Impaired complex I assembly in a Leigh syndrome patient with a novel missense mutation in the ND6 gene.
ABSTRACT We describe a novel mutation in the ND6 gene (T14487C) in a patient with Leigh syndrome. Biochemical analyses indicated a low complex I activity in the patient's fibroblasts but normal values in muscle and liver. Cybrid clones showed a specific complex I defect that correlates with the mutant heteroplasmy levels. Additionally, we demonstrate an altered mobility and a decrease in the levels of fully assembled complex I in the patient's fibroblasts and cybrids, suggesting that the mutation has a profound effect on complex I assembly and/or stability.
- SourceAvailable from: Masakazu Kohda[Show abstract] [Hide abstract]
ABSTRACT: Objective Mitochondrial respiratory chain disorder (MRCD) is an intractable disease of infants with variable clinical symptoms. Our goal was to identify the causative mutations in MRCD patients.Methods The subjects were 90 children diagnosed with MRCD by enzyme assay. We analyzed whole mitochondrial DNA (mtDNA) sequences. A cybrid study was performed in two patients. Whole exome sequencing was performed for one of these two patients whose mtDNA variant was confirmed as non-pathogenic.ResultsWhole mtDNA sequences identified 29 mtDNA variants in 29 patients (13 were previously reported, the other 13 variants and three deletions were novel). The remaining 61 patients had no pathogenic mutations in their mtDNA. Of the 13 patients harboring unreported mtDNA variants, we excluded seven variants by manual curation. Of the remaining six variants, we selected two Leigh syndrome patients whose mitochondrial enzyme activity was decreased in their fibroblasts and performed a cybrid study. We confirmed that m.14439G>A (MT-ND6) was pathogenic, while m.1356A>G (mitochondrial 12S rRNA) was shown to be a non-pathogenic polymorphism. Exome sequencing and a complementation study of the latter patient identified a novel c.55C>T hemizygous missense mutation in the nuclear-encoded gene NDUFA1.InterpretationOur results demonstrate that it is important to perform whole mtDNA sequencing rather than only typing reported mutations. Cybrid assays are also useful to diagnose the pathogenicity of mtDNA variants, and whole exome sequencing is a powerful tool to diagnose nuclear gene mutations as molecular diagnosis can provide a lead to appropriate genetic counseling.Annals of Clinical and Translational Neurology. 04/2014;
- [Show abstract] [Hide abstract]
ABSTRACT: Alcoholic cardiomyopathy (ACM) presents as decreased myocardial contractility, arrhythmias and secondary non-ischemic dilated cardiomyopathy leading to heart failure. Mitochondrial dysfunction is known to have a significant role in the development and complications of ACM. This study investigated if chronic ethanol feeding promoted myocardial mitochondrial topoisomerase dysfunction as one underlying cause of mitochondrial DNA (mtDNA) damage and mitochondrial dysfunction in ACM.Alcohol and alcoholism (Oxford, Oxfordshire). Supplement 05/2014;
- [Show abstract] [Hide abstract]
ABSTRACT: A simple electrochemical biosensor was developed for the detection of mitochondrial NADH dehydrogenase 6 gene (MT-ND6) and its enzymatic digestion by BamHI enzyme. This biosensor was fabricated by modification of glassy carbon electrode with gold nanoparticles (AuNPs/GCE) and a probe oligonucleotide (ssDNA/AuNPs/GCE). The probe, which is a thiolated segment of the MT-ND6 gene, was deposited by self-assembling immobilization on AuNPs/GCE. Two indicators including methylene blue (MB) and neutral red (NR) was used as the electro-active indicators and electrochemical response of the modified electrode was measured by differential pulse voltammetry. The proposed biosensor can detect the complementary sequences of MT-ND6 gene. Also the modified electrode was used for the detection of enzymatic digestion process by BamHI enzyme. The electrochemical biosensor can detect the MT-ND6 gene and its enzymatic digestion in polymerase chain reaction (PCR) amplified DNA extracted from human blood. Also the biosensor was used directly for detection of MT-ND6 gene in all of human genome.Analytical Biochemistry 06/2014; · 2.31 Impact Factor