OBJECTIVES: To describe and validate a new protocol for molecular diagnosis of spinal muscular atrophy (SMA), a frequent neuromuscular disease of childhood. DESIGN AND METHODS: SMA is caused in most cases by homozygous deletion of the SMN1 gene. We describe a triplex quantitative real-time PCR method in which fragments of SMN1, SMN2 (a nearly-identical neighboring gene with markedly reduced function) and of a control gene, CFTR, are amplified in the same tube. RESULTS: We validated this method in three ways. First, testing the same samples ten times yielded CV values <4.6%. Second, in 104 previously-genotyped individuals, SMN copy numbers identical to those of the previously-determined genotype was unambiguously obtained in all cases. Finally, results using the technique in practice are described and analyzed for reproducibility of amplification efficiency and for inter-run variability. CONCLUSIONS: In over 1200 samples, this technique has proven accurate, fast, economical and reproducible.
OBJECTIVES: To describe and validate a new protocol for molecular diagnosis of spinal muscular atrophy (SMA), a frequent neuromuscular disease of childhood. DESIGN AND METHODS: SMA is caused in most cases by homozygous deletion of the SMN1 gene. We describe a triplex quantitative real-time PCR method in which fragments of SMN1, SMN2 (a nearly-identical neighboring gene with markedly reduced function) and of a control gene, CFTR, are amplified in the same tube. RESULTS: We validated this method in three ways. First, testing the same samples ten times yielded CV values <4.6%. Second, in 104 previously-genotyped individuals, SMN copy numbers identical to those of the previously-determined genotype was unambiguously obtained in all cases. Finally, results using the technique in practice are described and analyzed for reproducibility of amplification efficiency and for inter-run variability. CONCLUSIONS: In over 1200 samples, this technique has proven accurate, fast, economical and reproducible.
Authors: Guillaume M Hautbergue; Lydia M Castelli; Laura Ferraiuolo; Alvaro Sanchez-Martinez; Johnathan Cooper-Knock; Adrian Higginbottom; Ya-Hui Lin; Claudia S Bauer; Jennifer E Dodd; Monika A Myszczynska; Sarah M Alam; Pierre Garneret; Jayanth S Chandran; Evangelia Karyka; Matthew J Stopford; Emma F Smith; Janine Kirby; Kathrin Meyer; Brian K Kaspar; Adrian M Isaacs; Sherif F El-Khamisy; Kurt J De Vos; Ke Ning; Mimoun Azzouz; Alexander J Whitworth; Pamela J Shaw Journal: Nat Commun Date: 2017-07-05 Impact factor: 14.919