PROJECT SUMMARY / ABSTRACT This NIH K08 proposal, describes a five-year career development program in epilepsy genetics research. Through this program, Dr. Khoshkhoo will receive training in human genetics, the experimental and analytic aspects of next generation sequencing, and single cell genomics. This new skillset will complement Dr. Khoshkhoo’s prior research and clinical training, and ideally position him to transition to an independent investigator position studying the functional and molecular mechanisms of genetic variants in epilepsy. The institutional resources available through Brigham and Women’s Hospital (BWH), Boston Children’s Hospital (BCH), and Harvard Medical School (HMS) are world class and they provide the ideal environment to foster the career developmental of young physician-scientists. Dr. Khoshkhoo’s mentor for this proposal, Dr. Christopher Walsh (a Professor of Neurology at HMS and HHMI Investigator at BCH), is a leader in genetics and genomics of human neurologic diseases. Dr. Walsh has a long track record for mentoring other trainees to successful careers in biomedical research. In addition, Dr. Khoshkhoo has assembled a group of collaborators with complementary expertise, and an Advisory Committee with extensive experience in mentoring physician- scientists to develop independent research programs. The primary scientific objective of this proposal is to identify the role of pathogenic post-zygotic (somatic) mutations (variants) in temporal lobe epilepsy (TLE), and to characterize the cell-type specific and transcriptional mechanisms through which these variants contribute to the development of epilepsy. Dr. Khoshkhoo provides pilot data indicating that a subset of sporadic TLE cases harbor likely pathogenic somatic variants, which supports his central hypothesis that genetic and transcriptional dysregulation caused by somatic variants plays a key role in TLE pathogenesis. This proposal will systemically examine surgical TLE resections for the presence of these somatic variants and investigate their cellular and transcriptional mechanisms. To achieve these objectives, a combination of ultra-deep gene panel sequencing, Parallel RNA and DNA analysis after Deep sequencing (PRDD-seq), and single nucleus RNA sequencing (snRNA-seq) will be employed. These state of the art strategies will aim to: (1) identify pathogenic somatic variants in surgically resected hippocampal tissue from TLE patients and post-mortem neurotypical individuals; (2) determine the cell-type(s) of mutant cells in TLE cases with known pathogenic somatic variants; and (3) examine the downstream gene expression changes caused by these variants in TLE. These studies will not only help establish somatic variants as a novel mechanism for TLE pathogenesis, but also investigate their cellular and molecular mechanisms in situ. Overall, this proposal introduces a new conceptual and experimental framework for studying TLE and the findings may have immediate d...