Second occurrence of the PAK3-R67C variation and multiscale analysis of the corresponding knock-in mice reveal novel phenotypic features and functional synaptic defects

Variations in PAK3, a gene located on the X chromosome, are known to contribute to intellectual disability (ID) and are associated with a broad clinical spectrum. The correlation between genotype and phenotype in PAK3-related disorders remains incompletely understood. In this study, we focused on the PAK3-R67C variant, the most amino-terminal variation identified to date, which was initially associated with a non-syndromic form of X-linked ID. We describe the clinical picture of its second occurrence, and present additional clinical insights. Our findings indicate that the R67C mutation is associated with moderate to severe ID, along with speech and gait abnormalities, and features consistent with autism spectrum disorders. To explore these associations further, we conducted a comprehensive phenotypic analysis using a corresponding knock-in mouse model. The Pak3-R67C mice, which present significant microcephaly, displayed mild motor deficits, social avoidance behaviour, and impaired emotional learning and memory. At the physiological level, Pak3-R67C mice exhibited reduced hippocampal synaptic efficacy and defects in long-term potentiation, despite unaltered synapse structure. In vitro biochemical assays revealed that the PAK3-R67C variant enhances interactions with PAK-interacting exchange factors (PIX). Notably, the expression of this variant in COS7 cells, altered the distribution of focal adhesions, while cell migration capacity remained unaffected. Collectively, these data, which include the second reported occurrence of the R67C variant, a detailed characterization of the corresponding knock-in mouse line, and a multi-tiered analysis of its biological and biochemical properties, contribute to a better understanding of the genotype-phenotype relationship in PAK3-related disorders.