Development and validation of a one-step SMN assay for genetic testing in spinal muscular atrophy via MALDI-TOF MS

Spinal muscular atrophy (SMA) is a fatal neuromuscular disorder primarily attributed to the homozygous deletion of the survival motor neuron 1 (SMN1) gene, with disease severity closely correlated to the copy number variations (CNV) of SMN2. Conventional methodologies, however, fail to provide a comprehensive gene overview of SMN and are often both time-intensive and costly. In this study, we present a novel one-step MALDI-TOF MS assay for SMA gene testing. To accurately quantify CNV, we incorporated RPPH1 as an internal control alongside normal samples and competing templates targeting SMN1, SMN2, and RPPH1 for multiple corrections. The CNV assay enables precise quantification of exons 7/8 in both SMN1 and SMN2 genes, achieving a kappa value of 0.935 (P < 0.001) when compared with multiple ligation-dependent probe amplification (MLPA) during its development phase. This accuracy was further corroborated in a cohort comprising 78 individuals. To identify patients harboring compound heterozygous mutations or silent carriers, prevalent pathogenic variants along with sequence variants of SMN1 were integrated into our analysis framework; plasmids were constructed for methodological validation purposes. Utilizing these combinatorial assays for SMN detection, we identified one patient exhibiting a compound heterozygous mutation characterized by genotype [0 + 1^d] and another subject presenting genotype [2 + 1], who harbored simultaneous variants of g.27134T > G and g.27706_27707delAT. The CNV assessment combined with pathogenic variants analysis developed through MALDI-TOF MS provides a comprehensive gene profile of SMN within a single analytical run. Given its unparalleled cost-effectiveness and time efficiency, this approach holds significant promise for further application in clinical diagnosis as well as newborn screening for SMA.