RESUMO
Frontotemporal lobar degeneration (FTLD) is a genetically heterogenous syndrome and has been associated most recently with a hexanucleotide repeat expansion within the C9ORF72 gene. Pathogenic TDP-43 gene (TARDBP) mutations have been identified in amyotrophic lateral sclerosis, but the role of TARDBP mutations in FTLD is more contradictory. To investigate the role of TARDBP mutations in a clinical series of Finnish FTLD patients, we sequenced TARDBP exons 1 to 6 in 77 FTLD patients. No evident pathogenic mutations were found. We identified a novel heterozygous c.876_878delCAG sequence variant in 2 related patients with behavioral variant frontotemporal dementia without amyotrophic lateral sclerosis. The variant is predicted to cause an amino acid deletion of serine at position 292 (p.Ser292del). However, p.Ser292del was also found in 1 healthy middle-aged control. Interestingly, both patients carried the C9ORF72 expansion. Therefore, the TARDBP variant p.Ser292del might be considered a rare polymorphism and the C9ORF72 repeat expansion the actual disease-causing mutation in the family. Our results suggest that TARDBP mutations are a rare cause of FTLD. However, the interaction of several genetic factors needs to be taken into account when investigating neurodegenerative diseases.
Assuntos
Proteínas de Ligação a DNA/genética , Demência Frontotemporal/genética , Proteínas/genética , Irmãos , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína C9orf72 , Estudos de Casos e Controles , Estudos de Coortes , Expansão das Repetições de DNA , Éxons , Feminino , Variação Genética , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNARESUMO
Defects in complex I due to mutations in mitochondrial DNA are associated with clinical features ranging from single organ manifestation like Leber hereditary optic neuropathy (LHON) to multiorgan disorders like mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome. Specific mutations cause overlap syndromes combining several phenotypes, but the mechanisms of their biochemical effects are largely unknown. The m.3376G>A transition leading to p.E24K substitution in ND1 with LHON/MELAS phenotype was modeled here in a homologous position (NuoH-E36K) in the Escherichia coli enzyme and it almost totally abolished complex I activity. The more conservative mutation NuoH-E36Q resulted in higher apparent K(m) for ubiquinone and diminished inhibitor sensitivity. A NuoH homolog of the m.3865A>G transition, which has been found concomitantly in the overlap syndrome patient with the m.3376G>A, had only a minor effect. Consequences of a primary LHON-mutation m.3460G>A affecting the same extramembrane loop as the m.3376G>A substitution were also studied in the E. coli model and were found to be mild. The results indicate that the overlap syndrome-associated m.3376G>A transition in MTND1 is the pathogenic mutation and m.3865A>G transition has minor, if any, effect on presentation of the disease. The kinetic effects of the NuoH-E36Q mutation suggest its proximity to the putative ubiquinone binding domain in 49kD/PSST subunits. In all, m.3376G>A perturbs ubiquinone binding, a phenomenon found in LHON, and decreases the activity of fully assembled complex I as in MELAS.
Assuntos
Complexo I de Transporte de Elétrons/genética , Proteínas de Escherichia coli/química , Síndrome MELAS/genética , Proteínas de Membrana/química , NADH Desidrogenase/genética , Atrofia Óptica Hereditária de Leber/genética , Ubiquinona/metabolismo , Sequência de Aminoácidos , Animais , Complexo I de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação/fisiologia , NADH Desidrogenase/metabolismo , Ligação Proteica/genética , Ligação Proteica/fisiologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Amiodarone is a commonly prescribed and one of the most effective anti-arrhythmic drugs available. However, its use is limited by serious toxic adverse effects including optic neuropathy. Previously, amiodarone-associated optic neuropathy has been reported at an incidence of 1.3%-1.8%. Nearly, one-third of patients with amiodarone-induced toxic optic neuropathy are asymptomatic and typically visual acuity improves after drug cessation. We describe the case of a 75-year-old woman who experienced severe optic neuropathy with bilateral optic disc edema and hemorrhages, irreversible loss of vision, and severe defects in visual fields after 1.5 months use of amiodarone. The optic disc edema resolved promptly after discontinuation of the drug, but the patient remained blind permanently. This is the first report of only 6.5 weeks of amiodarone treatment resulting in bilateral optic neuropathy with bilateral and irreversible loss of vision. To ideally establish a connection between amiodarone and optic neuropathy, re-exposure of the patient to the drug should reproduce the symptoms. As a limitation of the study, this was not done in the present case because it would have been unethical. The worldwide growth of the elderly population in number is expected to increase age-related conditions including cardiac diseases. The use of cardiovascular drugs, also anti-arrhythmic agents such as amiodarone, may increase. Thus, clinicians need to be aware of the possibility of drug-induced toxic optic neuropathy, especially if a patient receiving a regimen of amiodarone complains of visual problems.
Assuntos
Amiodarona/efeitos adversos , Antiarrítmicos/efeitos adversos , Cegueira/induzido quimicamente , Doenças do Nervo Óptico/induzido quimicamente , Idoso , Amiodarona/uso terapêutico , Antiarrítmicos/uso terapêutico , Feminino , Humanos , Doenças do Nervo Óptico/patologia , Papiledema/induzido quimicamente , Papiledema/patologia , Índice de Gravidade de Doença , Fatores de Tempo , Campos Visuais/efeitos dos fármacosRESUMO
LHON (Leber hereditary optic neuropathy) is a maternally inherited disease that leads to sudden loss of central vision at a young age. There are three common primary LHON mutations, occurring at positions 3460, 11778 and 14484 in the human mtDNA (mitochondrial DNA), leading to amino acid substitutions in mitochondrial complex I subunits ND1, ND4 and ND6 respectively. We have now examined the effects of ND6 mutations on the function of complex I using the homologous NuoJ subunit of Escherichia coli NDH-1 (NADH:quinone oxidoreductase) as a model system. The assembly level of the NDH-1 mutants was assessed using electron transfer from deamino-NADH to the 'shortcut' electron acceptor HAR (hexammine ruthenium), whereas ubiquinone reductase activity was determined using DB (decylubiquinone) as a substrate. Mutant growth in minimal medium with malate as the main carbon source was used for initial screening of the efficiency of energy conservation by NDH-1. The results indicated that NuoJ-M64V, the equivalent of the common LHON mutation in ND6, had a mild effect on E. coli NDH-1 activity, while nearby mutations, particularly NuoJ-Y59F, NuoJ-V65G and NuoJ-M72V, severely impaired the DB reduction rate and cell growth on malate. NuoJ-Met64 and NuoJ-Met72 position mutants lowered the affinity of NDH-1 for DB and explicit C-type inhibitors, whereas NuoJ-Y59C displayed substrate inhibition by oxidized DB. The results are compatible with the notion that the ND6 subunit delineates the binding cavity of ubiquinone substrate, but does not directly take part in the catalytic reaction. How these changes in the enzyme's catalytic properties contribute to LHON pathogenesis is discussed.
Assuntos
Bactérias/metabolismo , Complexo I de Transporte de Elétrons/genética , Mitocôndrias/metabolismo , Mutação , Atrofia Óptica Hereditária de Leber/genética , Ubiquinona/química , Catálise , DNA Mitocondrial/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Escherichia coli/metabolismo , Feminino , Humanos , Concentração Inibidora 50 , Cinética , Masculino , MutagêneseRESUMO
PURPOSE: To investigate the association of mutations in the mitochondrial DNA (mtDNA) or nuclear candidate genes with mitochondrial disease-related ophthalmic manifestations (nystagmus, ptosis, ophthalmoplegia, optic neuropathy and retinopathy) in children. METHODS: A retrospective cohort of children (n = 98) was identified from the medical record files of a tertiary care hospital. The entire mtDNA and nuclear genes POLG1, OPA1 and PEO1 were analysed from the available DNA samples (n = 38). Furthermore, some nuclear candidate genes were investigated based on family history and phenotype. Rare mtDNA mutations were evaluated using in silico predictors and sequence alignment. RESULTS: Three patients had previously identified mutations in mtDNA that are associated with optic neuropathy (in MT-ND6 and MT-ND1) and nystagmus (in tRNA Arg). Nine rare mutations in MT-ATP6 were identified in seven patients, of whom four manifested with retinopathy and three had clusters of MT-ATP6 mutations. Nuclear PEO1 and OPA1 were unchanged in all samples, but a patient with nystagmus had a heterozygous POLG1 mutation. The analysis of nuclear candidate genes revealed mutations in NDUF8 (patient with nystagmus), TULP1 (patient with optic neuropathy, nystagmus and retinopathy) and RP2 (patient with retinopathy) genes. CONCLUSIONS: Children with retinopathy, nystagmus or optic neuropathy, especially together with developmental delay or positive family history, should be considered for mitochondrial disease. MT-ATP6 should be taken into account for children with retinopathy of unknown aetiology.
Assuntos
DNA Mitocondrial/genética , Oftalmopatias/genética , Genoma Mitocondrial , Doenças Mitocondriais/genética , Mutação , Adolescente , Blefaroptose/genética , Criança , Pré-Escolar , Análise Mutacional de DNA , DNA Polimerase gama , DNA Polimerase Dirigida por DNA/genética , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , ATPases Mitocondriais Próton-Translocadoras/genética , Nistagmo Patológico/genética , Oftalmoplegia/genética , Doenças do Nervo Óptico/genética , Doenças Retinianas/genética , Estudos RetrospectivosRESUMO
Effects of Complex I mutations were studied by modeling in NuoH, NuoJ or NuoK subunits of Escherichia coli NDH-1 by simultaneous optical monitoring of deamino-NADH oxidation and proton translocation and fitting to the data a model equation of transmembrane proton transport. A homolog of the ND1-E24 LHON/MELAS mutation caused 95% inhibition of d-NADH oxidation and proton translocation. The NuoJ-Y59F replacement decreased proton translocation. The NuoK-E72Q mutation lowered the enzyme activity, but proton pumping could be rescued by the double mutation NuoK-E72Q/I39D. Moving the NuoK-E72/E36 pair one helix turn towards the periplasm did not affect redox activity but decreased proton pumping.
Assuntos
Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , Prótons , Transporte Biológico , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , OxirreduçãoRESUMO
PURPOSE: Leber hereditary optic neuropathy (LHON) is regarded as the most common mitochondrial disease. We have previously reported comprehensive population-based epidemiological data on common mitochondrial DNA (mtDNA) mutations including m.3243A>G, m.8344A>G and large-scale mtDNA deletions in Northern Finland. Our aim was to investigate the prevalence of primary LHON mutations and mutations in the four mtDNA genes considered hot spots for LHON in the same population. METHODS: The study population consisted of 42 adult patients with an aetiologically undefined bilateral optic atrophy. The major LHON mutations m.3460G>A, m.11778G>A and m.14484T>C were analysed by restriction fragment length polymorphism (RFLP), and MTND1, MTND6 and MTATP6 genes were sequenced. MTND5 gene was analysed by conformation-sensitive gel electrophoresis (CSGE). RESULTS: No major LHON mutations were found in the population of the province of Northern Ostrobothnia giving the prevalence of these mutations 0-1.36:100 000 (95% CI). However, two main mutations were found elsewhere in Northern Finland, homoplasmic m.11778G>A from Kainuu and heteroplasmic m.3460G>A from Central Ostrobothnia. Furthermore, tobacco-alcohol amblyopia was diagnosed in five patients in the study population and one of them had m.11778G>A. CONCLUSION: The prevalence of the three major LHON mutations is lower in Northern Finland than elsewhere in Finland or in Western Europe. As LHON and tobacco-alcohol amblyopia have a similar phenotype, we recommend analysing the known LHON-associated mutations before setting tobacco-alcohol amblyopia diagnosis.
Assuntos
Alcoolismo/complicações , Ambliopia/etiologia , DNA Mitocondrial/genética , Nicotiana/efeitos adversos , Atrofia Óptica Hereditária de Leber/epidemiologia , Atrofia Óptica Hereditária de Leber/genética , Mutação Puntual , Adulto , Ambliopia/diagnóstico , Feminino , Finlândia/epidemiologia , Humanos , Masculino , ATPases Mitocondriais Próton-Translocadoras/genética , Epidemiologia Molecular , NADH Desidrogenase/genética , Atrofia Óptica , Polimorfismo de Fragmento de Restrição , Prevalência , Acuidade Visual , Adulto JovemRESUMO
MtDNA sequence variation is presumed to be neutral in effect, but associations with diseases and mtDNA haplogroups have been reported. The aim here was to evaluate the functional consequences of m.4216T>C present in haplogroup J. Furthermore, we evaluated m.3866T>C in MT-ND1, a variant detected in a child belonging to haplogroup J and with an isolated complex I deficiency. Homologous substitutions were introduced into Escherichia coli. NADH dehydrogenase domain activity of NDH-1 with either one or both mutations was markedly decreased suggesting that m.4216T>C and m.3866T>C may have an effect on the structural integrity of complex I.
Assuntos
DNA Mitocondrial/genética , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Escherichia coli/genética , Mutagênese , NADH Desidrogenase/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Mutação PuntualRESUMO
Seven of the 45 subunits of mitochondrial NADH:ubiquinone oxidoreductase (complex I) are mitochondrially encoded and have been shown to harbor pathogenic mutations. We modeled the human disease-associated mutations A4136G/ND1-Y277C, T4160C/ND1-L285P and C4171A/ND1-L289M in a highly conserved region of the fourth matrix-side loop of the ND1 subunit by mutating homologous amino acids and surrounding conserved residues of the NuoH subunit of Escherichia coli NDH-1. Deamino-NADH dehydrogenase activity, decylubiquinone reduction kinetics, hexammineruthenium (HAR) reductase activity, and the proton pumping efficiency of the enzyme were assayed in cytoplasmic membrane preparations. Among the human disease-associated mutations, a statistically significant 22% decrease in enzyme activity was observed in the NuoH-L289C mutant and a 29% decrease in the double mutant NuoH-L289C/V297P compared with controls. The adjacent mutations NuoH-D295A and NuoH-R293M caused 49% and 39% decreases in enzyme activity, respectively. None of the mutations studied significantly affected the K(m) value of the enzyme for decylubiquinone or the amount of membrane-associated NDH-1 as estimated from the HAR reductase activity. In spite of the decrease in enzyme activity, all the mutant strains were able to grow on malate, which necessitates sufficient NDH-1 activity. The results show that in ND1/NuoH its fourth matrix-side loop is probably not directly involved in ubiquinone binding or proton pumping but has a role in modifying enzyme activity.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Substituição de Aminoácidos , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Humanos , Cinética , Malatos/metabolismo , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , NADH Desidrogenase/metabolismo , Estrutura Quaternária de Proteína , Bombas de Próton/metabolismo , Compostos de Rutênio/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/metabolismoRESUMO
The ND1 subunit gene of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) is a hot spot for mutations causing Leber hereditary optic neuropathy and several mutations causing the mitochondrial encephalopathy, lactic acidosis and stroke-like episodes syndrome (MELAS). We have used Escherichia coli and Paracoccus denitrificans as model systems to study the effect of mutations 3946 and 3949, which change conserved residues in ND1 and cause MELAS. The vicinity of these mutations was also explored with a series of mutations in charged residues. The 3946 mutation results in E214K substitution in human ND1. Replacement of the equivalent residue in E. coli with lysine or glutamine detracted from enzyme assembly and the assembled enzyme was inactive. However, the equivalent E234Q mutant enzyme in P. denitrificans failed to assemble completely (or was rapidly degraded). Also the corresponding substitution with aspartate decreased the enzyme activity in P. denitrificans and E. coli. The 3949-equivalent substitution, Y229H in E. coli, lowered the catalytic activity by 30%. In addition, an activation of the enzyme during catalytic turnover was seen in this bacterial NDH-1, something that was even more pronounced in another mutant in the same loop, D213E. Several other mutations in this region decreased the enzyme activity. The studied MELAS mutations are situated in a matrix-side loop, which appears to be highly sensitive to structural perturbations. The results provide new information on the function of the region affected by the MELAS mutations 3946 and 3949 that is not obtainable from patient samples or current eukaryote models.
Assuntos
Sequência Conservada , DNA Mitocondrial , Complexo I de Transporte de Elétrons/genética , Proteínas de Escherichia coli/química , Síndrome MELAS/genética , Paracoccus denitrificans/genética , Mutação Puntual , Sequência de Aminoácidos , Catálise , Membrana Celular/química , Membrana Celular/genética , Complexo I de Transporte de Elétrons/química , Proteínas de Escherichia coli/genética , Humanos , Dados de Sequência Molecular , Paracoccus denitrificans/química , Paracoccus denitrificans/enzimologia , Subunidades Proteicas/química , Ubiquinona/químicaRESUMO
The ND4L subunit of mitochondrial NADH:ubiquinone oxidoreductase (complex I) is an integral membrane protein that contains two highly conserved glutamates within putative trans-membrane helices. We employed complex I from Escherichia coli (NDH-1) to study the role of these residues by site-directed mutagenesis. The conserved glutamates of the NuoK subunit, E36 and E72, were replaced by either Asp or Gln residues, and the effects of the mutations on cell growth and catalysis of electron transfer from deamino-NADH to ubiquinone analogues were examined. Additional mutants that carried acidic residues at selected positions within this domain were also prepared and analyzed. The results indicated that two closely located membrane-embedded acidic residues in NuoK are essential for high rates of ubiquinone reduction, a prerequisite for the growth of cytochrome bo-deficient E. coli cells on malate as the main carbon source. The two acidic residues do not have to be on adjacent helices, and mutual location on the same helix, either helix 2 or 3, at an interval of three amino acids (about one turn of the putative helix), resulted in high activity and good growth phenotypes. Nevertheless, shifting only one of them, either E36 or E72, toward the periplasmic side of the membrane by about one turn of the helix severely hampered activity and growth, whereas moving both acidic residues together to that deeper membrane position stimulated the ubiquinone reductase activity of the enzyme but not cell growth on malate, suggesting impaired energy conservation in this mutant.
Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/química , Ácido Glutâmico/química , Proteínas de Membrana/química , NADH Desidrogenase/química , Subunidades Proteicas/química , Sequência de Aminoácidos , Sequência Conservada , Complexo I de Transporte de Elétrons/genética , Ativação Enzimática , Deleção de Genes , Teste de Complementação Genética , Ácido Glutâmico/genética , Proteínas de Membrana/genética , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína/genética , Subunidades Proteicas/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The chemiluminescent superoxide indicators lucigenin and coelenterazine were compared in rat liver submitochondrial particles and cytoplasmic membranes from Paracoccus denitrificans. Qualitative monitoring is possible with both probes, but quantitative work with lucigenin is hampered by its dependence on one-electron reduction before the photon-emitting reaction. Therefore, calibration of measurements on complex I, capable of efficient lucigenin prereduction with reduced nicotinamide adenine dinucleotide, against xanthine oxidase, which in the presence of hypoxanthine is not able to reduce the probe to a significant rate compared to complex I, may give results in error by one order of magnitude. Coelenterazine, although susceptible of storage-dependent high background chemiluminescence, does not require prereduction and is thus a more reliable probe.