Functional characterization of a novel RAC1 variant causing a neurodevelopmental condition with Noonan syndrome features

RAC1 is a member of the Rho GTPase subfamily within the RAS superfamily of small GTP-binding proteins, regulating the actin cytoskeleton architecture, cell spreading, migration and proliferation. De novo missense variants in RAC1 are associated with rare neurodevelopmental disorders characterized by DD/ID and brain abnormalities and a wide range of additional features [1]. Previous studies documented a possible role of RAC1 in contributing to the ectodermal features associated with Noonan syndrome [2,3]. Here, we describe a familial case (affected mother and daughter) with a previously unreported missense RAC1 variant (p.A13D). We functionally demonstrate its pathogenicity proving a gain-of-function (GoF) effect. In particular, the mutation increases RAC1 activation and spreading in HEH293T and COS1 overexpressing cells. A significant enhancement of cell spreading was also documented in primary fibroblasts isolated from the patient carrying the heterozygous RAC1A13D, compared to control fibroblasts. The individuals carrying the mutation present with DD and facial features resembling Noonan syndrome. Consistently, we demonstrate a strong hyperactiving effect of the variant with respect to MAPK signaling, providing a possible explanation for the RASopathy-like features of the mutated subjects.