dystromirs. miR-1, miR-133a and miR-206 were found to be highly abundant in mdx serum, suggesting that these miRNAs are promising disease biomarkers. Indeed, the relative serum levels of these miRNAs were normalised in response to peptide-PMO mediated dystrophin restoration therapy. This study has revealed further complexity in the miRNA transcriptome of the mdx mouse, an understanding of which will be valuable for the development of novel DMD therapeutics and for monitoring their efficacy. Myostatin is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as DMD. This study describes a novel myostatin inhibition approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in cultured myotubes. Silencing was sensitive to treatment with the histone deacetylase inhibitor Trichostatin A, and the silent state chromatin mark H3K9me2 was enriched at the myostatin promoter following siRNA transfection, suggesting epigenetic remodelling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for myostatin and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders. The work in this thesis therefore demonstrates the potential of small RNAs as therapeutic agents and as disease biomarkers in the context of DMD.

No description available.

Les myopathies inflammatoires sont des maladies auto-immunes rares dont le dénominateur commun est la faiblesse musculaire et l'inflammation. Leur origine n’est pas connue et les traitements sont conventionnels partiellement efficaces. Par une approche épidémiologique, nous avons montré que l’étude de l’incidence et de la prévalence est un outil utile pour mettre en évidence des déterminants des myopathies inflammatoires. Une meilleure identification et une meilleure classification des patients atteints de ces maladies sont cependant nécessaires pour préciser l’épidémiologie des myopathies inflammatoires.Par une approche translationnelle, nous avons montré que, par rapport aux autres myopathies inflammatoires, des dysfonctions mitochondriales périfasciculaires sont une caractéristique des dermatomyosites, qui jouent un rôle dans l’intolérance à l’effort et le maintien de l’inflammation. Ces résultats ouvrent des nouvelles voies pour mieux comprendre et traités les myopathies inflammatoires. / Inflammatory myopathies are rare autoimmune diseases whose common denominator is muscle weakness and inflammation. Their origin is not known and conventional treatments are partially effective. Using an epidemiological approach, we have shown that the study of incidence and prevalence is an useful tool for identifying determinants of inflammatory myopathies. However, better identification and classification of patients is mandatory to refine the epidemiology of inflammatory myopathies. Using a translational approach, we have shown that, compared with other inflammatory myopathies, perifascicular mitochondrial dysfunctions are a characteristic of dermatomyositis, which play a role in exercise intolerance and in the maintenance of inflammation. These results open up new avenues to better understand and treat inflammatory myopathies.

Les myopathies congénitales sont des maladies neuromusculaires dont le diagnostic est établi grâce aux données cliniques, histologiques et génétique. Cependant, le diagnostic génétique est manquant pour la moitié des patients, ce qui suggère de nouveaux gènes impliqués. Le but de mon projet était d'identifier de nouveaux gènes de myopathies congénitales et de valider l'impact des mutations trouvées. En utilisant une stratégie d'analyse de séquençage d'exomes de patients déjà exclus pour les gènes connus, nous avons mis en évidence deux nouveaux gènes impliqués dans les myopathies congénitales. Des mutations récessives dans le gène PYROXD1, codant pour une oxydoréductase, causent une myopathie apparaissant à l'enfance avec des défauts spécifiques en histologie. Grâce à un modèle animal, nous avons montré que les mutations impactaient l'activité enzymatique de la protéine. Des mutations dominantes ou récessives dans le gène CACNA1S causent une myopathie avec un phénotype similaire pour toutes les mutations. Les études fonctionnelles ont montré que les mutations causaient un défaut dans le couplage excitation-contraction. / Congenital myopathies are neuromuscular diseases diagnosed by clinical, histological and genetic data. However, the genetic diagnosis is missing for half of the patients, suggesting new genes involved. The goal of my project was to identify new genes of congenital myopathies and validate the impact of the mutations. Using a strategy of analyzing DNA sequencing of patients already excluded for known genes, we have identified two new genes involved in congenital myopathies. Recessive mutations in the PYROXD1 gene, encoding an oxidoreductase, cause a myopathy with childhood-onset and a histology specific spectra. Functionnal studies showed that the mutations have an effect on the enzymatic activity of the protein. We showed that dominant or recessive mutations in the CACNA1S gene cause a neonatal onset myopathy with a similar phenotype for all found mutations.

Actuellement, plus de soixante-dix dystrophies musculaires différentes ont été caractérisées cliniquement. La pathogenèse des différents types de dystrophies musculaire implique de nombreuses voies biologiques et s'accompagne d'une grande variabilité de signes cliniques, mais toutes ces dystrophies ont une caractéristique commune : la perte progressive des fibres musculaires. Pour les formes plus communes de ces dystrophies musculaires, les stratégies thérapeutiques visent principalement à restaurer la fonction du gène altéré. Bien que ces stratégies soient encourageantes pour un grand nombre de patients, elles ne peuvent être retenues comme traitement universel pour l'ensemble des dystrophies musculaires. L'identification de voies biologiques et de défauts secondaires communs à des dystrophies musculaires représente un intérêt majeur pour le développement de traitements palliatifs généralistes applicables à un large éventail de dystrophies musculaires. Pendant mes travaux de doctorat, j'ai initié une étude comparative approfondie sur dix-sept mutants différents chez le nématode Caenorhabditis elegans présentant une dégénérescence musculaire progressive, dont sept sont des modèles de dystrophies musculaires chez le nématode. La recherche de voies biologiques et de défauts secondaires communs, nous a permis d'établir différentes classes de dégénérescence musculaire chez le nématode et de révéler l'existence d'au moins deux mécanismes subcellulaires différents. De plus, nos résultats démontrent que des perturbations de la voie autophagique semblent systématiquement associées à la dégénérescence musculaire. Enfin, ce travail aboutit sur l'établissement d'un modèle proposant une chronologie des évènements sub-cellulaires menant à la dégénérescence progressive dystrophine-dépendante / Currently, more than sevently different muscular dystrophies have been clinically characterized. The pathogenesis of these different types of muscular dystrophies involves numerous cellular pathways and leads to a large variability of the clinical features. However, all the muscular dystrophies share a common feature: the progressive muscle cell loss. For the most common forms of muscular dystrophies, therapeutic strategies consist in restoring the function of the affected gene. Even if these strategies are encouraging for a lot of patients, they cannot be used as a universal treatment for all the muscular dystrophies. The identification if common pathways or common secondary defects to different muscular dystrophies is of great interest for the discovery of new palliative treatment, able to decrease the pathology of a large panel of muscular dystrophies. I have initiated a comparative analysis of seventeen C. elegans mutants presenting with muscle degeneration. Seven these mutants are considered as models for muscular dystrophies. Thanks to these comparative analysis, we were able to define different classes of muscle degeneration in C. elegans and to reveal the existence of at leats two different pathways. Moreover, our results show that perturbations in the autophagic pathway are systematically associated with muscle degeneration. This study leads to the establishment of a putative chronology of events occurring before the muscle cell loss, during the dystrophin-dependent degeneration process