Résumé :
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Mutations in the gene encoding calpain-3 (CAPN3) cause autosomal recessive Limb-Girdle Muscular Dystrophy type 2A (LGMD2A) and idiopathic Eosinophilic Myositis. Accurate diagnosis and genetic counselling is based on the identification of disease-causing mutations on both alleles of CAPN3 in the patients. Unfortunately, in 20 to 30% of patients, routine genomic analysis fails to identify both disease-causing alleles. These patients should benefit from complementary approaches towards completing mutation's identification. Here, we performed transcriptional analyses in five patients suspected of being affected with LGMD2A, in whom initial DHPLC/genomic mutation screening evidenced no, or a single-allele CAPN3 disease-causing mutation thus not sufficient to firmly confirm diagnosis. This allowed to identify and characterise cDNA deletions. Further genomic characterisation allowed to determinate the origin of these deletions, either as splicing defects caused by intronic mutations or an internal multi-exonic deletion. Indeed, we report in the CAPN3 gene two novel mutations (c.1745+4_1745+7delAGTG produced from IVS13 and c.2185-16A>G produced from IVS20) and a recurrent large sized-genomic deletion including exons 2 to 8 for which genomic breakpoints have been characterised, in the CAPN3 gene. In addition, our results indicate nonsense-mediated mRNA decay as a mechanism for under-expression of CAPN3 associated to some specific variations. All abnormalities evidenced at the transcriptional level have been accurately predicted using Splicing Sequences Finder, a novel algorithm for the prediction of deleterious effects on normal splicing. (Note CP and MK contributed equally to this work)
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