Clinical, biochemical and genetic spectrum of 70 patients with ACAD9 deficiency: is riboflavin supplementation effective?

BACKGROUND: Mitochondrial acyl-CoA dehydrogenase family member 9 (ACAD9) is essential for the assembly of mitochondrial respiratory chain complex I. Disease causing biallelic variants in ACAD9 have been reported in individuals presenting with lactic acidosis and cardiomyopathy. RESULTS: We describe the genetic, clinical and biochemical findings in a cohort of 70 patients, of whom 29 previously unpublished. We found 34 known and 18 previously unreported variants in ACAD9. No patients harbored biallelic loss of function mutations, indicating that this combination is unlikely to be compatible with life. Causal pathogenic variants were distributed throughout the entire gene, and there was no obvious genotype-phenotype correlation. Most of the patients presented in the first year of life. For this subgroup the survival was poor (50% not surviving the first 2 years) comparing to patients with a later presentation (more than 90% surviving 10 years). The most common clinical findings were cardiomyopathy (85%), muscular weakness (75%) and exercise intolerance (72%). Interestingly, severe intellectual deficits were only reported in one patient and severe developmental delays in four patients. More than 70% of the patients were able to perform the same activities of daily living when compared to peers. CONCLUSIONS: Our data show that riboflavin treatment improves complex I activity in the majority of patient-derived fibroblasts tested. This effect was also reported for most of the treated patients and is mirrored in the survival data. In the patient group with disease-onset below 1 year of age, we observed a statistically-significant better survival for patients treated with riboflavin.

Effect of resveratrol on cultured skin fibroblasts from patients with oxidative phosphorylation defects.

Few therapeutic options are available to patients with oxidative phosphorylation disorders. Administering pharmacological agents that are able to stimulate mitochondrial biogenesis have been put forward as a possible treatment, yet the approach remains in need of thorough testing. We investigated the effect of resveratrol in an in vitro setting. Mitochondrial enzymatic activities were tested in cultured skin fibroblasts from patients harboring a nuclear defect in either complex II or complex IV (n = 11), and in fibroblasts from healthy controls (n = 11). In the latter, preincubation with resveratrol resulted in a significant increase of citrate synthase, complex II and complex IV enzyme activity. In patients with complex II or complex IV deficiency, however, activity of the deficient complex could not be substantially augmented, and response was dependent upon the residual activity. We conclude that resveratrol is not capable of normalizing oxidative phosphorylation activities in deficient cell lines.

Characterization of CoQ10 biosynthesis in fibroblasts of patients with primary and secondary CoQ10 deficiency.

Primary coenzyme Q10 (CoQ10) deficiencies are associated with mutations in genes encoding enzymes important for its biosynthesis and patients are responsive to CoQ10 supplementation. Early treatment allows better prognosis of the disease and therefore, early diagnosis is desirable. The complex phenotype and genotype and the frequent secondary CoQ10 deficiencies make it difficult to achieve a definitive diagnosis by direct quantification of CoQ10. We developed a non-radioactive methodology for the quantification of CoQ10 biosynthesis in fibroblasts that allows the identification of primary deficiencies. Fibroblasts were incubated 72 h with 28 µmol/L (2)H3-mevalonate or 1.65 mmol/L (13)C6-p-hydroxybenzoate. The newly synthesized (2)H3- and (13)C6- labelled CoQ10 were analysed by high performance liquid chromatography-tandem mass spectrometry. The mean and the reference range for (13)C6-CoQ10 and (2)H3-CoQ10 biosynthesis were 0.97 (0.83-1.1) and 0.13 (0.09-0.17) nmol/Unit of citrate synthase, respectively. We validated the methodology through the study of one patient with COQ2 mutations and six patients with CoQ10 deficiency secondary to other inborn errors of metabolism. Afterwards we investigated 16 patients' fibroblasts and nine showed decreased CoQ10 biosynthesis. Therefore, the next step is to study the COQ genes in order to reach a definitive diagnosis in these nine patients. In the patients with normal rates the deficiency is probably secondary. In conclusion, we have developed a non-invasive non-radioactive method suitable for the detection of defects in CoQ10 biosynthesis, which offers a good tool for the stratification of patients with these treatable mitochondrial diseases.

Early myoclonic epilepsy, hypertrophic cardiomyopathy and subsequently a nephrotic syndrome in a patient with CoQ10 deficiency caused by mutations in para-hydroxybenzoate-polyprenyl transferase (COQ2).

BACKGROUND: Primary coenzyme Q10 (CoQ10) deficiencies are heterogeneous autosomal recessive disorders. CoQ2 mutations have been identified only rarely in patients. All affected individuals presented with nephrotic syndrome in the first year of life. METHODS: An infant is studied with myoclonic seizures and hypertrophic cardiomyopathy in the first months of life and developed a nephrotic syndrome in a later stage. RESULTS: At three weeks of age, the index patient developed myoclonic seizures. In addition, he had hypertrophic cardiomyopathy and increased CSF lactate. A skeletal muscle biopsy performed at two months of age disclosed normal activities of the oxidative phosphorylation complexes. The child was supplemented with CoQ10 (5 mg/kg/day). At the age of four months, brain MR images showed bilateral increased signal intensities in putamen and cerebral cortex. After that age, he developed massive proteinuria. The daily dose of CoQ10 was increased to 30 mg/kg. Renal biopsy showed focal segmental glomerulosclerosis. Biochemical analyses of a kidney biopsy sample revealed a severely decreased activity of succinate cytochrome c reductase [complex II + III] suggesting ubiquinone depletion. Incorporation of labelled precursors necessary for CoQ10 synthesis was significantly decreased in cultured skin fibroblasts. His condition deteriorated and he died at the age of five months. A novel homozygous mutation c.326G > A (p.Ser109Asn) was found in COQ2. CONCLUSIONS: In contrast to previously reported patients with CoQ2 the proband presented with early myoclonic epilepsy, hypertrophic cardiomyopathy and only in a later stage developed a nephrotic syndrome. The phenotype of this patient enlarges the phenotypical spectrum of the multisystem infantile variant.

Cellular pathophysiological consequences of BCS1L mutations in mitochondrial complex III enzyme deficiency.

Mutations in BCS1L, an assembly factor that facilitates the insertion of the catalytic Rieske Iron-Sulfur subunit into respiratory chain complex III, result in a wide variety of clinical phenotypes that range from the relatively mild Björnstad syndrome to the severe GRACILE syndrome. To better understand the pathophysiological consequences of such mutations, we studied fibroblasts from six complex III-deficient patients harboring mutations in the BCS1L gene. Cells from patients with the most severe clinical phenotypes exhibited slow growth rates in glucose medium, variable combined enzyme deficiencies, and assembly defects of respiratory chain complexes I, III, and IV, increased H(2)O(2) levels, unbalanced expression of the cellular antioxidant defenses, and apoptotic cell death. In addition, all patients showed cytosolic accumulation of the BCS1L protein, suggestive of an impaired mitochondrial import, assembly or stability defects of the BCS1L complex, fragmentation of the mitochondrial networks, and decreased MFN2 protein levels. The observed structural alterations were independent of the respiratory chain function and ROS production. Our results provide new insights into the role of pathogenic BCS1L mutations in mitochondrial function and dynamics.