PhD Candidate Shanghai Jiao Tong University, China
Abstract: Developmental and epileptic encephalopathy (DEE) is a severe neurodevelopmental disorder in children, characterized by high genetic heterogeneity. Despite advances, the mechanisms underlying DEE remain complex and poorly elucidated. In this study, we identified a novel de novo CYFIP2 nonsense mutation, c.2473C>T (p.Arg825Ter), in a DEE65 family, presenting milder phenotypes compared to other gain-of-function (GoF) mutations, suggesting a distinct mechanism. CYFIP2 is crucial for actin polymerization, yet its loss-of-function (LoF) effects are largely unexplored. To address this, we generated induced pluripotent stem cells (iPSCs) from the Arg825 patient and used CRISPR to create heterozygous and homozygous p.Arg87Cys cell lines, representing the most severe DEE65 mutation, in wild-type iPSCs. Additionally, to elucidate the mechanism of DEE65 caused by the Arg825 mutation, we performed CRISPR-mediated correction in Arg825 iPSCs to obtain an isogenic control iPSC line. Analysis showed that CYFIP2 expression was decreased in Arg825 iPSCs but remained at wild-type levels in Arg87 iPSCs. All lines were differentiated into neural progenitor cells, forebrain neurons, and excitatory cortical projection neurons. Multi-electrode array analysis revealed that the Arg825 neurons exhibited reduced epileptiform activity compared to the Arg87 neurons. Arg825 neurons also showed reduced SYN1 synaptic signaling and lower dendritic spine density compared to Arg87 neurons, consistent with patient phenotypes. RNA-seq analysis indicated that the Arg825 mutation impairs axon development and causes transcriptional dysregulation. Overexpression studies in HEK293T cells demonstrated that the Arg825 mutation significantly reduces CYFIP2 interaction with WAVE1 and increases nuclear localization, indicating a potential genetic compensation mechanism affecting mRNA transcription. Overall, this study developed human iPSC models of CYFIP2 mutations, revealing a mechanism where nonsense mutations lead to mRNA truncation, disrupting the assembly of the WAVE1 regulatory complex and actin polymerization in DEE. Future research will employ this iPSC platform for deeper mechanistic insights and drug screening, offering new avenues for prenatal diagnosis and personalized treatment of DEE.
Funding Source: This study was supported by National Key Research and Development Program (2024YFC2707002), Innovation Program of Shanghai Municipal Education Commission (2023ZKZD16) and National Natural Science Foundation of China (82071262).
