{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,20]],"date-time":"2026-05-20T03:27:32Z","timestamp":1779247652927,"version":"3.51.4"},"reference-count":51,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2023,12,7]],"date-time":"2023-12-07T00:00:00Z","timestamp":1701907200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Neurol."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>Pure hereditary spastic paraplegia (SPG) type 4 (SPG4) is caused by mutations of <jats:italic>SPAST<\/jats:italic> gene. This study aimed to analyze <jats:italic>SPAST<\/jats:italic> variants in SPG4 patients to highlight the occurrence of splicing mutations and combine functional studies to assess the relevance of these variants in the molecular mechanisms of the disease.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>We performed an NGS panel in 105 patients, <jats:italic>in silico<\/jats:italic> analysis for splicing mutations, and <jats:italic>in vitro<\/jats:italic> minigene assay.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results and discussion<\/jats:title><jats:p>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 <jats:italic>SPAST<\/jats:italic> 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 <jats:italic>in silico<\/jats:italic> and <jats:italic>in vitro<\/jats:italic> analysis through minigene assay and RNA, if available. For two splicing variants (i.e., c.1245+1G&amp;gt;A and c.1414-2A&amp;gt;T), functional assays confirm the types of molecular alterations suggested by the <jats:italic>in silico<\/jats:italic> 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 <jats:italic>in silico<\/jats:italic> analysis to decipher the pathogenic mechanism.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fneur.2023.1296924","type":"journal-article","created":{"date-parts":[[2023,12,8]],"date-time":"2023-12-08T12:42:43Z","timestamp":1702039363000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":5,"title":["Cohort analysis of novel SPAST variants in SPG4 patients and implementation of in vitro and in vivo studies to identify the pathogenic mechanism caused by splicing 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