Résumé :
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Primary dysferlinopathies are a heterogeneous group of autosomal recessive muscular dystrophies, caused by mutations in the large-sized DYSF gene encoding dysferlin. Dysfunction of dysferlin causes deficient sarcolemmal repair and leads to muscle fiber degeneration. In a patient born from a distantly consanguineous union, presenting with moderately severe primary dysferlinopathy, we identified a homozygous multi-exonic internal deletion using genomic, transcriptional and F.I.S.H. analyses. Currently, this patient presents with proximo-distal weakness, but is still ambulant without a cane at 41 years. Bioinformatic analysis of putative Open-Reading-Frames within the resulting, deleted transcript predicts a possible translation into a truncated protein maintaining two C2 domains and the transmembrane domain. While muscle biopsy samples were not available, dysferlin protein analyses on monocytes obtained from the patient evidenced expression of a truncated molecule at the expected size, which localizes to the plasma membrane. Ectopic expression of the predicted Open-Reading-Frame using transfection into fibroblasts confirmed targeting to the plasma membrane. To further evaluate the functionality of this Mini-dysferlin, we constructed an AAV-vector containing the predicted Open-Reading-Frame identified in the patient under a muscle specific promoter. Intra-muscular injection of the Mini-DYSF-AAV vector in wild-type and dysferlin-deficient mice led to high-level expression of the truncated protein. Using a membrane repair assay, based on membrane wounding of transduced isolated muscle fibers with a two-photon laser-scanning microscope, we demonstrated that the Mini-dysferlin efficiently reseals the damaged sarcolemma. Therefore, this Mini-dysferlin is at least partially functional. Importantly, this demonstrates the modularity of dysferlin. In Duchenne muscular dystrophy, the identification of functional Mini-dystrophins led to the development of therapeutic strategies such as gene transfer of Mini-dystrophin and exon-skipping approaches. Our findings constitute a prerequisite for similar therapeutic strategies for a subset of primary dysferlinopathies. (MK and NW; NL and IR contributed equally to this work)
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