CHARACTERIZATION AND FUNCTIONAL STUDIES OF NOVEL VARIANTS IDENTIFIED THROUGH NGS TECHNOLOGY IN INHERITED THROMBOCYTOPENIAS

Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by reduced platelet count and an increased bleeding tendency. Despite advances in genetic technologies, approximately 50% of IT cases remain without a definitive molecular diagnosis. Moreover, the identification of causative variants is still challenging. In fact, Next Generation Sequencing (NGS) -based diagnostic screenings often reveal as variants of uncertain significance (VUS). In such cases, the development of gene-specific functional studies is essential to determine their pathogenicity. For this reason, this PhD thesis focuses on functional characterization of novel genetic variants identified through NGS in a cohort of patients with inherited thrombocytopenia. Taking advantage of in vitro functional assays, we investigated several VUS identified in ITs causative genes. These variants were subjected to in-depth molecular analysis to assess their impact on protein function, subcellular localization, and transcriptional activity. Our findings demonstrate that many of the variants tested significantly disrupt normal protein function and, for this reason, possibly lead to impaired thrombopoiesis and abnormal platelet formation. In particular, we focused on novel mutations in MECOM, ETV6 and FLI1 genes, three transcription factors which mutations affect their transcriptional activity. Since ITs caused by these genes are very rare and poorly studied, we have also investigated the pathogenic mechanisms underlying these disorders. We hypothesised that the deleterious effect of MECOM mutations is at least partly mediated by loss of repression of the MPL gene, and we demonstrated that ETV6 mutations act through a dominant-negative effect that also excludes the wild-type form from the nucleus. These studies highlight the importance of functional validation in determining the pathogenicity of genetic variants in ITs and contributes to a better understanding of the molecular mechanisms underlying these disorders. Understanding the molecular basis of these disorders is essential for improving diagnostic accuracy, genetic counselling and developing potential therapeutic strategies for patients affected by ITs.