Molecular and functional study of genes associated with epilepsy and movement disorders

Paroxysmal neurological disorders, including early-onset epilepsies and movement disorders, arise from alterations in neuronal excitability and network stability. The identification of disease-associated genes has provided crucial insights into the molecular mechanisms underlying these conditions. A powerful approach to investigate such mechanisms is the functional characterization of pathogenic variants identified in patients. In this PhD project, I studied disease-causing variants in SCN2A, PRRT2, and TMEM151A, three genes implicated in epilepsy and paroxysmal movement disorders, with the aim of elucidating their pathogenic mechanisms, refining genotype–phenotype correlations, and gaining insight into their physiological roles in neuronal function. Pathogenic variants in SCN2A, encoding the voltage-gated sodium channel NaV1.2, are associated with a broad spectrum of neurodevelopmental disorders. I report on the clinical and functional characterization of two de novo SCN2A missense variants, c.4976C>T (p.A1659V) and c.710T>A (p.I237N), identified in patients with neonatal-onset, drug-resistant epilepsy and severe neurodevelopmental impairment, characterized by somatic mosaicism. Despite clinical features suggestive of a gain-of-function mechanism and in silico predictions supporting pathogenicity, electrophysiological analyses revealed a marked reduction in sodium current density for both variants and altered gating properties for the A1659V variant, consistent with an overall loss-of-function effect. These findings highlight the complexity of SCN2A genotype–phenotype relationships and have important implications for precision treatment strategies. PRRT2 is a neuronal protein that regulates intrinsic excitability and synaptic function through modulation of voltage-gated sodium channels. Pathogenic variants in PRRT2 are a major cause of Benign Familial Infantile Seizures (BFIS) and Paroxysmal Kinesigenic Dyskinesia (PKD), two paroxysmal disorders that frequently co-occur or segregate within the same families. Focusing on missense variants within its transmembrane domain, we demonstrated that PRRT2 variants can differentially affect NaV1.2 channel regulation, resulting in either loss- or gain-of-function phenotypes at the channel level, despite similar clinical presentations. In silico docking analyses further identified putative interaction interfaces between PRRT2 and NaV1.2, guiding targeted mutagenesis to functionally validate residues involved in channel modulation. These data underscore the central role of PRRT2–NaV interactions in disease pathogenesis and suggest that paroxysmal manifestations arise when PRRT2 function deviates from its physiological range. TMEM151A, recently identified as a causative gene for PKD, remains poorly characterized. Notably, TMEM151A-associated PKD overlaps with the same paroxysmal disorder classically caused by PRRT2 variants, indicating a shared disease phenotype and potentially a shared disease mechanism. Using molecular dynamics simulations, immunocytochemistry, and electron microscopy, we defined its membrane topology, revealing a protein predominantly oriented toward the cytosol with a structured transmembrane domain. Functional analyses showed that pathogenic TMEM151A variants impair protein expression and subcellular localization, supporting a loss-of-function mechanism. TMEM151A overexpression promoted neurite outgrowth and dendritic arborization in primary neurons, whereas pathogenic variants impaired neuronal maturation. Moreover, analysis of a human TMEM151A heterozygous knockout iNeuron model revealed impaired network formation and reduced structural complexity, confirming a key role for TMEM151A in neuronal development and excitability. Finally, electrophysiological studies demonstrated a selective modulation of NaV1.1 channels by TMEM151A. Overall, this work provides converging molecular, cellular, and functional evidence that alterations in sodium channel regulation and neuronal network maturation represent shared pathogenic mechanisms linking epilepsy and paroxysmal movement disorders. By integrating the study of SCN2A, PRRT2, and TMEM151A, this thesis contributes to a deeper understanding of disease mechanisms and highlights potential targets for genotype-informed therapeutic strategies.