AUTHOR=Ferese Rosangela , Scala Simona , Suppa Antonio , Campopiano Rosa , Asci Francesco , Zampogna Alessandro , Chiaravalloti Maria Antonietta , Griguoli Annamaria , Storto Marianna , Pardo Alba Di , Giardina Emiliano , Zampatti Stefania , Fornai Francesco , Novelli Giuseppe , Fanelli Mirco , Zecca Chiara , Logroscino Giancarlo , Centonze Diego , Gambardella Stefano 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 mutations JOURNAL=Frontiers in Neurology VOLUME=14 YEAR=2023 URL=https://www.frontiersin.org/journals/neurology/articles/10.3389/fneur.2023.1296924 DOI=10.3389/fneur.2023.1296924 ISSN=1664-2295 ABSTRACT=Introduction

Pure hereditary spastic paraplegia (SPG) type 4 (SPG4) is caused by mutations of SPAST gene. This study aimed to analyze SPAST 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.

Methods

We performed an NGS panel in 105 patients, in silico analysis for splicing mutations, and in vitro minigene assay.

Results and 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.