Résumé :
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Currently, in most laboratories, molecular explorations in neuromuscular disorders (NMDs) are based on a differential molecular genotyping by a complex and time consuming gene by gene approach. As a consequence, it is estimated that 35-50 % of patients do not have a precise genetic diagnostic. On the one hand, many genes whose mutations cause NMD remain to be identified but, on the other hand, it became evident that the current approaches are not sufficiently powerful and accurate to perform exhaustive screenings even in known genes, and that around 50% of patients without a diagnosis carry unidentified mutations in these genes. In the recent years, technological tools have been developed towards performing massive molecular analyses using DNA Chips. We have taken advantage of these technologies to develop specific tools to explore NMD known and candidate genes.Several sets of DNA chips were designed towards revisiting the molecular pathology associated with known NMD genes. Both comparative genomic hybridization (CGH) arrays to detect CNV (duplications or deletions), as well as Sequence Capture (SC) arrays for massive genomic selection, sequencing and point mutation detection were used in our studies. We used the HD2 generation chips developed by Roche-Nimblegen and comtaining up to 2 Millions of probes. For CGH analyses, specific designs were performed to cover the full exonic and intron-exon boundaries for each of the targeted genes at high density and backbone probes were added to cover deep intronic regions and intergenic sequences throughout the entire genome. As examples, this strategy allowed to re-visit the molecular pathology in DMD/BMD, Dysferlinopathies, Calpapathies, Emerinopathies, Sarcoglycanopathies and several congenital muscular dystrophies including those associated to LAMA2 and COL6A genes defects. Beside, pathogenic duplications and deletions ranging from 7kb to 1.5 Mb have been identified in Charcot-Marie-Tooth neuropathies. In summary, single- or multiple exons deletions and/or duplications were identified for the first time in some of these genes and unexpected or novel re-arrangements were observed. Our approach also identified several "missing" mutations in autosomal recessive diseases for which only one mutation had been previously identified. Moreover, all of the corresponding breakpoints were precisely mapped and further characterized by a single junction fragment PCR. SC-chips coupled to subsequent massive sequencing are being tested and their results will also be presented.Altogether, our results provide a solid basis for using DNA micro-arrays as a robust and accurate approach towards exploring multiple genes simultaneously in large cohorts of patients affected with NMD. *Some of these data have been generated in the context of "NMD-Chip"*, a project funded by the European FP7 Health call. The NMD-Chip Consortium comprises 13 European institutions involved in the clinical management and molecular explorations in NMDs, from 8 countries.Team 1 : Marc Bartoli, Patrice Bourgeois, Vale Delague, Martin Krahn, Nicolas L*Team 2 : Mireille Coss Lucie Orhant, France Leturcq, Jamel Chelly*Team 3 : Caroline Guittard, Christophe Bud*Team 4 : Vale Allamand, Gis Bonne, Isabelle Nelson, Pascale Richard, Thomas Voit *Equal contribution.
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