Severe Neonatal Presentation of Mitochondrial Citrate Carrier (SLC25A1) Deficiency

Abstract

Mutations of the mitochondrial citrate carrier (CIC) SLC25A1 cause combined D-2- and L-2-hydroxyglutaric aciduria (DL-2HGA; OMIM #615182), a neurometabolic disorder characterized by developmental delay, hypotonia, and seizures. Here, we describe the female child of consanguineous parents who presented neonatally with lactic acidosis, periventricular frontal lobe cysts, facial dysmorphism, recurrent apneic episodes, and deficient complex IV (cytochrome c oxidase) activity in skeletal muscle. Exome sequencing revealed a homozygous SLC25A1 missense mutation [NM_005984.4: c.593G>A; p.(Arg198His)] of a ubiquitously conserved arginine residue putatively situated within the substrate-binding site I of CIC. Retrospective review of the patient's organic acids confirmed the D- and L-2-hydroxyglutaric aciduria typical of DL-2HGA to be present, although this was not appreciated on initial presentation. Cultured patient skin fibroblasts showed reduced survival in culture, diminished mitochondrial spare respiratory capacity, increased glycolytic flux, and normal mitochondrial bulk, inner membrane potential, and network morphology. Neither cell survival nor cellular respiratory parameters were improved by citrate supplementation, although oral citrate supplementation did coincide with amelioration of lactic acidosis and apneic attacks in the patient. This is the fifth clinical report of CIC deficiency to date. The clinical features in our patient suggest that this disorder, which can potentially be recognized either by molecular means or based on its characteristic organic aciduria, should be considered in the differential diagnosis of pyruvate dehydrogenase deficiency and respiratory chain disorders. One-Sentence Summary A novel homozygous missense substitution in SLC25A1 was identified in a neonate presenting with lactic acidosis, intracerebral cysts, and an apparent mitochondrial complex IV defect in muscle.

Keywords: Citrate transporter; Cytochrome c oxidase; Exome sequencing; Lactic acidosis; Mitochondrial disease; SLC25A1.

Fig. 1

Cranial MRI appearance at 5 days (a, b) and 3 months of age (c–f). (a) and (b) Diffuse deficiency of cerebral white matter with enlarged fluid spaces and marked hypoplasia of the corpus callosum. Arrow indicates connatal (frontal lobe) cyst; a similar (smaller) right-sided cyst was present but is not well shown on this axial slice. (c) Lactate doublet is present on MR spectroscopy. (d) Evolution of changes in panels a and b. Myelination is markedly reduced for age. Basal ganglia, brainstem, and cerebellum have a relatively normal appearance. (f) Small optic nerves and optic chiasm (not shown)

Fig. 2

SLC25A1-deficient patient cells exhibit lessened spare respiratory capacity and increased glycolytic flux. Micro-oximetry was performed, as per Antoun et al. (2015), on cells grown in standard medium and on cells grown in medium supplemented with 4 mM citrate. Oxygen consumption rate (OCR) was corrected for non-mitochondrial oxygen consumption and normalized to protein content. (a) Basal OCR of patient fibroblasts does not differ significantly from that of control fibroblasts. (b) Spare respiratory capacity is reduced in the patient fibroblasts; this was not ameliorated by citrate supplementation. (c) Glucose-stimulated extracellular acidification rate (ECAR), reflecting glycolytic flux, is markedly increased in the patient cells (n = 3). (d) OCR/ECAR ratio (basal OCR divided by glucose-driven ECAR) is markedly reduced in the patient, i.e., the patient cells are relatively reliant on glycolysis (n = 3). Values are presented as mean ± SEM, analyzed by unpaired, two-tailed student’s t-test

Fig. 3

Normal patient mitochondrial networking, drug-induced fragmentation, total mitochondrial bulk, and inner membrane charge in skin-derived fibroblasts. (a, b) High-throughput confocal imaging of fixed control (left) and patient (right) fibroblasts ± acute 60 μM FCCP treatment; immunostained for the outer membrane transporter TOMM-20 (n = 4). (c) Mitochondrial content (MitoTracker Green) (n = 3) and (d) inner membrane potential (TMRE) (n = 3) measured by flow cytometry. Values are presented as mean ± SEM; analyzed by (b) two-way ANOVA or (c) nonparametric Mann-Whitney test for unequal variance and (d) unpaired, two-tailed student’s t-test