FIGURE 2.

Metabolic abnormalities in patient-derived fibroblasts with the EARS2 G317C variant. A, Immunoblot analysis of primary human fibroblasts from five healthy subjects and patient P3-041. B, Principal component analysis (PCA) of the metabolomes of healthy control and P3-041 fibroblasts. The metabolomic profiling was performed on primary human fibroblasts from six healthy subjects and patient P3-041. C, Metabolite set enrichment analysis (MSEA) of metabolites differentially changed in the P3-041 fibroblasts compared to the control primary fibroblasts. The significantly altered metabolites were filtered by p < 0.05 and fold changes ≥1.5 or ≤0.67. D, Comparison of several metabolites related to the TCA cycle between control and P3-041 primary fibroblasts. **p < 0.01, *p < 0.05, calculated by unpaired 2-tailed t test. E, Metabolic abnormalities related to purine and pyrimidine pathways in P3-041 fibroblasts. Metabolites are ordered by mean log2-transformed P3-041 versus control ratio. F, Acylcarnitine abnormalities in P3-041 fibroblasts compared to controls. Metabolites are ordered by mean log2-transformed P3-041 versus control ratio. G, Pathway enrichment of metabolites that are normalized by ectopic expression of WT EARS2 in P3-041 fibroblasts. The significantly downregulated metabolites by WT EARS2 expression (p < 0.05 and fold changes ≤0.67) were used in MetaboAnalyst pathway analysis. The circle size corresponds to impact values (x-axis) and the color reflects −log(p) values (y-axis). H,Representative metabolites of the significantly affected pathways in (C) but normalized by expression of WT EARS2. The control (ctrl-3) in this experiment is a human neonatal fibroblast line from ATCC