Résumé :
|
Dysferlinopathies are a group of autosomal recessive muscular dystrophies, due to defects in the DYSF gene. The aim of our project is to set-up functional test in monocytes and to explore therapeutic approaches in dysferlinopathies. Presently, after clinical exploration, initial diagnosis of dysferlinopathies is based on Western blot (WB) from proteins obtained after muscle biopsy or blood monocytes, and our results suggest these latter representing an excellent and easy to obtain cellular model: RT-PCR, WB and IF (with NCL-Hamlet antibody) have demonstrated that monocytes express a DYSF mRNA (6.9kb) and produce Dysferlin protein (237kDa). However Dysferlin cannot be detected by WB performed on monocytes after a 48h blood storage, whereas Hamlet antibody still detects a lower yet uncharacterized band (MW = 200 Kda). IF have shown that Dysferlin is present at plasma membrane in resting monocytes while a cytoplasmic staining is shown when monocytes are submitted to mechanical stress or electroporation. Furthermore, we have designed a new diagnosis test using flow cytometry from monocytes. This approach allowed detecting both Dysferlin and monocytes within only 100µl of whole blood from healthy individuals. We are now screening a cohort of patient using flow cytometry and WB analyses in order to valid this method. Finally we are setting up functional tests towards studying monocytes phagocytosis. Our hypothesis is based on the following data: 1/ In monocytes, Dysferlin is associated, as in skeletal muscle cells, with caveolin and annexins, both proteins being involved in monocytes phagocytosis; 2/ phagocytosis requires compensatory plasma membrane supply through exocytosis; 3/ if Dysferlin could play a role in plasma membrane supply after phagocytosis, then phagocytosis could be altered in monocytes from patients affected with dysferlinopathy. In such case, monocyte phagocytosis could be used to evaluate the disease severity and allow the efficiency of potential therapeutic strategies.
|