Functional cellular analyses reveal energy metabolism defect and mitochondrial DNA depletion in a case of mitochondrial aconitase deficiency

Abstract

Defects in the tricaboxylic acid cycle (TCA) are associated with a spectrum of neurological phenotypes that are often difficult to diagnose and manage. Whole-exome sequencing (WES) led to a rapid expansion of diagnostic capabilities in such disorders and facilitated a better understanding of disease pathogenesis, although functional characterization remains a bottleneck to the interpretation of potential pathological variants. We report a 2-year-old boy of AfroCaribbean ancestry, who presented with neuromuscular symptoms without significant abnormalities on routine diagnostic evaluation. WES revealed compound heterozygous missense variants of uncertain significance in mitochondrial aconitase (ACO2), which encodes the TCA enzyme ACO2. Pathogenic variants in ACO2 have been described in a handful of families as the cause of infantile cerebellar-retinal degeneration syndrome. Using biochemical and cellular assays in patient fibroblasts, we found that ACO2 expression was quantitatively normal, but ACO2 enzyme activity was less than 20% of that observed in control cells. We also observed a deficiency in cellular respiration and, for the first time, demonstrate evidence of mitochondrial DNA depletion and altered expression of some TCA components and electron transport chain subunits. The observed cellular defects were completely restored with ACO2 gene rescue. Our findings demonstrate the pathogenicity of two VUS in ACO2, provide novel mechanistic insights to TCA disturbances in ACO2 deficiency, and implicate mitochondrial DNA depletion in the pathogenesis of this recently described disorder.

Proteome Imbalance of Mitochondrial Electron Transport Chain in Brown Adipocytes Leads to Metabolic Benefits

“…The XFe24 extracellular flux analyzer from Seahorse Biosciences was used to measure the rate of mitochondrial oxygen consumption, as previously described (Sadat et al, 2016). Mitochondria were isolated from 6-8-week-old male and female mice and immediately plated on the XFe24 cell culture microplate at a density of 2μg per well.…”

Clinical, radiological, and genetic characteristics of 16 patients with ACO2 gene defects: Delineation of an emerging neurometabolic syndrome

“…Since then additional reports described less than a dozen additional ACO2 deficient patients . Interestingly, these reports further expanded the clinical spectrum of ACO2 gene defects to include milder phenotypes such as isolated late onset optic atrophy recently termed optic atrophy 9 (OPA9, OMIM #616289).…”

Expanding the phenotypic spectrum of Succinyl-CoA ligase deficiency through functional validation of a new SUCLG1 variant

“…SUCLG1 cDNA was cloned into pLenti6.3 destination vector and fibroblasts were transduced with the SUCLG1 -lentivirus as previously described [11]. The control fibroblasts lines were established from five different healthy individuals as previously described [11]. Transduced cells were put under Blasticidin selection (5 µg/mL) for a period of 10 days before using them for analysis.…”

“…mtDNA content of the fibroblasts was analyzed using real-time qPCR method as described before [11]. The beta 2 microglobulin gene ( B2M ) was used as the nuclear gene (nDNA) normalizer for the calculation of mtDNA/nDNA ratio.…”

“…XF24 extracellular flux analyzer from Seahorse Biosciences was used to measure the rates of fibroblasts oxygen consumption, as previously described [11]. Cells were plated the previous day of experiment on the XF24 cell culture microplates at a density of 60,000 cells per well.…”