Functional characterization of de novo CDC42 missense mutations underlying immune-hematolological or otopalatodigital phenotypes

CDC42 (cell division cycle 42) is a small GTPase of the RAS superfamily regulating key developmental processes, including hematopoiesis, immunity and craniofacial/skeletal formation. We recently characterized different CDC42 missense mutations associated with a broad spectrum of rare conditions, ranging from neurodevelopmental disorders, with craniofacial, skeletal and heart anomalies, including phenotypes resembling RASopathies, to immune-hematological diseases, (e.g., NOCARH syndrome). Currently, the pathogenic mechanisms underlying such a wide clinical heterogeneity are not fully clarified. Here, we profile four de novo pathogenic CDC42 variants (p.C81Y, p.V44F, p.L55P and p.R66G) associated with phenotypically distinct conditions. The clinical characterization of the patient carrying the p.C81Y mutant showed a NOCARH-like phenotype [Szczawinska-Poplonyk A, et al. 2020]. Concerning the other three variants, they are associated with variable skeletal dysplasia (spinal and limbs malformations, joint contractures), heart anomalies, deafness and cleft palate, resembling phenotypes of the otopalatodigital (OPD) syndromic spectrum. GTPase activity, binding to effectors/regulators, intracellular localization and spreading were assessed in vitro. The mutant associated with the immune-hematologic related condition shows decreased CDC42 stability, impaired PAK1, N-WASP and RHOGDI binding and aberrant CDC42 intracellular localization, consistently with other NOCARH-like associated variants. On the other hand, the variants related to the OPD-like phenotype do not impact protein localization, while they affect cell spreading, indicating different pathogenic mechanisms underlying the distinct phenotypes. Our study expands the molecular and clinical spectrum of CDC42 pathogenic variants, confirms the relevance of functional validation of unclassified variants to assess their possible role in disease pathogenicity and suggests divergent pathogenic mechanisms underlying CDC42-related NOCARH and OPD-like phenotypes.