Résumé :
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The myotonic dystrophies (DM) include DM1 (or Steinerts disease), caused by expansion of a CTG triplet repeat in the 3' UTR of the DM protein kinase (DMPK) gene, and DM2 (myotonic dystrophy type 2), caused by expansion of a CCTG tetranucleotide repeat in the first intron of ZNF9 gene. Despite distinctive clinical features, both DM types share in common autosomal dominant inheritance, muscle weakness, myotonia, posterior capsular cataracts, and multiorgan involvement affecting the heart, brain, and endocrine system. In DM, the gene carrying the unstable expanded repeats is transcribed, producing RNAs with expanded CUG or CCUG that interfere with RNA processing and nucleocytoplasmic transfer. These mutant transcripts accumulate in the cell nucleus in form of focal aggregates, termed "foci", that sequester essential RNA binding protein such as muscleblind proteins. Sequestration of RNA binding proteins likely alter splicing of a large set of pre-mRNAs, including transcripts of insulin receptor, ClC-1 chloride channel and other genes. These pathogenic ribonuclear inclusions can be detected by fluorescent in situ hybridization (FISH) method. Considering foci as a key DM biomarker, we have set up an automated FISH procedure which allows the detection and quantification of the proportion of expanded DMPK transcripts retained in the nucleus. We first demonstrated the reliability of the methods which was then used to evaluate various skeletal muscles and other tissues of 1 month and 4 month-DMSXL mice, a transgenic DM1 mouse model carrying >1000 CTG. We showed that the foci load was high in all muscle tissues (skeletal, smooth, cardiac) and in the brain, whereas lowest signal levels were observed in the liver and epithelia. The results were compared to muscle biopsies of DM1 and DM2 patients. We further evidenced that MBNL1 co-localize with RNA inclusions in DMSXL mice. A major interest of this approach is that it may represent a powerful tool for monitoring preclinical efficacy of molecular therapies in the context of systemic delivery.
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