Clinical biochemistry · 2011
Spinal muscular atrophy: clinical validation of a single-tube multiplex real time PCR assay for determination of SMN1 and SMN2 copy numbers.
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Maranda Bruno, Fan Li, Soucy Jean-François, Simard Louise, Mitchell Grant A
Аннотация
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.
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.