Mitochondrial energetic impairment in a patient with late-onset glutaric acidemia Type 2

Abstract

Glutaric acidemia type 2 (GA2), also called multiple acyl-CoA dehydrogenase deficiency, is an autosomal recessive disorder of fatty acid, amino acid, and choline metabolism resulting in excretion of multiple organic acids and glycine conjugates as well as elevation of various plasma acylcarnitine species (C4-C18). It is caused by mutations in the ETFA, ETFB, or ETFDH genes which are involved in the transfer of electrons from 11 flavin-containing dehydrogenases to Coenzyme Q₁₀ (CoQ₁₀ ) of the mitochondrial electron transport chain (ETC). We report a patient who was originally reported as the first case with primary myopathic CoQ₁₀ deficiency when he presented at 11.5 years with exercise intolerance and myopathy that improved after treatment with ubiquinone and carnitine. At age 23, his symptoms relapsed despite increasing doses of ubiquinone and he was shown to have biallelic mutations in the ETFDH gene. Treatment with riboflavin was started and ubiquinone was changed to ubiquinol. After 4 months, the patient recovered his muscle strength with normalization of laboratory exams and exercise tolerance. Functional studies on fibroblasts revealed decreased levels of ETFDH as well as of very long-chain acyl-CoA dehydrogenase and trifunctional protein α. In addition, the mitochondrial mass was decreased, with increased formation of reactive oxygen species and oxygen consumption rate, but with a decreased spared respiratory capacity, and decreased adenosine triphosphate level. These findings of widespread dysfunction of fatty acid oxidation and ETC enzymes support the impairment of a larger mitochondrial ETC supercomplex in our patient.

Keywords: ETFDH; electron transport chain; glutaric acidemia type 2; mitochondria.

FIGURE 1

Concentration of ETFDH, very long-chain acyl-CoA dehydrogenase (VLCAD), trifunctional protein (TFPα), and TFPβ protein relative to GAPDH control in cells from our patient versus healthy control. (a) ETFDH protein (upper panel) concentration was decreased by 73% as compared to control. Cell free extract (CFE) 10 μg loaded in lanes marked as Control and ETFDH-deficient (ETFDHD). Lower panel shows GAPDH as loading control. (b) Compared to control, protein levels of TFPα and β both diminished (48 and 10%, respectively) in ETFDHD patient CFE, 40 μg loaded. Lower panel shows GAPDH as loading control. (c) Compared to control, VLCAD diminished 31% in ETFDHD patient CFE, 40 μg loaded. Lower panel shows GAPDH as loading control

FIGURE 2

Measurements of various markers of mitochondrial function in cells from our patient versus healthy control. (a) Superoxide levels detected by MitoSOX Red assay in patient and control cells. Data shown here are means ± SD and are normalized to mean value of control cells. ****p < .0001. (b) Mitochondrial mass detected by MitoTracker Green assay in ETFD-deficient and healthy control cells. Data shown here are means ± SD and are normalized to mean value of control cells. **p < .01. (c) Mitochondrial basal respiration of healthy control compared to ETFDHD fibroblast cell line. Data shown here are means ± SEM and are normalized to protein amount mean value from a single biological replicate and seven technical replicates. ***p value <.001. (d) Mitochondrial spare respiratory capacity of healthy control compared to ETFDHD fibroblast cell line. Data shown here are means ± SD and are normalized to protein amount mean value from a single biological replicate and seven technical replicates. (e) Mitochondrial adenosine triphosphate (ATP) of ETFDHD (FB862) compared to healthy (FB826) fibroblast cells. Data shown here are means ± SD and are normalized to protein amount mean value from a single biological replicate and four technical replicates. ****p value <.0001