RAC1 is a widely studied Rho GTPase, a class of molecules that modulate numerous cellular functions essential for normal development. RAC1 is highly conserved across species and is under strict mutational constraint. We report seven individuals with distinct de novo missense RAC1 mutations and varying degrees of developmental delay, brain malformations, and additional phenotypes. Four individuals, each harboring one of c.53G>A (p.Cys18Tyr), c.116A>G (p.Asn39Ser), c.218C>T (p.Pro73Leu), and c.470G>A (p.Cys157Tyr) variants, were microcephalic, with head circumferences between -2.5 to -5 SD. In contrast, two individuals with c.151G>A (p.Val51Met) and c.151G>C (p.Val51Leu) alleles were macrocephalic with head circumferences of +4.16 and +4.5 SD. One individual harboring a c.190T>G (p.Tyr64Asp) allele had head circumference in the normal range. Collectively, we observed an extraordinary spread of ∼10 SD of head circumferences orchestrated by distinct mutations in the same gene. In silico modeling, mouse fibroblasts spreading assays, and in vivo overexpression assays using zebrafish as a surrogate model demonstrated that the p.Cys18Tyr and p.Asn39Ser RAC1 variants function as dominant-negative alleles and result in microcephaly, reduced neuronal proliferation, and cerebellar abnormalities in vivo. Conversely, the p.Tyr64Asp substitution is constitutively active. The remaining mutations are probably weakly dominant negative or their effects are context dependent. These findings highlight the importance of RAC1 in neuronal development. Along with TRIO and HACE1, a sub-category of rare developmental disorders is emerging with RAC1 as the central player. We show that ultra-rare disorders caused by private, non-recurrent missense mutations that result in varying phenotypes are challenging to dissect, but can be delineated through focused international collaboration.
RAC1 is a highly conserved Rho GTPase critical for many cellular and developmental processes. De novo missense RAC1 variants cause a highly variable neurodevelopmental disorder. Some of these variants have previously been shown to have a dominant negative effect. Most previously reported patients with this disorder have either severe microcephaly or severe macrocephaly. Here, we describe eight patients with pathogenic missense RAC1 variants affecting residues between Q61 and R68 within the switch II region of RAC1. These patients display variable combinations of developmental delay, intellectual disability, brain anomalies such as polymicrogyria and cardiovascular defects with normocephaly or relatively milder micro- or macrocephaly. Pulldown assays, NIH3T3 fibroblast spreading assays and staining for activated PAK1/2/3 and WAVE2 suggest that these variants increase RAC1 activity and over-activate downstream signalling targets. Axons of neurons isolated from Drosophila embryos expressing the most common of the activating variants are significantly shorter, with an increased density of filopodial protrusions. In vivo, these embryos exhibit frequent defects in axonal organization. Class IV dendritic arborization neurons expressing this variant exhibit a significant reduction in the total area of the dendritic arbour, increased branching and failure of self-avoidance. RNAi knock down of the WAVE regulatory complex component Cyfip significantly rescues these morphological defects. These results establish that activating substitutions affecting residues Q61-R68 within the switch II region of RAC1 cause a developmental syndrome. Our findings reveal that these variants cause altered downstream signalling, resulting in abnormal neuronal morphology and reveal the WAVE regulatory complex/Arp2/3 pathway as a possible therapeutic target for activating RAC1 variants. These insights also have the potential to inform the mechanism and therapy for other disorders caused by variants in genes encoding other Rho GTPases, their regulators and downstream effectors.