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.

中文翻译：

---

RAC3 功能的变异特异性变化破坏了神经发育表型中的皮质生成。

RAC3 的变体编码一种小 GTP 酶 RAC3，它对调节肌动蛋白细胞骨架和细胞内信号转导至关重要，与一种罕见的神经发育障碍相关，伴有脑结构异常和面部畸形。我们调查了一组 10 名无关的参与者，这些参与者表现出整体精神运动迟缓、肌张力减退、行为障碍、刻板动作、畸形特征、癫痫发作和肌肉骨骼异常。大脑 MRI 显示了多种脑畸形的复杂模式，包括胼胝体异常、白质变薄、灰质异位、多小脑回/脑回异常、脑干异常和小脑发育不良。这些患者携带八种不同的新生 RAC3 变体，包括六种新变体 (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 和 c.348G > C p.K116N。然后我们检查了这些新的和以前报道的致病变异 p.P29L、p.P34R、p.A59G、p.Q61L 和 p.E62K 的病理生理学意义。体外分析显示，所有测试的 RAC3 变体都具有不同程度的生化和生物学活性，并且对包括 p21 激活的激酶 1 在内的下游效应子表现出一系列不同的亲和力。然后我们重点关注四种变体 p.Q61L、p.E62del、 Switch II 区域的 p.D63N 和 p.Y64C，这对小 GTP 酶的生化活性至关重要，也是其他 Rho 家族基因 RAC1 和 CDC42 共有的变异热点。使用子宫内电穿孔在胚胎小鼠大脑中急性表达四种变体导致皮质神经元形态和迁移的缺陷，最终在皮质发生过程中形成簇。值得注意的是，p.E62del、p.D63N 和 p.Y64C 的迁移缺陷被 p21 激活激酶 1 的显性负性版本挽救。我们的结果表明，RAC3 变体在大脑发育过程中通过变体导致皮质神经元的形态和功能缺陷-特异性机制，最终导致异质性神经发育表型。