Large Scale Profiling of Fibroblasts from Pediatric Patients with Inborn Errors of Metabolism Results in the Identification of Siblings with L-2-Hydroxyglutaric Aciduria

Inborn errors of metabolism (IEMs) are caused by germline mutations that interfere with the normal physiological functioning of single metabolic enzymes or nutrient transporters1. Discerning an effective treatment for IEMs requires identifying, validating and understanding the impact of disease-causing mutations that are specific to the patient1. Whole-exome sequencing (WES) is the most common strategy for identifying unknown genetic aberrations1. Metabolomics and metabolic flux analysis (MFA) can help functionally validate whether suspected mutations are causative as well as direct the development of diagnostic and therapeutic approaches1. More specifically, mass spectrometry-based metabolomics enables quantification of metabolite abundance, and isotopically labeled MFA enables assessment of the precise manner in which patient cells utilize nutrients for various catabolic pathways1,2. In this study, we conducted metabolomics and MFA to assess metabolic abnormalities in fibroblasts derived from 27 patients, who possessed both known and unknown IEMs, and to gain mechanistic insight into these diseases. To characterize the metabolic abnormalities, we performed a metabolomics experiment that involved 27 patient-derived fibroblast lines and 4 control lines, which eventually yielded a detailed metabolic profile (681 metabolites) for each sample. Additionally, 13C-labeled glucose and 13C-labeled glutamine were introduced to various cell lines in order to evaluate the flux of glucose and glutamine metabolism. We confirmed the quality of our metabolomics results by conducting unsupervised clustering analysis, which showed close grouping of biological replicates, as well as by validating the metabolic changes in a previously reported patient1. Differential analysis of metabolites for each of the 27 patients provided further insight into their specific metabolic anomalies. Notably, we discovered a pair of siblings with dramatically increased 2-hydroxyglutarate (2-HG) levels. By using a derivatization-based mass spectrometry method, we determined that L-2-HG rather than D-2-HG was elevated in the patient samples. WES of both children identified a homozygous mutation that introduced a stop codon in the L-2-HG dehydrogenase gene. This was consistent with the diagnosis of L-2-hydroxyglutaric aciduria in the children3. Metabolic flux analysis provided mechanistic insight into the disease, showing that L-2-HG was primarily being made from glutamine rather than glucose. The unbiased metabolomics approach enabled the discovery and characterization of an important metabolic deficiency and will likely contribute to the discovery of additional IEMs in the cohort.