Germline inactivating mutations of the SLC25A1 gene contribute to various human disorders, including Velocardiofacial (VCFS), DiGeorge (DGS) syndromes and combined D/L-2-hydroxyglutaric aciduria (D/L-2HGA), a severe systemic disease characterized by the accumulation of 2-hydroxyglutaric acid (2HG). The mechanisms by which SLC25A1 loss leads to these syndromes remain largely unclear. Here, we describe a mouse model of SLC25A1 deficiency that mimics human VCFS/DGS and D/L-2HGA. Surprisingly, inactivation of both Slc25a1 alleles results in alterations in the development of multiple organs, and in a severe proliferation defect by activating two senescence programs, oncogene-induced senescence (OIS) and mitochondrial dysfunction-induced senescence (MiDAS), which converge upon the induction of the p53 tumor suppressor. Mechanistically, cells and tissues with dysfunctional SLC25A1 protein undergo metabolic and transcriptional rewiring leading to the accumulation of 2HG via a non-canonical pathway and to the depletion of nicotinamide adenine dinucleotide, NAD+, which trigger senescence. Replenishing the pool of NAD+ or promoting the clearance of 2HG rescues the proliferation defect of cells with dysfunctional SLC25A1 in a cooperative fashion. Further, removal of p53 activity via RNA interference restores proliferation, indicating that p53 acts as a critical barrier to the expansion of cells lacking functional SLC25A1. These findings reveal unexpected pathogenic roles of senescence and of p53 in D/L-2HGA and identify potential therapeutic strategies to correct salient molecular alterations driving this disease.

To identify therapies for combined D, L-2-hydroxyglutaric aciduria (C-2HGA), a rare genetic disorder caused by recessive variants in the SLC25A1 gene. Patients C-2HGA were identified and diagnosed by whole exome sequencing and biochemical genetic testing. Patient derived fibroblasts were then treated with phenylbutyrate and the functional effects assessed by metabolomics and RNA-sequencing. In this study, we demonstrated that C-2HGA patient derived fibroblasts exhibited impaired cellular bioenergetics. Moreover, Fibroblasts form one patient exhibited worsened cellular bioenergetics when supplemented with citrate. We hypothesized that treating patient cells with phenylbutyrate (PB), an FDA approved pharmaceutical drug that conjugates glutamine for renal excretion, would reduce mitochondrial 2-ketoglutarate, thereby leading to improved cellular bioenergetics. Metabolomic and RNA-seq analyses of PB-treated fibroblasts demonstrated a significant decrease in intracellular 2-ketoglutarate, 2-hydroxyglutarate, and in levels of mRNA coding for citrate synthase and isocitrate dehydrogenase. Consistent with the known action of PB, an increased level of phenylacetylglutamine in patient cells was consistent with the drug acting as 2-ketoglutarate sink. Our pre-clinical studies suggest that citrate supplementation has the possibility exacerbating energy metabolism in this condition. However, improvement in cellular bioenergetics suggests phenylbutyrate might have interventional utility for this rare disease.

Germline inactivating mutations of the SLC25A1 gene contribute to various human developmental disorders, including combined D/L-2-hydroxyglutaric aciduria (D/L-2HGA), a severe systemic syndrome characterized by the accumulation of both enantiomers of 2-hydroxyglutaric acid (2HG). The mechanisms by which SLC25A1 deficiency leads to this disease and the role of 2HG are unclear and no therapies exist. We now show that mice lacking both Slc25a1 alleles display a spectrum of alterations that resemble human D/L-2HGA. Mechanistically, SLC25A1 loss results in a proliferation defect and activates two distinct senescence pathways, oncogene-induced senescence (OIS) and mitochondrial dysfunction-induced senescence (MiDAS), both involving the p53 tumor suppressor and driven by two discernible signals: the accumulation of 2HG, inducing OIS, and mitochondrial dysfunction, triggering MiDAS. Inhibiting these senescence programs or blocking p53 activity reverses the growth defect caused by SLC25A1 dysfunction and restores proliferation. These findings reveal novel pathogenic roles of senescence in human disorders and suggest potential strategies to correct the molecular alterations caused by SLC25A1 loss.

Combined D, L-2-Hydroxyglutaric Aciduria (D,L-2HGA) is a rare genetic disorder caused by recessive mutations in the SLC25A1 gene that encodes the mitochondrial citrate carrier protein (CIC). SLC25A1 deficiency leads to a secondary increase in mitochondrial 2-ketoglutarate that, in turn, is reduced to neurotoxic 2-hydroxyglutarate. Clinical symptoms of Combined D,L-2HGA include neonatal encephalopathy, respiratory insufficiency and often with death in infancy. No current therapies exist, although replenishing cytosolic stores by citrate supplementation to replenish cytosolic stores has been proposed. In this study, we demonstrated that patient derived fibroblasts exhibited impaired cellular bioenergetics that were worsened with citrate supplementation. We hypothesized treating patient cells with phenylbutyrate, an FDA approved pharmaceutical drug, would reduce mitochondrial 2-ketoglutarate, leading to improved cellular bioenergetics including oxygen consumption and fatty acid oxidation. Metabolomic and RNA-seq analyses demonstrated a significant decrease in intracellular 2-ketoglutarate, 2-hydroxyglutarate, and in levels of mRNA coding for citrate synthase and isocitrate dehydrogenase. Consistent with the known action of phenylbutyrate, detected levels of phenylacetylglutamine was consistent with the drug acting as 2-ketoglutarate sink in patient cells. Our pre-clinical studies suggest citrate supplementation is unlikely to be an effective treatment of the disorder. However, cellular bioenergetics suggests phenylbutyrate may have interventional utility for this rare disease.

Patients with an inborn error of metabolism (IEM) are deficient of an enzyme involved in metabolism, and as a consequence metabolism reprograms itself to reach a new steady state. This new steady state underlies the clinical phenotype associated with the deficiency. Hence, we need to know the flux of metabolites through the different metabolic pathways in this new steady state of the reprogrammed metabolism. Stable isotope technology is best suited to study this. In this review the progress made in characterizing the altered metabolism will be presented. Studies done in patients to estimate the residual flux through the metabolic pathway affected by enzyme deficiencies will be discussed. After this, studies done in model systems will be reviewed. The focus will be on glycogen storage disease type I, medium-chain acyl-CoA dehydrogenase deficiency, propionic and methylmalonic aciduria, urea cycle defects, phenylketonuria, and combined D,L-2-hydroxyglutaric aciduria. Finally, new developments are discussed, which allow the tracing of metabolic reprogramming in IEM on a genome-wide scale. In conclusion, the outlook for flux analysis of metabolic derangement in IEMs looks promising.