title: Spinal muscular atrophy: clinical validation of a single-tube multiplex real time PCR assay for determination of SMN1 and SMN2 copy numbers.
abstract: OBJECTIVE
  To describe and validate a new protocol for molecular diagnosis of spinal muscular atrophy (SMA), a frequent neuromuscular disease of childhood.
  
  
  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.affiliation: Department of Genetics, CHUS and Université de Sherbrooke, Sherbrooke, Québec, Canada.
authors.forename: Bruno
authors.initials: B
authors.lastname: Maranda
authors.forename: Li
authors.initials: L
authors.lastname: Fan
authors.forename: Jean-François
authors.initials: JF
authors.lastname: Soucy
authors.forename: Louise
authors.initials: L
authors.lastname: Simard
authors.forename: Grant A
authors.initials: GA
authors.lastname: Mitchell
mesh_terms: D000483:Alleles; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D017353:Gene Deletion; D005786:Gene Expression Regulation; D005838:Genotype; D006720:Homozygote; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D014897:Spinal Muscular Atrophies of Childhood / Q000175:diagnosis* / Q000235:genetics*; D055533:Survival of Motor Neuron 1 Protein / Q000096:biosynthesis* / Q000235:genetics*; D055540:Survival of Motor Neuron 2 Protein / Q000235:genetics
chemical_list: D017931:DNA Primers; C526471:SMN1 protein, human; C526477:SMN2 protein, human; D055533:Survival of Motor Neuron 1 Protein; D055540:Survival of Motor Neuron 2 Protein