Résumé :
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Communication n° 531 INTRODUCTION: autosomal recessive spinal muscular atrophy (SMA) is clinically classified in type I (severe), II (intermediate), III (mild). SMA is caused by a loss of function of the Survival of Motor Neuron (SMN) protein. The full-length protein is encoded by the SMN1 gene on chromosome 5q. The homologous SMN2 gene, adjacent to SMN1 towards the centromere, differs only for 5 nucleotides. A critical difference between SMN1 and SMN2, due to a single exon nucleotide change, is the skipping of exon 7 in SMN2 (Lorson C.L. et al., 1999). The absence of SMN transcripts containing exon 7 is found in 95% of SMA patients and is the consequence of homozygous deletions of SMN1. The remaining 5% of patients have a heterozygous deletion of SMN1 and a point mutation in the non-deleted allele. OBJECTIVES: in our diagnostic laboratory, we developed an improved Real Time Quantitative PCR method to determine the number of copies of SMN1 and SMN2. This analysis was successfully used to identify SMA carriers among at risk-family members. Moreover, this method allows to determine the SMN2 copy number, which is variable among individuals and known to affect the clinical phenotype of SMA (Feldkotter M. et al, 2002). MATERIALS AND METHODS: genomic DNA was isolated from blood by the salting-out without phenol method. The diagnostic test for SMN1 deleted patients was made with DraI and DdeI digestion of exons 7 and 8 respectively (van der Steege et al.1995). The number of copies of SMN1 and SMN2 gene was detected by using TaqMan quantitative PCR assay (ABI Prism 7000,).using TaqMan RNaseP Assay Kit (Applied Biosystems) for reference. RESULTS: here we report the case of a 5 year-old girl with a typical SMA III phenotype, but no homozygous SMN1 deletion, as evaluated by the enzymatic digestion method. Real time assay and mutation analysis (work in progress) are using to get a complete diagnosis for this patient. By Real Time Quantitative PCR, an heterozygous deletion of SMN1 was demonstrated, along with the presence of a high number (four) of copies of SMN2. CONCLUSIONS: our dates would confirm the diagnosis of SMA III in this girl and clarify its molecular basis. This case report clearly illustrates how the integration of different technologies may be necessary for a complete diagnostic work-up in some cases of SMA.
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