Functional characterization of schizophrenia rare variants using genetically engineered human iPSCs

项目摘要 精神分裂症（Schizophrenia，SCZ）是一种常见的神经发育障碍。然而，基因 SCZ的结构非常复杂，阻碍了研究进展。最近，Scz外显子组荟萃分析 （SCHEMA）Consortium已经确定了具有超罕见编码变体的基因，每种基因都会对以下疾病产生重大风险： 嘘我们假设，这些罕见的变异体在人类诱导多能干细胞（hiPSC）中的存在， 将导致与Scz相关的神经元表型。PI潘力博士在基因编辑方面拥有丰富的经验 和iPSC，包括开发一种改进的方法，用于无缝引入和去除突变， 转化为hiPSC。她以前专注于神经退行性疾病，但在这项申请中，她提出， 变更为SCZ研究。她的实验室位于神经生物学系， 它有着长期的重大精神疾病研究记录， iPSC（如HD iPSC联盟）。在特定目标1中，具有蛋白质截短变体的同基因hiPSC系 将通过CRISPR辅助的同源重组进行基因工程改造， iPSC系的组，其中每个等基因系携带不同的N-末端蛋白截短点突变。 敲低和过表达拯救实验将确定是否存在蛋白表达的损失 从而丧失基因功能。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测序和定量蛋白质组学进行研究。在具体目标3中，突变对突触的影响 和电生理表型将被描绘。我们假设，了解细胞 SCHEMA突变引起的表型和改变的信号通路将阐明细胞内 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.