Dottorato in GENETICA E BIOLOGIA MOLECOLARE

Titolo della tesi: Molecular bases of RAS superfamily-related diseases

The RAS GTPase pathway is involved in several key processes, essential in development and growth, so it is understandable that genetic mutations leading to dysregulated RAS function cause various types of diseases. Specifically, somatic mutations in RAS-related genes are associated with several forms of cancer, while RAS-related germline mutations induce a heterogeneus group of rare syndromic conditions, named RASopathies. In the last fifteen years, thanks to Whole Exome Sequencing (WES) analyses, novel disease genes and variants were associated with previously undiagnosed diseases. Moreover, mutations of genes belonging to the RAS superfamily were associated to novel syndromic conditions, including RASopathy-like diseases. In this context, I focused my work on studying the molecular basis of the diseases caused by mutations affecting three genes of the RAS superfamily, ARF3, RAC1 and CDC42, associated with a heterogeneous spectrum of pediatric neurodevelopmental or autoinflammatory disorders. Particularly, in this PhD Thesis I have addressed the pathogenic mechanisms induced by a total of fourteen variants as well as the genotype-phenotype correlations. To characterize the consequences of the pathogenic mutations, I chose to perform in vitro studies in cell culture models. I induced ectopic expression of the putative pathogenic variants compared to the wild-type proteins by transient transfection of engineered constructs in mammalian cell lines, then analyzed protein and cell function dysregulation. Specifically, I have analyzed the localization, stability, activation, effector binding of each pathogenetic variant and their cellular effects (i.e., polarized migration, cellular organization, MAP kinase activation). The results obtained in this thesis have enabled me to demonstrate an impairment of diverse functional processes induced by the variants, possibly underlying the heterogeneity of the clinical features associated with the mutations.