Variant-specific changes in RAC3 function disrupt corticogenesis in neurodevelopmental phenotypes

September 14, 2022

Marcello Scala 1 2 3Masashi Nishikawa 3Hidenori Ito 3Hidenori Tabata 3Tayyaba Khan 4Andrea Accogli 1Laura Davids 5Anna Ruiz 6Pietro Chiurazzi 7 8Gabriella Cericola 9Björn Schulte 10Kristin G Monaghan 11Amber Begtrup 11Annalaura Torella 12 13Michele Pinelli 12Anne Sophie Denommé-Pichon 14 15 16Antonio Vitobello 14 15Caroline Racine 15 16Maria Margherita Mancardi 17Courtney Kiss 18Andrea Guerin 18Wendy Wu 4 19Elisabeth Gabau Vila 20Bryan C Mak 21Julian A Martinez-Agosto 21 22 23Michael B Gorin 21 24 25Bugrahan Duz 26Yavuz Bayram 27 28Claudia M B Carvalho 29 30Jaime E Vengoechea 5David Chitayat 31 32 33Tiong Yang Tan 34Bert Callewaert 35Bernd Kruse 9Lynne M Bird 36 37Laurence Faivre 14 16Marcella Zollino 7 8Saskia Biskup 10 38Undiagnosed Diseases NetworkTelethon Undiagnosed Diseases ProgramPasquale Striano 1 2Vincenzo Nigro 12 13Mariasavina Severino 39Valeria Capra 40Gregory Costain 4 32 33 41Koh Ichi Nagata 3 42


Variants in RAC3, encoding a small GTPase RAC3 which is critical for the regulation of actin cytoskeleton and intracellular signal transduction, are associated with a rare neurodevelopmental disorder with structural brain anomalies and facial dysmorphism. We investigated a cohort of 10 unrelated participants presenting with global psychomotor delay, hypotonia, behavioural disturbances, stereotyped movements, dysmorphic features, seizures and musculoskeletal abnormalities. MRI of brain revealed a complex pattern of variable brain malformations, including callosal abnormalities, white matter thinning, grey matter heterotopia, polymicrogyria/dysgyria, brainstem anomalies and cerebellar dysplasia. These patients harboured eight distinct de novo RAC3 variants, including six novel variants (NM_005052.3): c.34G > C p.G12R, c.179G > A p.G60D, c.186_188delGGA p.E62del, c.187G > A p.D63N, c.191A > G p.Y64C and c.348G > C p.K116N. We then examined the pathophysiological significance of these novel and previously reported pathogenic variants p.P29L, p.P34R, p.A59G, p.Q61L and p.E62K. In vitro analyses revealed that all tested RAC3 variants were biochemically and biologically active to variable extent, and exhibited a spectrum of different affinities to downstream effectors including p21-activated kinase 1. We then focused on the four variants p.Q61L, p.E62del, p.D63N and p.Y64C in the Switch II region, which is essential for the biochemical activity of small GTPases and also a variation hot spot common to other Rho family genes, RAC1 and CDC42. Acute expression of the four variants in embryonic mouse brain using in utero electroporation caused defects in cortical neuron morphology and migration ending up with cluster formation during corticogenesis. Notably, defective migration by p.E62del, p.D63N and p.Y64C were rescued by a dominant negative version of p21-activated kinase 1. Our results indicate that RAC3 variants result in morphological and functional defects in cortical neurons during brain development through variant-specific mechanisms, eventually leading to heterogeneous neurodevelopmental phenotypes.

Keywords: RAC3; axon guidance; brain development; neuronal migration; small GTPase.