Spinal muscular atrophy (SMA), a leading genetic cause of infant mortality, is caused by the loss of the survival motor neuron1 (SMN1) gene. The SMN1 gene is duplicated in humans with the duplicate gene, survival motor neuron 2 (SMN2), being partially functional. Interestingly, there is strong inverse relationship between SMN2 copy number and disease severity where SMA patients with higher SMN2 copy numbers generally have a less severe disease course. As such, accurate quantification of SMN1 and SMN2 copy numbers provides critical diagnostic and prognostic values for the disease. SMA has become an actionable disease with the recent advancement of treatment options. Newborn screening of SMA is increasingly practiced in many states and other countries. Current screening methods are complex and time consuming. Here we present a simple, high-level multiplexing digital PCR (dPCR) solution on a novel platform to meet the growing demand for SMA genetic testing and treatment options.

The hydrolysis probe-based multiplex digital PCR assay quantifies SMN1(FAM) and SMN2(HEX) copy numbers by targeting the single nucleotide difference between these genes at exon 7 as well as the total SMN(SMN1+ SMN2) (TYE665) copy number targeting the intron 1 region. RPPH1(TAMRA), which is always present at 2 copies, was used as an internal reference control. We first evaluated the 4-plex assay on characterized control DNA samples and then we validated the assay with 15 blinded clinical samples. To simplify the workflow, we eliminated the restriction enzyme digestion step usually recommended for copy number measurements. All experiments were conducted on a novel digital PCR instrument with 5 optical channels and a walk-away workflow identical to real-time PCR. The digital PCR system integrated partitioning of PCR mixture, thermocycling, and data acquisition into a single benchtop instrument. The copy numbers were determined by analysis software using Poisson statistics of the total, negative, and positive partitions.

Our multiplex digital SMA screening assay showed high specificity and sensitivity using the control samples with SMN1/SMN2 copy numbers at 0:3 (NA23255), 3:0 (NA17117) and 1:1 (NA03815). The copy numbers of the blinded clinical samples were correctly identified with 100% concordance to a previously published study including one sample with high SMN2 copy number. Additionally, the total SMN from 14 blinded clinical samples agreed with the sum of the copy numbers from SMN1 and SMN2 assays, indicating there is no additional deletion downstream of exon 7. One clinical sample showed the total copy number (4 copies) is more than the copy numbers from SMN1 (zero copies) and SMN2 (2 copies) due to a microdeletion. This sample has two SMN1 alleles that are lacking exons 7 and 8 (SMN[DELTA]78) resulting from a 6.3 kb microdeletion of SMN1 instead of a full deletion of SMN1. These microdeletions were verified by regular PCR. Additionally, the multiplex dPCR SMA copy number screening assay can be accomplished within 90 minutes on the integrated platform, potentially allowing the assay to be translated into a clinic setting.

We developed a truly rapid multiplex SMA screening assay with a simple workflow on a fully integrated digital PCR instrument. This multiplex digital PCR assay provides a highly accurate, sensitive, and powerful solution for SMA genetic testing and therapeutic treatment decisions.