Functional characterization of schizophrenia rare variants using genetically engineered human iPSCs

项目总结 精神分裂症(SCZ)是一种常见的、使人衰弱的神经发育障碍。然而，基因 SCZ的体系结构高度复杂，阻碍了研究进展。最近，SCZ Exome Meta分析 (SCHEMA)联盟已经确定了具有超罕见编码变体的基因，每个基因都会带来相当大的风险 SCZ.我们假设在人类诱导的多能干细胞(Hipscs)中存在这些罕见的变异。 将导致与SCZ相关的神经元表型。私家侦探潘力博士在基因编辑方面拥有丰富经验 和IPSCs，包括开发一种改进的无缝引入和消除突变的方法 转化为HiPSCs。她之前一直专注于神经退行性疾病，但通过这项申请，她建议 改成对SCZ的研究。她的实验室位于神经生物学分部，这促进了这一转变， 它在重大精神疾病的研究和高度合作的研究方面有着长期的记录 IPSC(如高清iPSC联盟)。在特定目标1中，具有蛋白质截断变体的等基因HiPSC系 与SCZ相关的基因工程将通过CRISPR辅助的同源重组产生 IPSC品系面板，每个等基因品系携带不同的N-末端蛋白质截断点突变。 基因敲除和过表达挽救实验将确定是否存在蛋白质表达缺失 从而导致基因功能丧失。在特定目标2中，突变对基因和蛋白质表达的影响将 使用RNA序列和定量蛋白质组学进行研究。在特定目标3中，突变对突触的影响 并将描绘电生理表型。我们假设理解细胞 由模式突变引起的表型和改变的信号通路将阐明细胞 精神分裂症亚型的发病机制，并提供潜在的治疗靶点。

PROJECT SUMMARY Schizophrenia (Scz) is a common and debilitating neurodevelopmental disorder. However, the genetic architecture of Scz is highly complex, impeding research progress. Recently, the Scz Exome Meta-Analysis (SCHEMA) Consortium has identified genes with ultra-rare coding variants that each confer substantial risk for Scz. We hypothesize that the presence of these rare variants in human induced pluripotent stem cells (hiPSCs) will lead to neuronal phenotypes relevant to Scz. The PI, Dr. Pan Li, has extensive experience with gene editing and iPSCs, including development of an improved method for seamless introduction and removal of mutations into hiPSCs. She has previously focused on neurodegenerative disorders, but with this application proposes to change to study of Scz. This transition is facilitated by the location of her lab in the Division of Neurobiology, which has a long track record of research into major mental illnesses and of highly collaborative research using iPSCs (eg the HD iPSC Consortium). In Specific Aim 1, isogenic hiPSC lines with protein truncating variants associated with Scz will be genetically engineered by CRISPR-assisted homologous recombination to generate panels of iPSC lines, with each isogenic line bearing a different N-terminal protein truncating point mutation. Knockdown and overexpression rescue experiments will determine whether there is loss of protein expression and thus loss of gene function. In Specific Aim 2, the effect of mutations on gene and protein expression will be studied using RNA seq and quantitative proteomics. In Specific Aim 3, the effects of mutations on synaptic and electrophysiological phenotypes will be delineated. We hypothesize that understanding the cellular phenotypes and altered signaling pathways evoked by the SCHEMA mutations will clarify the cellular pathogenesis of subtypes of schizophrenia, and provide potential therapeutic targets.