Development of a method for determining the deletion of exon 7 in the SMN1 gene along with the copy number of the SMN1 and SMN2 genes using digital PCR.

Abstract

Pathogenic variants in the SMN1 gene are the underlying cause of autosomal recessive spinal muscular atrophy (SMA), a condition that is included in the list of expanded neonatal screening. SMA is diagnosed primarily by qPCR, a method that is subject to several limitations. The objective of this study was to develop an approach that would enhance the accuracy of SMA diagnosis by employing digital PCR. To this end, a novel technique was developed that facilitates the concurrent evaluation of exon 7 deletion in the SMN1 gene and the simultaneous calculation of the SMN1, SMN2, and RPP30 gene dosages, employing a mere two fluorescent channels as a reference. The study utilized anonymized DNA samples of newborns obtained from dried blood spots, in accordance with standard protocols employed for expanded neonatal screening. The study’s findings yielded the development of specific primers, a digital PCR design, and a method for analyzing the results, including a comparative analysis with the generally recognized “gold standard” MLPA. The developed method enables the accurate evaluation of exon 7 deletion in the SMN1 gene, as well as the dosage of the SMN1 and SMN2 genes, within a single digital PCR reaction. This advancement has the potential to serve as a diagnostic tool for SMA, particularly in cases that are subject to controversy during neonatal screening.