CLINICAL AND FUNCTIONAL PROFILING OF CDC42 VARIANTS AFFECTING DEVELOPMENT

The small RAS superfamily GTPase CDC42 (cell division cycle 42) functions as an intracellular signaling node, regulating diverse developmental processes (e.g., adhesion and migration). In the last years, our group has causally linked pathogenic variants in CDC42 to various rare neurodevelopmental and inflammatory disorders and functionally characterized their impact on CDC42 function. Here, we describe the functional characterization of a large set of newly identified pathogenic variants, profile the associated clinical phenotype, and delineate the contributing signaling pathways. In silico and in vitro analyses demonstrate that mutations variably perturb CDC42 function by multiple mechanisms, perturbing the equilibrium between the active and inactive states of the GTPase, altering CDC42 intracellular localization, and/or affecting its interaction with effectors (i.e., IQGAP1, RHOGDI, and N-WASP and PAK1). We also show that this variable impact on protein function is specifically associated with a subset of clinical features. Specifically, CDC42 membrane mislocalization and altered N-WASP binding are associated with hematologic/immunologic phenotypes, while dysregulation of the RASmitogen-activated protein kinase (MAPK) pathway appear to be associated with RASopathy-like features. Notably, a subset of variants are associated with cleft lip/palate and other otopalatodigital syndrome-like features, and share dysregulation of cellular focal adhesions. Dandy Walker malformations are characteristic anomalies of patients bearing strongly hyperactive mutants, whereas the Takenouchi Kosaki syndrome-associated variant (p.Y64C) shows distinctive molecular dysregulation. These findings reveal a remarkably variable impact of dominantly acting CDC42 mutations on cell function, and phenotypes. These findings emphasize the importance of functional profiling for syndrome recognition and delineation.