ABSTRACT
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.
Subject(s)
Ataxia/metabolism , Mitochondrial Diseases/metabolism , Muscle Weakness/metabolism , Ubiquinone/analogs & derivatives , Ubiquinone/deficiency , Cell Line , Cell Survival/drug effects , Fibroblasts/drug effects , Humans , Hydroxybenzoates/pharmacology , Mitochondria/metabolism , Mitochondrial Proteins/deficiency , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Oxidative Phosphorylation , Tandem Mass Spectrometry , Ubiquinone/biosynthesis , Ubiquinone/metabolism , Vanillic Acid/pharmacologyABSTRACT
Inherited methylation disorders are a group of rarely reported, probably largely underdiagnosed disorders affecting transmethylation processes in the metabolic pathway between methionine and homocysteine. These are methionine adenosyltransferase I/III, glycine N-methyltransferase, S-adenosylhomocysteine hydrolase and adenosine kinase deficiencies. This paper provides the first consensus recommendations for the diagnosis and management of methylation disorders. Following search of the literature and evaluation according to the SIGN-methodology of all reported patients with methylation defects, graded recommendations are provided in a structured way comprising diagnosis (clinical presentation, biochemical abnormalities, differential diagnosis, newborn screening, prenatal diagnosis), therapy and follow-up. Methylation disorders predominantly affect the liver, central nervous system and muscles, but clinical presentation can vary considerably between and within disorders. Although isolated hypermethioninemia is the biochemical hallmark of this group of disorders, it is not always present, especially in early infancy. Plasma S-adenosylmethionine and S-adenosylhomocysteine are key metabolites for the biochemical clarification of isolated hypermethioninemia. Mild hyperhomocysteinemia can be present in all methylation disorders. Methylation disorders do not qualify as primary targets of newborn screening. A low-methionine diet can be beneficial in patients with methionine adenosyltransferase I/III deficiency if plasma methionine concentrations exceed 800 µmol/L. There is some evidence that this diet may also be beneficial in patients with S-adenosylhomocysteine hydrolase and adenosine kinase deficiencies. S-adenosylmethionine supplementation may be useful in patients with methionine adenosyltransferase I/III deficiency. Recommendations given in this article are based on general principles and in practice should be adjusted individually according to patient's age, severity of the disease, clinical and laboratory findings.