Genes to Proteins

基因到蛋白质

基本信息

批准号：
10265441
负责人：
MARGARET ELIZABETH ROSS
金额：
$ 29.66万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-15 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10265441
关键词：
3-Dimensional ASCL1 gene Algorithmic Software Algorithms Animal Model Animals Benign Biochemical Biological Biological Assay Biological Models Cell Differentiation process Cell Line Cell Survival Cells Cellular Assay Cellular Morphology Characteristics Clinical Co-Immunoprecipitations Complex D Cells DNA Data Diagnostic Differentiated Gene Dimensions Doxycycline Elements Epilepsy Evaluation FRAP1 gene Fractionation Frequencies Future Gene Expression Gene Expression Regulation Gene Proteins Genes Genetic Glutamates Goals Human Impairment In Vitro Individual Interdisciplinary Study Intervention Knock-out Modeling Mutation Neurons Open Reading Frames Parents Pathogenesis Pathogenicity Pathway interactions Patients Pharmacology Phenotype Pre-Clinical Model Precision therapeutics Property Proteins RNA Splicing Research Personnel Rodent Model SNAP receptor Signal Transduction Single Nucleotide Polymorphism System Testing Variant Zebrafish cell motility cell type clinically significant differentiation protocol experimental study genetic variant human pluripotent stem cell human reference genome improved in silico in vitro Model in vivo inhibitory neuron insertion/deletion mutation loss of function loss of function mutation member multi-electrode arrays mutant next generation sequencing overexpression progenitor protein expression protein function protein protein interaction protein transport screening synaptogenesis syntaxin binding protein 1 therapeutic evaluation tool transcriptome sequencing two-dimensional variant of unknown significance

项目摘要

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.!

DNA 到蛋白质：癫痫中的基因调控、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，个体之间序列变异的高频率对归因错义变异提出了挑战如引起癫痫。这个高度跨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、神经元网络和整个动物的后果一系列癫痫类型患者中遇到的不确定意义的遗传变异（VUS）。这最终目标是制定建立遗传诊断标准和识别潜在目标的策略进行干预。为此，项目 1 将与项目 2 调查员一起审讯 1 至 10 天内的多个 VUS。每年 2 个经常遇到的非离子通道编码癫痫基因（5 年内最多 10 个基因）。项目 1 采用每个基因 12-15 个 VUS 的中等通量评估，在 3 个里程碑中进行检查：里程碑 1 将评估用于 VUS 功能分析的计算机和体外模型系统： 1a。借助基因和变异管理核心 (GVCC)，生成和评估计算机工具以改进 VUS 建模致病性； 1b.在每个研究的 VUS 的生化测试中使用 HEK293T 细胞的二维 (2-D) 培养物检查突变蛋白稳定性、已知的蛋白相互作用和聚集； 1c.测试一个的影响 VUS 涉及亚细胞定位、蛋白质运输和/或翻译后处理。里程碑 2 将建立体外细胞自主效应的测定方法。人类癫痫工具核心（HETC）将使选定的基因失活并产生表达 dox- 的人类多能干细胞（hPSC）诱导性 Neurogenin 2 (iNeurons) 或 ASCL1 & DLX2 (iGNs) 用于直接诱导神经元。敲除神经元将过度表达含有蛋白质的 WT 或 VUS 及其在敲除中拯救 LOF 表型的能力通过评估祖细胞增殖、细胞存活、潜力，在二维培养物中对细胞进行检查使用细胞形态、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 或包含 VUS 的 STXBP1）的突触形成、更新使用多电极阵列 (MEA) 测量（塑性）、传输和烧制特性。这些二维文化将筛选分化为谷氨酸能兴奋性或 GABA 能抑制表型的细胞，挑选项目 1 中最有前途的细胞系，项目 2 将在 2-D 和 3-D 混合细胞类型的细胞系统中使用。总体而言，该 U54 提供：1) 多个优化、交叉验证的 hPSC 平台来检查癫痫基因； 2）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； 4）未来精准治疗测试的平台。！