Newborn screening for spinal muscular atrophy: The potential of digital polymerase chain reaction technique

Spinal muscular atrophy (SMA) is a degenerative neuromuscular disorder caused by a homozygous SMN1 loss-of-function variant. Early detection of SMA at the pre-symptomatic stage is essential for effective therapy. Consequently, Japan initiated newborn screening (NBS) for SMA in 2021 in the Kumamoto Prefecture, following global recommendations and implementations. The current NBS protocol involves a two-step process: first, quantitative real-time polymerase chain reaction (qPCR) for SMN1, followed by SMN1 and SMN2 copy number analysis using multiplex ligation-dependent probe amplification (MLPA). However, this approach is time-intensive, and qPCR alone cannot distinguish a single copy of SMN1 exon 7. The current NBS protocol is designed to detect approximately 96 % of SMA cases, specifically those with homozygous SMN1 exon 7 deletions. This study developed a digital PCR system for simultaneous analysis of SMN1 and SMN2 copy numbers to reduce the diagnostic time and improve diagnostic accuracy. Digital PCR was tested on dried blood spot (DBS) samples from 6 SMA patients (P-1 - P-6) and 386 healthy newborns. Additionally, the SMN1 and SMN2 copy numbers of the 6 patients were evaluated using MLPA. The results demonstrate that digital PCR enables simultaneous analysis of SMN1 and SMN2 copy numbers, with the outcomes for all six patients matching those obtained through MLPA. Moreover, digital PCR was more cost-effective than qPCR. Thus, digital PCR offers a practical and efficient alternative for SMA screening in NBS, enabling simultaneous analysis of SMN1 and SMN2 copy numbers while also improving the diagnostic speed and accuracy.