Molecular characterization of unsolved cases involving patients affected by RASopathies

RASopathies include a heterogeneous group of genetic syndromes caused by alterations in the RAS/MAPK signal transduction pathway and are characterized by a pronounced phenotypic overlap. These conditions include Noonan syndrome, cardio-facio-cutaneous syndrome, Costello syndrome, and neurofibromatosis type 1. Despite the widespread implementation of standard next-generation sequencing (NGS), molecular diagnosis and the characterization of genotype-phenotype correlations remain challenging due to the presence of variants of uncertain significance (VUS), non-canonical splicing variants, structural rearrangements and somatic mosaicism. This thesis focuses on the study of some unsolved cases in patients affected by RASopathies through the integrated analysis of clinical-genetic databases and the application of advanced genomic and functional strategies. Specifically, the study evaluates the efficacy of ultra-depth NGS in identifying low-frequency mosaic variants, alongside the utility of hybrid minigene assays to characterize the impact of genomic variants on RNA splicing. Overall, this work emphasizes that the integration of these methodologies helps in resolving previously negative or ambiguous cases and in redefining the pathogenicity of unclassified variants, ultimately setting the basis for more precise genetic counseling and personalized clinical management.