Genes to Proteins

DNA到蛋白质：癫痫的基因调控、蛋白质表达和功能 数百个基因的功能丧失(LOF)突变与人类癫痫有关。然而， 个体间的高频率序列变异对归因于错义变异提出了挑战 会导致癫痫。这个高度多学科的研究团队开发了一种模块化平台方法 加速确定功能、药理学、神经网络和整个动物的后果 在一系列癫痫类型的患者中遇到的不确定意义的遗传变异(VUS)。这个 最终目标是制定建立遗传诊断标准和确定潜在靶标的策略 进行干预。为此，项目1将与项目2调查人员一起审问1中的多个VU以 2个常见的、每年编码癫痫基因的非离子通道(5年内最多10个基因)。项目 1采用中等吞吐量评估，每个基因12-15个VUS，在3个里程碑中进行检查： 里程碑1将评估用于VUS功能分析的硅胶和体外模型系统：1a。 使用基因和变量管理核心(GVCC)，在Silico工具中生成和评估以改进VUS的建模 致病性；1b。使用HEK293T细胞的二维(2-D)培养对每个研究的VU进行生化测试 检测突变蛋白质的稳定性、已知蛋白质的相互作用和聚集；测试一种 VU关于亚细胞定位、蛋白质运输和/或翻译后加工。 里程碑2将在体外建立细胞自主效应的分析。人类癫痫工具的核心 (HETC)将使选定的基因失活并产生表达DOX-2的人类多能干细胞(HPSC)。 诱导神经原蛋白2(INeurons)或ASCL1和DLX2(IGNS)用于直接诱导神经元。基因敲除神经元 将使其过表达含有WT或VUS的蛋白质及其在基因敲除中拯救LOF表型的能力 细胞将在2-D培养中通过评估祖细胞增殖、细胞存活、潜在的 利用细胞形态、运动性和RNAseq进行分化和基因表达。 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iNeurons(例如，表达WT或含有VUS的STXBP1)的突触形成、转换 使用多电极阵列(MEA)的(可塑性)、传输和点火特性。这些2-D文化将 筛选细胞向谷氨酸能兴奋表型或GABA能抑制表型分化 项目1中最有希望的线，项目2将在2-D和3-D混合细胞类型的细胞系统中使用。 作为一个整体，U54提供了：1)多个优化的、交叉验证的hPSC平台，用于审问癫痫 基因；2)人体VUS对10例非离子通道癫痫的体外和体内致病性测定 基因；3)每个癫痫基因的优化模型；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.!