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Genes to Proteins

基因到蛋白质

基本信息

批准号：
10670363
负责人：
MARGARET ELIZABETH ROSS
金额：
$ 28.5万
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/10670363
关键词：
3-Dimensional ASCL1 gene Acceleration 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 DNA Data Doxycycline Elements Epilepsy Evaluation FRAP1 gene Fractionation Frequencies Future Gene Expression Gene Expression Regulation 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 Phenotype Pre-Clinical Model Precision therapeutics Proliferating 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 diagnostic criteria 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 pharmacologic predictive tools 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