Genes to Proteins

DNA到蛋白质：基因调控、蛋白质表达及其在癫痫中的作用 数百个基因的功能丧失（LOF）突变与人类癫痫有关。然而，在这方面， 个体间高频率的序列变异对将错义变异归因于 导致癫痫这个高度多学科的研究团队开发了一种模块化平台方法， 加速功能、药理学、神经网络和整个动物后果的确定 在一系列癫痫类型患者中遇到的不确定意义的遗传变异（VUS）。的 最终的目标是设计出建立遗传诊断标准和识别潜在靶点的策略 进行干预。为此，项目1将与项目2的调查人员一起，在1至10年内对多个VUS进行审讯。 每年2个常见的、非离子通道编码癫痫基因（5年内最多10个基因）。项目 1采用中等通量评估，每个基因12-15个VUS，在3个千年期内检查： 里程碑1将评估用于VUS功能分析的计算机模拟和体外模型系统，以：1a. 利用基因和变异体策展核心（GVCC），生成和评估计算机模拟工具，以改进VUS建模 致病性; 1b.在每个研究的VUS的生化试验中使用HEK 293 T细胞的二维（2-D）培养物 检查突变蛋白质稳定性、已知蛋白质相互作用和聚集; 1c.测试a的影响 VUS对亚细胞定位、蛋白质运输和/或翻译后加工的影响。 里程碑2将建立体外细胞自主效应的测定。人类癫痫的核心工具 （HETC）将扩增选定的基因，并产生表达dox-1的人多能干细胞（hPSC）。 诱导的神经生成素2（iNeurons）或ASCL 1和DLX 2（iGN）用于直接诱导神经元。敲除神经元 将使其过表达含有WT或VUS的蛋白及其在敲除中拯救LOF表型的能力。 将通过评估祖细胞增殖、细胞存活、 使用细胞形态学、运动性和RNAseq的分化和基因表达。 里程碑3. VUS对突触形成和网络特性的功能影响。这些 将检查iNeuron（例如表达WT或含VUS的STXBP 1）的突触形成、转换和突触形成。 （可塑性）、传输和烧制性能。这些二维培养物 筛选向谷氨酸能兴奋性或GABA能抑制性表型分化的细胞， 项目1中最有前途的细胞系，项目2将用于2-D和3-D的混合细胞类型的细胞系统。 作为一个整体，这个U 54提供：1）多个优化的，交叉验证的hPSC平台，以询问癫痫 基因; 2）在体外和体内测定人VUS对多达10种非离子通道癫痫的致病性 基因; 3）每个癫痫基因的优化模型;以及4）未来精确治疗测试的平台。

DNA TO PROTEINS: GENE REGULATION, PROTEIN EXPRESSION AND FUNCTION IN EPILEPSY Loss of function (LOF) mutations in hundreds of genes are associated with human epilepsy. However, the high frequency of sequence variation among individuals presents a challenge to ascribe missense variants as causing epilepsy. This highly multidisciplinary research team develops a modular platform approach to accelerate determination of the functional, pharmacological, neuronal network and whole animal consequences of genetic variants of uncertain significance (VUS) encountered in patients with a range of epilepsy types. The ultimate goal is to devise strategies for establishing genetic diagnostic criteria and identifying potential targets for intervention. To this end, Project 1 will join Project 2 investigators in the interrogation of multiple VUS in 1 to 2 frequently encountered, non-ion channel encoding epilepsy genes per year (up to 10 genes in 5 years). Project 1 employs moderate-throughput assessment of 12-15 VUSs per gene, examined in 3 Milestones: Milestone 1 will assess in silico and in vitro model systems for VUS functional analyses to: 1a. with the gene and variant curation core (GVCC), generate and assess In silico tools to improve modeling of VUS pathogenicity; 1b. use 2-dimentional (2-D) cultures of HEK293T cells in biochemical tests of each studied VUS to examine mutant protein stability, known protein interactions and aggregation; 1c. test the impact of a VUS on subcellular localization, protein trafficking and/or post translational processing. Milestone 2 will establish assays of cell autonomous effects in vitro. The human epilepsy tool core (HETC) will inactivate selected genes and generate human pluripotent stem cells (hPSC) that express dox- induced Neurogenin 2 (iNeurons) or ASCL1 & DLX2 (iGNs) for direct induction of neurons. Knockout neurons will be made to overexpress WT or VUS containing protein and their ability to rescue LOF phenotypes in knockout cells will be examined in 2-D cultures through assessing progenitor proliferation, cell survival, potential for differentiation and gene expression using cell morphology, motility, and RNAseq. Milestone 3. Functional impact of VUS on synapse formation and network properties. These iNeurons (e.g. expressing WT or VUS-containing STXBP1) will be examined for synapse formation, turnover (plasticity), transport, and firing properties using multi-electrode arrays (MEA). These 2-D cultures will screen cells differentiated toward either glutamatergic excitatory or GABAergic inhibitory phenotypes, picking the most promising lines in Project 1 that Project 2 will use in cell systems of mixed cell types in 2-D and 3-D. As a whole, this U54 delivers: 1) multiple optimized, cross-validated hPSC platforms to interrogate epilepsy genes; 2) in vitro and in vivo determination of human VUS pathogenicity for up to 10 non-ion channel epilepsy genes; 3) optimized models for each epilepsy gene; and 4) platforms for future precision therapeutic testing.!