Résumé :
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Different therapeutic strategies are currently evaluated in spinal muscular atrophy (SMA) that are aimed at increasing full-length (FL) mRNA levels produced from the SMN2 gene. Of course, an inverse correlation between SMN2 copy number, SMN protein level and disease severity has been suggested. Therefore, accurate methods are needed for the quantification of SMN2 copies. We will describe the most widely used methods which are based on MLPA, real-time PCR or QMPSF. For all the strategies based on modulation of SMN expression at the transcriptional or at the RNA processing level, clinical trials that are envisaged for the somatic correction of SMA require sensitive and reliable assays, capable of monitoring the effects of the treatments on SMN mRNA levels. We have developed a sensitive, comparative assay based on multiplex fluorescent RT-PCR that is able to measure, in the same reaction, the levels of SMN mRNA with and without exon 7 sequences as well as those of total SMN mRNA. This assay allows to calculate directly the ratios of FL SMN mRNA to SMN mRNA without exon 7 (?7). We have used this assay to compare the levels of SMN transcripts in the blood of 75 unrelated normal subjects and of 48 SMA patients, and in muscle samples of 8 SMA patients. The SMN1 and the SMN2 genes produced very similar levels of total mRNA. Levels of transcripts lacking exon 7 were linearly dependent on the number of SMN2 copies, in SMA patients and in controls. In patients, FL mRNA levels correlated with SMN2 copy number. Other groups have developed alternative quantitative RT-PCR assays to quantify SMN transcripts. These methods can be used as sensitive biomarkers for monitoring the effects of various drugs in forthcoming clinical trials in SMA. 1 Department of Genetics, Rouen University Hospital & Inserm U614, Institute for Biomedical Research, University of Rouen, France 2 Inserm U798, University of Evry, Génopole, France 3 Department of Genetics, Tours University Hospital, France 4 Department of Genetics, Toulouse University Hospital, France 5 Department of Pediatrics, Bordeaux University Hospital, France 6 Department of Genetics, Necker-Enfants Malades University Hospital, Paris, France 7 Department of Neurology, Angers University Hospital, France.
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