Résumé :
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Communication n° 289. X-linked myotubular myopathy (XLMTM) is a severe congenital muscular disease characterized by generalized hypotonia and respiratory insufficiency at birth. The mutated gene, MTM1, is composed of 15 exons and encodes a protein named myotubularin, which acts as a phosphoinositide phosphatase implicated in endocytosis. MTM1 belongs to a large disease-associated gene family with 14 members in human that includes MTMR2 and MTMR13, mutated in Charcot-Marie-Tooth type 4B1 and 4B2, respectively. The molecular diagnosis of XLMTM is generally established by direct sequencing of MTM1 exons and flanking intronic sequences. Mutations are widespread throughout the coding sequence with more than 300 different MTM1 mutations described to date, and found in about 85% of clinically diagnosed XLMTM patients. We have amplified the full coding sequence by RT-PCR from cultured patients cells. Using the same method, we can also detect MTM1 RNA in peripheral blood. In one patient for whom no mutation was found by genomic DNA sequencing of the exons, we characterized a non-coding 94nt insertion in the MTM1 RNA which corresponds to the addition of a cryptic exon. The causative DNA change was an A to G transition in intron 7, in a sequence conserved at 80% in mouse, and created a consensus acceptor site preferentially used in lymphoblasts from the patient. In addition, we have recently generated a novel polyclonal antibody against myotubularin in rabbit that can detect the endogenous protein from various human cell lines by direct immunoblotting. We found abnormal levels of myotubularin in most analysed XLMTM cell lines, with either a decrease or absence of the protein, while autosomal centronuclear myopathy cases showed a normal level. We have established a protocol for immunodetection of endogenous myotubularin in leucocytes isolated from peripheral blood that can be used for rapid diagnosis. Furthermore, we show that myotubularin can be detected from human skeletal muscle biopsies, being absent in XLMTM patients carrying truncating MTM1 mutations. These novel approaches will be useful for molecular diagnosis, especially in cases in which MTM1 mutations have not been found by genomic DNA sequencing and also to confirm the pathogenicity of missense mutations.
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