Dottorato in NEUROSCIENZE CLINICO-SPERIMENTALI E PSICHIATRIA

Titolo della tesi: The importance of clinical and genetic diagnosis, systematic reporting, and biomarkers in the context of hereditary spastic paraparesis

Background Hereditary spastic paraparesis (HSP) is a group of central nervous system diseases primarily affecting the spinal upper motor neurons, with different inheritance patterns and phenotypes. As rare diseases, they require to be thoroughly assessed and reported, to find hallmark features and thus aiding the diagnostic process. The advent of next-generation sequencing (NGS) has led to huge improvements in knowledge of HSP genetics and made it possible to clarify the molecular aetiology of hundreds of “cold cases”. Different strategies based on NGS that are primarily utilized today typically include targeted resequencing panels (TRPs) and whole exome sequencing (WES) as first-tier methods, while whole genome sequencing (WGS) is often considered a second-tier option, due to its higher expenses. The debate over the optimal approach is ongoing. Extensive clinical and diagnostic evaluations, case reports and literature reviews are the most valid tools for identifying, describing, and expanding knowledge on HSPs, given their genetic and phenotypic variability as well as their growing number. To date, there is no effective therapy available for HSP. The most frequent form is due to loss of microtubule severing enzyme spastin, caused by mutations in the SPG4/SPAST gene; therapeutic approaches aimed at spastin enhancement are emerging. However, we lack cost-effective predictive biomarkers. Aim This thesis aims to: • remark the utility of NGS in HSP, uncovering novel mutations, exploring the concept of VUS and their interpretation in rare neurological diseases • point out the significance of comprehensive diagnostic assessment for better defining clinical characteristics and genotype-phenotype correlations, thus stressing comprehensive observational reports as valuable tools in providing information which enhance our understanding of rare neurological diseases • to find prognostic and predictive biomarkers for evaluate emerging spastin-enhancing therapeutic approaches in SPG4 Methods The present studies were performed in accordance with the Declaration of Helsinki and its later amendments. Written informed consent and ethical approval (CE Lazio) were obtained. Patients were enrolled and assessed in our Rare Neurological Diseases Centre (ICOT – Latina – Department of Neurorehabilitation), and other Italian centres for case series. In a single laboratory (Stella Maris Foundation, IRCCS, Calambrone, Pisa), we tested the patients with clinical evidence of HSP without genetic diagnosis, using multigene targeted resequencing panels and investigating the coding exons and flanking introns of the genes known to be associated with HSPs [5, 15, 30]. Results Study 1 - Power of NGS‑based tests in HSP diagnosis: analysis of massively parallel sequencing in clinical practice. At present times, primary NGS-based strategies predominantly comprise targeted resequencing panels and exome sequencing, whereas genome sequencing usually stands as secondary choice due to its elevated costs. The optimal approach remains a subject of considerable debate, influenced by numerous factors. Here, we aim to analyse the diagnostic power of different NGS techniques applied in HSP, by reviewing 38 selected studies in which different strategies were applied in different-sized cohorts of patients with genetically uncharacterized HSP. Our analysis showed that exome and genome sequencing have a higher diagnostic rate than the TRP method, that patients with complex HSP, onset at < 40 years, and a recessive family history appear more likely to receive a firm molecular diagnosis, that all the known disease-causing mutations are spread over 110 genes, and that three-quarters of patients harbor mutations in only 10 genes. Hence, testing with multigene panels encompassing all these identified genes could be advantageous in terms of saving time, by avoiding preliminary screenings and simplifying post-run analyses and the handling of large amounts of data. Case “TREX1-HSP” (Study in preparation, not submitted - Heterozygous variant in TREX1 associated with complex hereditary spastic paraparesis) In the present study, we present an Italian family with autosomal dominant inheritance of a TREX1 variant, which we assume in leading to a new HSP. A major limitation includes the lack of molecular studies in asymptomatic or apparently healthy relatives, lack of specific functional studies and limited longitudinal investigations. Nonetheless, multiple predictions in our family as well as in the previous report suggest to consider TREX1 as new potential aetiology in HSP. If one wants to maintain a uniform nomenclature and keep counting the list of HSP-genes, TREX1 should be considered as SPG92. In this condition he the phenotype is complicated, with sensorimotor neuropathy in LL alongside major LMN involvement. In our case we also documented intrafamilial variability, with the proband’s father showing a pure HSP with spastic dysarthria, which is absent in other members, and the proband’s brother showing a mild phenotype resembling CMT. Future and more precise functional studies should also consider to translate in clinical scenario the potential use of ER-stabilizing drugs, such as bromocriptine Study 3 – Clinical-Genetic Features Influencing Disability in Spastic Paraplegia Type 4 (A Cross-sectional Study by the Italian DAISY Network). Spastic paraplegia type 4 (SPG4, SPAST-HSP) is the most common form of HSP. We present both clinical and molecular findings of a large cohort of patients, with the aim of defining the clinical spectrum of SPAST-HSP in Italy, describing their molecular features, and assessing genotype-phenotype correlations to identify features associated with worse disability. A cross-sectional retrospective study with molecular and clinical data collected in an anonymized database was performed. A total of 723 Italian patients with SPAST-HSP (58% men) from 316 families, with a median age at onset of 35 years, were included. Penetrance was 97.8%, with men showing higher Spastic Paraplegia Rating Scale (SPRS) scores (19.67 ± 12.58 vs 16.15 ± 12.61, p = 0.009). In 26.6% of patients with SPAST-HSP, we observed a complicated phenotype, mainly including intellectual disability (8%), polyneuropathy (6.7%), and cognitive decline (6.5%). Late-onset cases seemed to progress more rapidly, and patients with a longer disease course displayed a more severe neurologic disability, with higher SPATAX (3.61 ± 1.46 vs 2.71 ± 1.20, p <0.001) and SPRS scores (22.63 ± 11.81 vs 12.40 ± 8.83, p < 0.001). Overall, 186 different variants in the SPAST gene were recorded, of which 48 were novel. Patients with SPAST-HSP harbouring missense variants displayed intellectual disability (14.5% vs 4.4%, p < 0.001) more frequently, whereas patients with truncating variants presented more commonly cognitive decline (9.7% vs2.6%, p= 0.001), cerebral atrophy (11.2% vs 3.4%, p = 0.003), lower limb spasticity (61.5% vs 44.5%), urinary symptoms (50.0% vs 31.3%, p < 0.001), and sensorimotor polyneuropathy (11.1% vs 1.1%, p < 0.001). Increasing disease duration (DD) and abnormal motor evoked potentials (MEPs) were also associated with increased likelihood of worse disability (SPATAX score>3). Study 4 – Hereditary spastic paraparesis type 46 (SPG46): new GBA2 variants in a large Italian case series and review of the literature. SPG46 is a rare, early-onset and autosomal recessive HSP, linked to biallelic GBA2 mutations. About thirty families have been described worldwide, with different phenotypes like complicated HSP, recessive cerebellar ataxia or Marinesco-Sjögren Syndrome. Herein, we report five SPG46 patients harbouring five novel GBA2 mutations, the largest series described in Italy so far. Probands were enrolled in five different centres and underwent neurological examination, clinical cognitive assessment, column imaging for scoliosis assessment, ophthalmologic examination, brain imaging, GBA2 activity in peripheral blood cells and genetic testing. Their phenotype was consistent with HSP, with notable features like upper gaze palsy and movement disorders. We review demographic, genetic, biochemical and clinical information from all documented cases in the existing literature, focusing on the global distribution of cases, the features of the syndrome, its variable presentation, new potential identifying features and the significance of measuring GBA2 enzyme activity. Study 5 – Hereditary spastic paraparesis type 18 (SPG18): new ERLIN2 variants in a series of Italian patients, shedding light upon genetic and phenotypic variability. SPG18 is a rare, early-onset, complicated HSP, firstly reported as linked to biallelic ERLIN2 mutations. Recent cases of late-onset, pure HSP with monoallelic ERLIN2 variants prompt inquiries into the zygosity of such genetic conditions. The observed relationship between phenotype and mode of inheritance suggests a potential dominant negative effect of mutated ERLIN2 protein, potentially resulting in a milder phenotype. This speculation suggests that a wider range of HSP genes could be linked to various inheritance patterns. With documented cases of HSP loci exhibiting both dominant and recessive patterns, this study emphasizes that the concept of zygosity is no longer a limiting factor in the establishment of molecular diagnoses for HSP. Recent cases have demonstrated phenoconversion in SPG18, from HSP to an amyotrophic lateral sclerosis (ALS)-like syndrome. This report highlights two cases out of five exhibiting HSP-ALS phenoconversion, discussing an observed prevalence in autosomal dominant SPG18. Additionally, the study emphasizes the relatively high incidence of the c.502G>A variant in monoallelic SPG18 cases. This mutation appears to be particularly common in cases of HSP-ALS phenoconversion, indicating its potential role as hotspot for a distinctive SPG18 phenotype with an ALS-like syndrome. Study 6 – New cellular imaging- based method to distinguish the SPG4 subtype of hereditary spastic paraplegia. An automated, simple, fast, and non- invasive cell imaging- based method was developed to quantify microtubule cytoskeleton organization changes in lymphoblastoid cells and peripheral blood mononuclear cells. It was observed that lymphoblastoid cells and peripheral blood mononuclear cells from individuals affected by SPG4- hereditary spastic paraplegia show a polarized microtubule cytoskeleton organization. In a pilot study on freshly isolated peripheral blood mononuclear cells, our method discriminates SPG4- hereditary spastic paraplegia from healthy donors and other hereditary spastic paraplegia subtypes. In addition, it is shown that our method can detect the effects of spastin protein level changes.