Lawrence, Chloe2025-07-302025-07-302025-07-30http://hdl.handle.net/10393/50707https://doi.org/10.20381/ruor-31284Epilepsy is a common neurological disorder with many underlying causes. The single gene contributions to the epilepsies have historically been considered rare and a challenge to diagnose. And yet, a molecular diagnosis in patients with epilepsy can be important for their treatment and management plans as well as for family planning. With the advent of next generation sequencing in the early-mid 2010s, there was an increase in the identification of disease-causing genes and variants in relation to epilepsy. Despite these advances, 50-80% of individuals with epilepsy do not have a genetic diagnosis after initial NGS testing. A straightforward and practical approach to improve the diagnostic rate is by reanalysis of the exome data after a period of time has elapsed. Studies in rare disease have shown that reanalysis can increase the number of diagnoses by 5-15%. The second chapter of this thesis demonstrates the benefit of using exome reanalysis for diagnostic purposes and for novel gene identification. The reanalysis of a cohort of 20 patients resulted in a 10% diagnostic rate (2/20) and candidate variants identified in an additional 35% (7/20) of cases. The third chapter of this project is a study of a candidate gene, UBE2R2, in a patient with genetic epilepsy and global developmental delay. To date, this gene has not been associated with disease or any form of epilepsy. Trio exome sequencing and research reanalysis identified a variant (c.21dup p.(Ser8GlnfsTer56)) in UBE2R2 that was rare (absent from gnomAD) and de novo and therefore UBE2R2 was considered a candidate gene for epilepsy and developmental delay. GeneMatcher was used to “match” with others interested in this gene, but no matches were, at this time, forthcoming. Functional studies were then performed that included assessment of cell cycle progression, cell growth, apoptosis, mitophagy, and interrogation of p53 and the β-catenin pathway. Results show that the expression of UBE2R2 was significantly decreased, mRNA expression of only one other E2 ubiquitin conjugating enzyme (UBE2R1) was increased, and expression of mRNA as well as protein expression of one of three Wnt target genes (AFF3) was significantly decreased in patient cells. The other functional work investigating proteins and pathways suspected to be impacted by the UBE2R2 variant (cell cycle progression, cell growth, apoptosis, mitophagy, p53 and β-catenin) did not demonstrate any statistically significant differences. Thus, while the functional assays performed were not able to provide significant evidence in favor of a disease-gene association, it also does not rule out UBE2R2 as the explanation for the phenotype of the patient. Taken together, these findings illustrate the benefits of whole exome sequencing and subsequent reanalysis for patients with genetic epilepsy that remain without a diagnosis and demonstrates the tools and methods available by which compelling candidates in putative novel disease genes can be further interrogated.enAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/EpilepsyGeneticsUBE2R2Exome ReanalysisFunctional AssayThesis