Spinal muscular atrophy: Clinical validation of a single-tube multiplex real time PCR assay for determination of SMN1 and SMN2 copy numbers

Abstract

ObjectivesTo describe and validate a new protocol for molecular diagnosis of spinal muscular atrophy (SMA), a frequent neuromuscular disease of childhood. Design and methodsSMA 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. ResultsWe 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. ConclusionsIn over 1200 samples, this technique has proven accurate, fast, economical and reproducible.