# Genes to Proteins > **NIH NIH U54** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2021 · $296,625 ## Abstract 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... ## Key facts - **NIH application ID:** 10265441 - **Project number:** 5U54NS117170-02 - **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR - **Principal Investigator:** MARGARET ELIZABETH ROSS - **Activity code:** U54 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2021 - **Award amount:** $296,625 - **Award type:** 5 - **Project period:** 2020-09-15 → 2025-07-31 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10265441 ## Citation > US National Institutes of Health, RePORTER application 10265441, Genes to Proteins (5U54NS117170-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10265441. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*