Fatal neonatal encephalopathy and lactic acidosis caused by a homozygous loss-of-function variant in COQ9

Abstract

Coenzyme Q10 (CoQ10) has an important role in mitochondrial energy metabolism by way of its functioning as an electron carrier in the respiratory chain. Genetic defects disrupting the endogenous biosynthesis pathway of CoQ10 may lead to severe metabolic disorders with onset in early childhood. Using exome sequencing in a child with fatal neonatal lactic acidosis and encephalopathy, we identified a homozygous loss-of-function variant in COQ9. Functional studies in patient fibroblasts showed that the absence of the COQ9 protein was concomitant with a strong reduction of COQ7, leading to a significant accumulation of the substrate of COQ7, 6-demethoxy ubiquinone10. At the same time, the total amount of CoQ10 was severely reduced, which was reflected in a significant decrease of mitochondrial respiratory chain succinate-cytochrome c oxidoreductase (complex II/III) activity. Lentiviral expression of COQ9 restored all these parameters, confirming the causal role of the variant. Our report on the second COQ9 patient expands the clinical spectrum associated with COQ9 variants, indicating the importance of COQ9 already during prenatal development. Moreover, the rescue of cellular CoQ10 levels and respiratory chain complex activities by CoQ10 supplementation points to the importance of an early diagnosis and immediate treatment.

Figure 1

(a) Brain ultrasound in a newborn with a pathogenic COQ9 variant. Left and middle panels: right and left parasagittal view, respectively, showing hyperechoic signal within the basal ganglia (arrow) and bilateral choroid plexus cysts (asterisk). Right panel: coronal view demonstrating symmetrical hyperechoic basal ganglia abnormalities suggesting a neonatal Leigh-like syndrome. (b) Schematic diagram showing the transcript generated in the presence of the c.521+1 deletion. Chromatogram shows that the deletion causes a skipping of exons 4 and 5 in the patient fibroblasts. (c) Western blotting on mitochondria-enriched fractions of patient and control fibroblasts (representative image). In patient cells no COQ9 is detected in the non-transduced condition (−). Signal is restored after lentiviral transduction with COQ9 wild-type cDNA (+). In addition, a COQ7 antibody was used, demonstrating a severe reduction of COQ7 protein in patient cells. Porin (VDAC) was used as a loading marker.

Figure 2

(a) Biochemical measurement of activities of complex II/III, complex IV of the respiratory chain and citrate synthase. Patient fibroblasts show a significant decrease of complex II/III activity. After re-expression of wild-type cDNA in patient fibroblasts, a significant increase in complex II/III activity relative to protein, to citrate synthase as well as to complex IV activity is observed. The activity of complex II/III remained unchanged in control cells. In addition, lentiviral expression of wild-type COQ9 cDNA in fibroblasts of a patient with a disease-causing homozygous variant in COQ2 showed no effect on the activity of complex II/III, demonstrating the specificity of our complementation strategy in the COQ9 patient. (b) Total amount of CoQ₁₀ was measured before and after lentiviral transduction as well as after treatment with CoQ₁₀. Both patient cell lines showed significantly reduced amounts of CoQ₁₀. After transduction patient fibroblasts with a variant in COQ9 showed a significant (about threefold) increase of CoQ₁₀, whereas in control cells and in patient cells suffering from COQ2 deficiency no significant difference could be observed. Treatment with CoQ₁₀ leads to an increase of the total amount of CoQ₁₀ in both patient cell lines. (c) MS/MS chromatograms of fibroblast lysate samples: (1) detected CoQ₁₀ in a control; (2) 6-DMQ₁₀ in a control is not present; (3) low detection of CoQ₁₀ in a COQ9 patient; (4) detection of 6-DMQ₁₀ metabolite in a COQ9 patient. (d) Quantification of the 6-DMQ₁₀ levels in COQ9-mutant fibroblasts before and after viral complementation with wild-type COQ9. The amount of 6-DMQ₁₀ is significantly reduced in COQ9-mutant fibroblasts after transduction. (e) Treatment with CoQ₁₀ in control and patient fibroblasts. Supplementation rescues complex II/III activity in fibroblasts derived from COQ9 and COQ2 patients whereas no differences were measured in control fibroblasts. Statistics: ***P<0.001, **P<0.01 and *P<0.05 relative to control or the indicated condition. All experimental data were obtained in at least three independent experiments.