Discussion
The NGS panel was applied to screen 105 patients carrying a clinical phenotype corresponding to upper motor neuron syndrome (UMNS), selectively affecting motor control of lower limbs. Pathogenic mutations in SPAST were identified in 12 patients (11.42%), 5 missense, 3 frameshift, and 4 splicing variants. Then, we focused on the patients carrying splicing variants using a combined approach of in silico and in vitro analysis through minigene assay and RNA, if available. For two splicing variants (i.e., c.1245+1G>A and c.1414-2A>T), functional assays confirm the types of molecular alterations suggested by the in silico analysis (loss of exon 9 and exon 12). In contrast, the splicing variant c.1005-1delG differed from what was predicted (skipping exon 7), and the functional study indicates the loss of frame and formation of a premature stop codon. The present study evidenced the high splice variants in SPG4 patients and indicated the relevance of functional assays added to in silico analysis to decipher the pathogenic mechanism.
Methods
We performed an NGS panel in 105 patients, in silico analysis for splicing mutations, and in vitro minigene assay.