Multiplex Droplet Digital PCR Method Applicable to Newborn Screening, Carrier Status, and Assessment of Spinal Muscular Atrophy.

BACKGROUND

Spinal muscular atrophy (SMA) is a progressive neuromuscular disorder with neuronal degeneration leading to muscular atrophy and respiratory failure. SMA is frequently caused by homozygous deletions that include exon 7 of the survival motor neuron gene , and its clinical course is influenced by the copy number of a nearby 5q paralog, . Multiple ligation probe amplification (MLPA) and real-time quantitative PCR (qPCR) can detect deletions. Yet, qPCR needs normalization or standard curves, and MLPA demands DNA concentrations above those obtainable from dried blood spots (DBSs). We developed a multiplex, droplet digital PCR (ddPCR) method for the simultaneous detection of deletions and copy number variation in DBS and other tissues. An Sanger sequencing process for DBS was also developed.

METHODS

, , and concentrations were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. A total of 1530 DBSs and 12 SMA patients were tested.

RESULTS

Population studies confirmed 1 to 5 exon 7 copies detected in unaffected specimens, whereas patients with SMA revealed 0 copies. Intraassay and interassay imprecisions were <7.1% CV for individuals with ≥1 copies. Testing 12 SMA-positive samples resulted in 100% sensitivity and specificity.

CONCLUSIONS

This ddPCR method is sensitive, specific, and applicable to newborn screening and carrier status determination for SMA. It can also be incorporated with a parallel ddPCR T-cell excision circles assay for severe combined immunodeficiencies.