Analytical validation of the droplet digital PCR assay for diagnosis of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a progressive motor neuron disease caused by homozygote loss of exon 7 on the survival motor neuron 1 (SMN1) gene. The severity of the SMA phenotype is influenced by copies of SMN2, a gene that is highly homologous with SMN1. We validated analytical performance of droplet digital polymerase chain reaction (ddPCR) for detection of copy number variation of SMN1 and SMN2 genes for diagnosis of SMA using clinical samples. For accuracy performance evaluation, ddPCR results were compared with those of multiplex ligation-dependent probe amplification (MLPA) as a reference standard. Copy numbers of SMN1/SMN2 exon 7 from 200 clinical samples were concordant between ddPCR and MLPA. For all samples, the copy number of SMN1/SMN2 exon 7 was concordant between MLPA and ddPCR. The ddPCR also showed acceptable degrees of repeatability and total imprecision. Therefore, ddPCR is expected to be useful for SMA diagnosis and to predict phenotypic severity of SMA patients by determining the copy number of SMN2 in clinical laboratories.