Functional characterization of genetic variants affecting the FGFR3 and the GPIb-IX receptors: molecular insights from in vitro models

Genetic variants can profoundly affect protein structure and function, often leading to inherited diseases with variable clinical expression. This PhD project aimed to investigate the molecular mechanisms by which specific amino acid substitutions alter protein activity, combining two experimental models representative of distinct disease contexts but united by a common methodological approach based on in vitro characterization. The first part of the study focused on FGFR3 Gly375Cys variant, a rare human pathogenic substitution associated with an achondroplasia-like phenotype. Although this mutation has been linked to skeletal dysplasia, its pathogenetic mechanisms remain only partially understood. Through expression studies and protein-level analyses of the murine homolog FGFR3 Gly369C, we identified a novel lower molecular weight isoform, specific to the mutant receptor only. Further investigations were carried out to characterize this isoform and to assess its potential contribution to the altered receptor processing and downstream signaling, providing new insights into the molecular heterogeneity of FGFR3-related disorders. The novel mutant isoform was found to miss the extracellular domain, but retaining transmembrane and intracellular kinase domain, suggesting a proteolytic cleavage had occurred. The molecule appeared highly phosphorylated indicative of a constitutive ligand-independent kinase activation, and delayed in turnover, suggesting a potential role in disease pathogenesis due to its prolonged activation. The second part of the project addressed some of the GP1BA variants associated with inherited macrothrombocytopenia, a group of disorders affecting platelet number and morphology. The in vitro expression of wild-type and mutant forms of the GPIb subunit was performed in a CHOβIX cell model expressing the GPIb-IX complex, enabling the evaluation of variant effects on receptor expression, assembly, surface localization, and interaction with the von Willebrand factor (vWF). This approach allowed us to distinguish pathogenic variants from likely benign or Variants of Uncertain Significance (VUSs), contributing to a more precise genotype-phenotype correlation in patients with inherited thrombocytopenias. Specifically, we functionally characterized seven variants. Of these, Ala172Val, Asn57His and Leu10del, emerged as pathogenic according to our assays on protein stability and expression and their interaction with vWF in both static and dynamic conditions. Overall, this work provides experimental evidence that supports the functional interpretation of disease-associated variants at the protein level. The combination of targeted in vitro assays and cellular models proved effective in elucidating the molecular consequences of genetic changes, ultimately contributing to a better understanding of variant pathogenicity and its impact on clinical phenotype.