Variant-specific changes in RAC3 function disrupt corticogenesis in neurodevelopmental phenotypes
Authors: Scala Marcello , Nishikawa Masashi , Ito Hidenori , Tabata Hidenori , Khan Tayyaba , Accogli Andrea , Davids Laura , Ruiz Anna , Chiurazzi Pietro , Cericola Gabriella , Schulte Björn , Monaghan Kristin G , Begtrup Amber , Torella Annalaura , Pinelli Michele , Denommé-Pichon Anne Sophie , Vitobello Antonio , Racine Caroline , Mancardi Maria Margherita , Kiss Courtney , Guerin Andrea , Wu Wendy , Gabau Vila Elisabeth , Mak Bryan C , Martinez-Agosto Julian A , Gorin Michael B , Duz Bugrahan , Bayram Yavuz , Carvalho Claudia M B , Vengoechea Jaime E , Chitayat David , Tan Tiong Yang , Callewaert Bert , Kruse Bernd , Bird Lynne M , Faivre Laurence , Zollino Marcella , Biskup Saskia ,   Undiagnosed Diseases Network, Brown Gabrielle , Butte Manish J , Dell'Angelica Esteban C , Dorrani Naghmeh , Douine Emilie D , Fogel Brent L , Gutierrez Irma , Huang Alden , Krakow Deborah , Lee Hane , Loo Sandra K , Mak Bryan C , Martin Martin G , Martínez-Agosto Julian A , McGee Elisabeth , Nelson Stanley F , Nieves-Rodriguez Shirley , Palmer Christina G S , Papp Jeanette C , Parker Neil H , Renteria Genecee , Sinsheimer Janet S , Wan Jijun , Wang Lee-kai , Perry Katherine Wesseling ,   Telethon Undiagnosed Diseases Program, Nigro Vincenzo , Brunetti-Pierri Nicola , Casari Giorgio , Cappuccio Gerarda , Torella Annalaura , Pinelli Michele , Musacchia Francesco , Mutarelli Margherita , Carrella Diego , Vitiello Giuseppina , Capra Valeria , Parenti Giancarlo , Leuzzi Vincenzo , Selicorni Angelo , Maitz Silvia , Banfi Sandro , Zollino Marcella , Montomoli Mario , Milani Donatelli , Romano Corrado , Tummolo Albina , De Brasi Daniele , Coppola Antonietta , Santoro Claudia , Peron Angela , Pantaleoni Chiara , Castello Raffaele , D’Arrigo Stefano , Striano Pasquale , Nigro Vincenzo , Severino Mariasavina , Capra Valeria , Costain Gregory , Nagata Koh ichi 
Published: March 2022

Abstract
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 &gt; C p.G12R, c.179G &gt; A p.G60D, c.186_188delGGA p.E62del, c.187G &gt; A p.D63N, c.191A &gt; G p.Y64C and c.348G &gt; 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.