A p.N92K variant of the GTPase RAC3 disrupts cortical neuron migration and axon elongation.

RAC3 encodes a small GTPase of the Rho family, crucial for actin cytoskeleton organization and signaling pathways. De novo deleterious variants in RAC3 cause neurodevelopmental disorder with structural brain anomalies and dysmorphic facies (NEDBAF). Disease-causing variants thus far reported are thought to impact key conserved regions within RAC3, such as the P-loop, switch I/II, and G boxes, which are essential for the interaction with regulatory proteins and effectors. Recently, however, a novel variant, c.276T>A, p.N92K, was identified in a prenatal case with complex brain malformations. This variant, located outside the core functional regions, represents a unique class of RAC3 pathogenic mutations. We investigated the variant's effects using in vitro, in silico, and in vivo approaches. Overexpression of RAC3-N92K in primary hippocampal neurons impaired differentiation, leading to round cell shape with lamellipodia, suggesting that RAC3-N92K is active. Biochemical studies showed that RAC3-N92K is 1) resistant to GAP-mediated inactivation, 2) responsive to GEF activation, and 3) capable of interacting with RAC effectors PAK1 and MLK2, as well as Rho-kinase 1, activating gene expression through SRF, NFκB, and AP1 pathways. Structural analyses suggest that N92K disrupts GAP interactions but preserves interactions with GEF, PAK1, and MLK2. In vivo, RAC3-N92K expression in embryonic mouse cortical neurons led to migration defects and periventricular clustering during corticogenesis, along with impaired axon elongation. These findings indicate that RAC3-N92K's activated state significantly disrupts cortical development, expanding the genetic and pathophysiological spectrum of NEDBAF.