Next Generation Molecular Studies of Hereditary Spastic Paraplegias in Men and Zebrafish

The term Hereditary spastic paraplegia (HSP) refers to a group of genetically heterogeneous neurodegenerative motor neuron disorders characterized by progressive age-dependent loss of corticospinal motor tract function, lower limb spasticity, and weakness. Recent clinical use of next generation sequencing (NGS) methodologies suggests that NGS facilitates the diagnostic approach to HSP, but the power of this method as a first-tier diagnostic procedure is unclear. The larger-than-expected genetic heterogeneity— there are over 80 potential disease-associated genes— and frequent overlap with other clinical conditions affecting the motor system make a molecular diagnosis in HSP cumbersome and time consuming. In a single-center, cross-sectional study, spanning 5 years, 242 subjects with a clinical diagnosis of HSP underwent molecular screening of a large set of genes, using two different customized NGS panels. The latest version of our targeted sequencing panel (SpastiSure3.0) comprises 118 genes known to be associated with HSP or syndromic conditions. Using an in-house validated bioinformatics pipeline and several in silico tools to predict mutation pathogenicity, we gathered a positive diagnostic yield of 30% (73/242), whereas variants of unknown significance (VUS) were found in 86 patients (36%), and 83 (34%) cases remained unsolved. This study is among the largest screening of consecutive HSP index cases enrolled in real-life clinical-diagnostic settings. Its results corroborate NGS as a modern, first-step procedure for molecular diagnosis of HSP. It also disclosed a significant number of new mutations in ultra-rare genes, expanding the clinical spectrum, and genetic landscape of HSP, at least in Italy. Interestingly, we identified new unreported mutations in the AP4S1 gene (SPG52), the smallest of the four subunits that form the AP-4 complex, a heterotetrameric protein complex which participates in membrane sorting between the trans-Golgi network and the endosomes, and plays a key role in signal-mediated tracking of integral membrane proteins. Mutations in AP4 subunits have been associated with alterations in neurodevelopment, epilepsy and complicated spastic paraplegia and the relative rarity of patients with mutations in subunits of the AP4 complex makes useful to report additional cases. In this study we described clinical presentations of three additional unrelated patients and their mutations. To improve our understating on to how AP4S1 operates during neurodevelopment, we knocked-down ap4s1 in zebrafish (Danio rerio), using morpholino antisense oligonucleotide technique. Our results showed that morphant embryos displayed an impairment of the neuronal excitability, locomotor defects, development delay, and altered neurogenesis, which are also phenotypic traits of AP4-HSP patients. Whilst we expanded the allelic heterogeneity in AP4-related diseases, we modeled in the simple vertebrate system zebrafish the early steps of abnormal neurodevelopment associated with AP4S1 defects offering a new tool for future therapeutic opportunities. Importantly, AP-complex served as an example for similar strategies in genes associated with HSP.