Human COQ4 deficiency: delineating the clinical, metabolic and neuroimaging phenotypes.

BACKGROUND: Human coenzyme Q4 (COQ4) is essential for coenzyme Q10 (CoQ10) biosynthesis. Pathogenic variants in COQ4 cause childhood-onset neurodegeneration. We aimed to delineate the clinical spectrum and the cellular consequences of COQ4 deficiency. METHODS: Clinical course and neuroradiological findings in a large cohort of paediatric patients with COQ4 deficiency were analysed. Functional studies in patient-derived cell lines were performed. RESULTS: We characterised 44 individuals from 36 families with COQ4 deficiency (16 newly described). A total of 23 different variants were identified, including four novel variants in COQ4. Correlation analyses of clinical and neuroimaging findings revealed three disease patterns: type 1: early-onset phenotype with neonatal brain anomalies and epileptic encephalopathy; type 2: intermediate phenotype with distinct stroke-like lesions; and type 3: moderate phenotype with non-specific brain pathology and a stable disease course. The functional relevance of COQ4 variants was supported by in vitro studies using patient-derived fibroblast lines. Experiments revealed significantly decreased COQ4 protein levels, reduced levels of cellular CoQ10 and elevated levels of the metabolic intermediate 6-demethoxyubiquinone. CONCLUSION: Our study describes the heterogeneous clinical presentation of COQ4 deficiency and identifies phenotypic subtypes. Cell-based studies support the pathogenic characteristics of COQ4 variants. Due to the insufficient clinical response to oral CoQ10 supplementation, alternative treatment strategies are warranted.

Detection of 6-demethoxyubiquinone in CoQ10 deficiency disorders: Insights into enzyme interactions and identification of potential therapeutics.

Coenzyme Q10 (CoQ10) is an essential cofactor of the mitochondrial oxidative phosphorylation (OXPHOS) system and its deficiency has important implications for several inherited metabolic disorders of childhood. The biosynthesis of CoQ10 is a complicated process, which involves at least 12 different enzymes. One of the metabolic intermediates that are formed during CoQ10 biosynthesis is the molecule 6-demethoxyubiquinone (6-DMQ). This CoQ precursor is processed at the level of COQ7 and COQ9. We selected this metabolite as a marker substance for metabolic analysis of cell lines with inherited genetic defects (COQ2, COQ4, COQ7 and COQ9) or siRNA knockdown in CoQ biosynthesis enzymes using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). In COQ4, COQ7 and COQ9 deficient cell lines, we detected significantly elevated levels of 6-DMQ. This suggests a functional interplay of these proteins. However, additional siRNA studies demonstrated that elevated 6-DMQ levels are not an exclusive marker of the COQ7/COQ9 enzymatic step of CoQ10 biosynthesis but constitute a more general phenomenon that occurs in disorders impairing the function or stability of the CoQ-synthome. To further investigate the interdependence of CoQ10 biosynthesis enzyme expression, we performed immunoblotting in various cell lines with CoQ10 deficiency, indicating that COQ4, COQ7 and COQ9 protein expression levels are highly regulated depending on the underlying defect. Supplementation of cell lines with synthetic CoQ precursor compounds demonstrated beneficial effects of 2,4-dihydroxybenzoic acid in COQ7 and COQ9 deficiency. Moreover, vanillic acid selectively stimulated CoQ10 biosynthesis and improved cell viability in COQ9 deficiency. However, compounds tested in this study failed to rescue COQ4 deficiency.

NAXE Mutations Disrupt the Cellular NAD(P)HX Repair System and Cause a Lethal Neurometabolic Disorder of Early Childhood.

To safeguard the cell from the accumulation of potentially harmful metabolic intermediates, specific repair mechanisms have evolved. APOA1BP, now renamed NAXE, encodes an epimerase essential in the cellular metabolite repair for NADHX and NADPHX. The enzyme catalyzes the epimerization of NAD(P)HX, thereby avoiding the accumulation of toxic metabolites. The clinical importance of the NAD(P)HX repair system has been unknown. Exome sequencing revealed pathogenic biallelic mutations in NAXE in children from four families with (sub-) acute-onset ataxia, cerebellar edema, spinal myelopathy, and skin lesions. Lactate was elevated in cerebrospinal fluid of all affected individuals. Disease onset was during the second year of life and clinical signs as well as episodes of deterioration were triggered by febrile infections. Disease course was rapidly progressive, leading to coma, global brain atrophy, and finally to death in all affected individuals. NAXE levels were undetectable in fibroblasts from affected individuals of two families. In these fibroblasts we measured highly elevated concentrations of the toxic metabolite cyclic-NADHX, confirming a deficiency of the mitochondrial NAD(P)HX repair system. Finally, NAD or nicotinic acid (vitamin B3) supplementation might have therapeutic implications for this fatal disorder.

Pre-exercise medium-chain triglyceride application prevents acylcarnitine accumulation in skeletal muscle from very-long-chain acyl-CoA-dehydrogenase-deficient mice.

Dietary modification with medium-chain triglyceride (MCT) supplementation is one crucial way of treating children with long-chain fatty acid oxidation disorders. Recently, supplementation prior to exercise has been reported to prevent muscular pain and rhabdomyolysis. Systematic studies to determine when MCT supplementation is most beneficial have not yet been undertaken. We studied the effects of an MCT-based diet compared with MCT administration only prior to exercise in very-long-chain acyl-CoA dehydrogenase (VLCAD) knockout (KO) mice. VLCAD KO mice were fed an MCT-based diet in same amounts as normal mouse diet containing long-chain triglycerides (LCT) and were exercised on a treadmill. Mice fed a normal LCT diet received MCT only prior to exercise. Acylcarnitine concentration, free carnitine concentration, and acyl-coenzyme A (CoA) oxidation capacity in skeletal muscle as well as hepatic lipid accumulation were determined. Long-chain acylcarnitines significantly increased in VLCAD-deficient skeletal muscle with an MCT diet compared with an LCT diet with MCT bolus prior to exercise, whereas an MCT bolus treatment significantly decreased long-chain acylcarnitines after exercise compared with an LCT diet. C8-carnitine was significantly increased in skeletal muscle after MCT bolus treatment and exercise compared with LCT and long-term MCT treatment. Increased hepatic lipid accumulation was observed in long-term MCT-treated KO mice. MCT seems most beneficial when given in a single dose directly prior to exercise to prevent acylcarnitine accumulation. In contrast, continuous MCT treatment produces a higher skeletal muscle content of long-chain acylcarnitines after exercise and increases hepatic lipid storage in VLCAD KO mice.