Functional Analysis of EARS2 Variants in a Combined OXPHOS Deficiency




Friedrich, Lauren

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Inborn errors of metabolism (IEMs) are a group of disorders characterized by abnormal metabolism. Historically, IEMs have been difficult to diagnose, as metabolism contains nonspecific systems. Whole Exome Sequencing (WES) may be used to identify causative mutations in some of these undiagnosed inborn errors, which may then be treatable. Metabolomic analysis enhances the interpretation and provides evidence for causality of some of these rare genetic variants. This project aims to analyze the functional difference in a patient's cell lines compared to normal fibroblast controls as well as to compare the patient's mutation to previously reported mutations. The case was referred to the Genetic and Metabolic Disease Program of a Mexican female who passed away at day 9 at San Antonio Children's hospital with a neurologic, hepatic, and metabolic phenotype. The lab received whole blood and fibroblasts. WES showed a homozygous Gly317Cys mutation in the EARS2 gene (Glutamyl-tRNA Synthetase 2). This nuclear gene encodes a mitochondrial tRNA synthetase which acts in mitochondrial translation. Variants were previously linked to Combined Oxidative Phosphorylation Deficiency 12, with one severe and one mild phenotype. The patient's cells were manipulated to produce cell lines with viral control, overexpression of wild type EARS2, overexpression of the patient's mutation, and expression of other mutations. The study investigates the respiration and metabolism of the patient cells and normal controls and between various mutations. A Western blot confirmed the presence of EARS2. Cell Titre Glo Assays showed decreased relative CTG activity in both the patient's mutation relative to wildtype overexpression (p = 0.0005) and in the more severe mutations from the literature compared to wildtype overexpression. Metabolomic analysis from QTOF showed metabolic differences including increased accumulated glycolytic intermediate metabolites when comparing the primary cell line with control fibroblasts. A continued pattern of increased intermediates in cells with overexpression mutation suggests a loss of function phenotype. When comparing mild and severe mutations with wildtype overexpression, upregulation in glycolysis was seen in the mutants, as well as downregulation in fatty acid biosynthesis and beta oxidation pathways. While the project is not conclusive in itself, it provides a platform from which to continue metabolomic analysis, perform glucose and glutamine tracing to better understand relevant metabolic pathways, and it suggests possible alterations in fatty acid metabolism pathways.

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The 58th Annual Medical Student Research Forum at UT Southwestern Medical Center (Tuesday, January 21, 2020, 3-6 p.m., D1.600)

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Friedrich, L., Ko, B., Pan, C., Solmonson, A., Vu, D., Yang, C., . . . DeBerardinis, R. (2020, January 21). Functional analysis of EARS2 variants in a combined OXPHOS deficiency. Poster session presented at the 58th Annual Medical Students Research Forum, Dallas, TX. Retrieved from

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