Functional characterization of novel germline mutations affecting CDC42 highlighted their differential impact on multiple signaling pathways.

CDC42 encodes a small RHO-family GTPase working as an intracellular signaling node. Recently, we identified germline mutations in this gene in a clinically heterogeneous group of disorders characterized by growth delay, facial dysmorphism and neurodevelopmental, immunological and hematological anomalies. In vitro and in vivo (C. elegans) functional studies established that mutations variably perturb CDC42 function and differentially affect cellular and developmental processes. Here, in the frame of a collaborative study, we identified three novel CDC42 dominant mutations (i.e., p.K16R, p.D170N and p.C188Y) causing diverse clinical phenotypes. Cellular studies established a hypomorphic effect of CDC42D170N and CDC42C188Y on polarized migration assessed by wound-healing assay, in line with previous data collected on mutations affecting the neighboring residues Glu171 and Arg186. These mutants also showed a significantly reduced expression levels. By contrast, the K16R substitution had a gain-of-function effect on polarized migration. Interestingly, confocal microscopy analysis revealed a nuclei-restricted expression of the CDC42C188Y mutant, which is no longer able to reach the plasma membrane. These findings were mirrored by in vivo data. In C. elegans, CDC-42 controls vulval induction and morphogenesis. Phenotypic characterization of transgenic lines conditionally expressing the wild-type or mutant CDC-42 alleles demonstrated a variable impact of mutations on multiple signaling pathways. Specifically, the C188Y change behaved as a loss-of-function mutation on both LET-60/RAS and WSP-1/WASP signaling cascades, leading to a less prevalent multivulva (Muv) and protruding vulva (Pvl) phenotypes, compared to the wild-type protein. Differently, while CDC42D170N had a disruptive effect on migration of vulval precursor cells (Pvl) but not on their induction (Muv), CDC42K16R displayed an opposite behavior. Finally, heat-shock experiments performed at different time-windows during C. elegans larval development established that Muv and Pvl are completely different phenotypes associated with dysregulation of multiple signaling cascades. Overall, these findings further highlight the extremely variable impact of CDC42 mutations on cellular processes and development.