RESUMO
Respiratory infections cause significant morbidity and mortality, yet it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning fatal disease. Transcriptomics strongly linked oleoyl-acyl-carrier-protein (ACP) hydrolase (OLAH), an enzyme mediating fatty acid production, with fatal A(H7N9) early after hospital admission, persisting until death. Recovered patients had low OLAH expression throughout hospitalization. High OLAH levels were also detected in patients hospitalized with life-threatening seasonal influenza, COVID-19, respiratory syncytial virus (RSV), and multisystem inflammatory syndrome in children (MIS-C) but not during mild disease. In olah-/- mice, lethal influenza infection led to survival and mild disease as well as reduced lung viral loads, tissue damage, infection-driven pulmonary cell infiltration, and inflammation. This was underpinned by differential lipid droplet dynamics as well as reduced viral replication and virus-induced inflammation in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza replication in macrophages and their inflammatory potential. Our findings define how the expression of OLAH drives life-threatening viral disease.
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Mitochondria possess a small genome that codes for core subunits of the oxidative phosphorylation system and whose expression is essential for energy production. Information on the regulation and spatial organization of mitochondrial gene expression in the cellular context has been difficult to obtain. Here we devise an imaging approach to analyze mitochondrial translation within the context of single cells, by following the incorporation of clickable non-canonical amino acids. We apply this method to multiple cell types, including specialized cells such as cardiomyocytes and neurons, and monitor with spatial resolution mitochondrial translation in axons and dendrites. We also show that translation imaging allows to monitor mitochondrial protein expression in patient fibroblasts. Approaching mitochondrial translation with click chemistry opens new avenues to understand how mitochondrial biogenesis is integrated into the cellular context and can be used to assess mitochondrial gene expression in mitochondrial diseases.
Assuntos
Proteínas Mitocondriais , Biossíntese de Proteínas , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Biogênese de Organelas , Fosforilação OxidativaRESUMO
PURPOSE: To analyze the influence of Caucasian mitochondrial haplogroups on controlled ovarian stimulation outcome (COS), embryo (E), and pregnancy success. METHODS: In a Caucasian population (n = 517) undergoing COS, mitochondrial haplogroups and physiological parameters were determined. Patients were classified, according to Bologna criteria, as good (>3)/poor ≤3) responder, on dependence of recruited oocytes (RO), and in pregnancy/non-pregnancy groups. Haplogroups were determined by sequencing mitochondrial hypervariable sequence I and confirmed by polymerase chain reaction (PCR), followed by restriction fragment length polymorphisms (RFLP). RESULTS: The rank of total dose of FSH (TD FSH) was similar in all clusters/haplogroups, except in JT, which is narrower (950-3,650 IU), particularly in T (1,350-3,650 IU). The statistical analysis showed higher RO and E in JT when compared to U, although it was only Uk which accumulated significantly in pregnancy respect to JT. Pearson's correlations between TD FSH and RO showed negative statistical significance in all population (P = 0.001), H (P = 0.03), JT (P = 0.01), and T (P = 0.03). The percentage of contribution of TD FSH on RO was almost nine times in the JT cluster as compared to all population one. CONCLUSIONS: JT cluster shows a different influence of TD FSH on RO. JT cluster shows higher RO and E than U, but it is Uk which exhibits a significant higher pregnancy rate than JT. The negative influence of the JT cluster on pregnancy success strongly suggests that the m.4216 T > C polymorphism could be responsible.
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Fertilização in vitro , Hormônio Foliculoestimulante Humano , Feminino , Gravidez , Humanos , Taxa de Gravidez , Fertilização in vitro/métodos , Indução da Ovulação/métodos , OócitosRESUMO
Leber hereditary optic neuropathy is a mitochondrial disease mainly due to pathologic mutations in mitochondrial genes related to the respiratory complex I of the oxidative phosphorylation system. Genetic, physiological, and environmental factors modulate the penetrance of these mutations. We report two patients suffering from this disease and harboring a m.15950G > A mutation in the mitochondrial DNA-encoded gene for the threonine transfer RNA. We also provide evidences supporting the pathogenicity of this mutation.
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Atrofia Óptica Hereditária de Leber , DNA Mitocondrial/genética , Complexo I de Transporte de Elétrons/genética , Humanos , Mutação , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/patologia , RNA de Transferência/genéticaRESUMO
Heredity of familial hypercholesterolemia (FH) can present as a dominant monogenic disorder of polygenic origin or with no known genetic cause. In addition, the variability of the symptoms among individuals or within the same families evidence the potential contribution of additional factors than monogenic mutations that could modulate the development and severity of the disease. In addition, statins, the lipid-lowering drugs which constitute the first-line therapy for the disease, cause associated muscular symptoms in a certain number of individuals. Here, we analyze the evidence of the mitochondrial genetic variation with a special emphasis on the role of CoQ10 to explain this variability found in both disease symptoms and statins side effects. We propose to use mtDNA variants and copy numbers as markers for the cardiovascular disease development of FH patients and to predict potential statin secondary effects and explore new mechanisms to identify new markers of disease or implement personalized medicine strategies for FH therapy.
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Aterosclerose , Inibidores de Hidroximetilglutaril-CoA Redutases , Hiperlipoproteinemia Tipo II , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Hiperlipoproteinemia Tipo II/complicações , Hiperlipoproteinemia Tipo II/tratamento farmacológico , Hiperlipoproteinemia Tipo II/genética , Aterosclerose/tratamento farmacológico , Aterosclerose/genética , Aterosclerose/complicações , Hipolipemiantes/uso terapêutico , Patrimônio GenéticoRESUMO
Thymidine kinase (TK2) deficiency causes mitochondrial DNA depletion syndrome. We aimed to report the clinical, biochemical, genetic, histopathological, and ultrastructural features of a cohort of paediatric patients with TK2 deficiency. Mitochondrial DNA was isolated from muscle biopsies to assess depletions and deletions. The TK2 genes were sequenced using Sanger sequencing from genomic DNA. All muscle biopsies presented ragged red fibres (RRFs), and the prevalence was greater in younger ages, along with an increase in succinate dehydrogenase (SDH) activity and cytochrome c oxidase (COX)-negative fibres. An endomysial inflammatory infiltrate was observed in younger patients and was accompanied by an overexpression of major histocompatibility complex type I (MHC I). The immunofluorescence study for complex I and IV showed a greater number of fibres than those that were visualized by COX staining. In the ultrastructural analysis, we found three major types of mitochondrial alterations, consisting of concentrically arranged lamellar cristae, electrodense granules, and intramitochondrial vacuoles. The pathological features in the muscle showed substantial differences in the youngest patients when compared with those that had a later onset of the disease. Additional ultrastructural features are described in the muscle biopsy, such as sarcomeric de-structuration in the youngest patients with a more severe phenotype.
Assuntos
Miopatias Mitocondriais , Timidina Quinase/metabolismo , DNA Mitocondrial/análise , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Miopatias Mitocondriais/genética , Miopatias Mitocondriais/patologia , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Succinato Desidrogenase , Timidina Quinase/genéticaRESUMO
BACKGROUND: Mitochondrial diseases (MD) are genetic metabolic disorders that impair normal mitochondrial structure or function. The aim of this study was to investigate the status of circulating cell-free mitochondrial DNA (ccfmtDNA) in cerebrospinal fluid (CSF), together with other biomarkers (growth differentiation factor-15 [GDF-15], alanine, and lactate), in a cohort of 25 patients with a molecular diagnosis of MD. METHODS: Measurement of ccfmtDNA was performed by using droplet digital PCR. RESULTS: The mean copy number of ccfmtDNA was approximately 6 times higher in the MD cohort compared to the control group; patients with mitochondrial deletion and depletion syndromes (MDD) had the higher levels. We also detected the presence of both wild-type mtDNA and mtDNA deletions in CSF samples of patients with single deletions. Patients with MDD with single deletions had significantly higher concentrations of GDF-15 in CSF than controls, whereas patients with point mutations in mitochondrial DNA presented no statistically significant differences. Additionally, we found a significant positive correlation between ccfmtDNA levels and GDF-15 concentrations (r = 0.59, P = 0.016). CONCLUSION: CSF ccfmtDNA levels are significantly higher in patients with MD in comparison to controls and, thus, they can be used as a novel biomarker for MD research. Our results could also be valuable to support the clinical outcome assessment of MD patients.
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Ácidos Nucleicos Livres , Doenças Mitocondriais , Biomarcadores/líquido cefalorraquidiano , Ácidos Nucleicos Livres/genética , DNA Mitocondrial/genética , Humanos , Mitocôndrias/genética , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genéticaRESUMO
PURPOSE: To determine the influence of different genotypes of Ala307Thr and Asn680Ser FSHr polymorphisms on controlled ovarian stimulation (COS) outcome and pregnancy. METHODS: This study collected blood and physiological and clinical parameters of 517 Caucasian patients (Statistical power ≥ 80%) that underwent COS treatment. Genotypes of Ala307Thr and Asn680Ser polymorphisms were determined using PCR amplification followed by Bsu36I and BsrI digestion, respectively. RESULTS: Ala307Ala and Ser680Ser genotypes associated to worse parameters of COS outcome (preovulatory follicles P = 0.05, in both), justifying their lower pregnancy rate than Non-Ala307Ala, P = 0.01 and Non-Ser680Ser, P = 0.004, respectively or together, (P = 0.003). Within the Non-Ala307Ala group, Thr307Thr genotype showed higher number of fertilized oocytes (P = 0.04) and embryos (P = 0.01) than Non-Thr307Thr, but no influence on pregnancy rate. Ala307Ala and Ser680Ser patients doubled probability of non-pregnancy than Non-Ala307Ala (odds ratio = 2.0) and Non-Ser680Ser (odds ratio = 2.11), respectively. Ala307Ala and Ser680Ser genotypes tend to appear together (P < 0.0001), which increases the probability of non-pregnancy. CONCLUSIONS: Ala307Ala and Ser680Ser genotypes of 307 and 680 FSHr polymorphisms associate to worse COS outcome than its respective Non-Ala307Ala and Non-Ser680Ser. Within the Non-Ala307Ala genotypes, Thr307Thr, although shows higher Fertilized Oocytes and Embryos, do not influence on pregnancy rate. Ala307Ala and Ser680Ser genotypes double the probability of Non-Pregnancy than their respective Non-Ala307Ala and Non-Ser680Ser genotypes. Furthermore, the strong tendency of these genotypes to appear together worsens the probability of pregnancy in these patients.
Assuntos
Infertilidade Feminina/terapia , Indução da Ovulação/estatística & dados numéricos , Polimorfismo de Nucleotídeo Único , Taxa de Gravidez , Receptores do FSH/genética , Técnicas de Reprodução Assistida/efeitos adversos , Adulto , Feminino , Humanos , Infertilidade Feminina/genética , Infertilidade Feminina/patologia , GravidezRESUMO
A 3-year-old girl presented with severe epilepsy in the context of Borrelia infection. After ceftriaxone/lidocaine administration, she showed secondarily generalized focal crises that led to neurological and motor sequelae. Genetic studies identified in the patient two heterozygous POLG mutations (c.2591A>G; p.Asn864Ser and c.3649G>C; p.Ala1217Pro). Through analysis of POLG activity in cultured fibroblasts, we confirmed that the mutations altered the mtDNA turnover. Moreover, patient fibroblasts were more sensitive than controls in the presence of a mitochondrial replication-affecting drug, the antiretroviral azidothymidine. To test if ceftriaxone treatment could worsen the deleterious effect of the patient mutations, toxicity assays were performed. Cell toxicity, without direct effect on mitochondrial respiratory function, was detected at different antibiotic concentrations. The clinical outcome, together with the different in vitro sensitivity to ceftriaxone among patient and control cells, suggested that the mitochondrial disease symptoms were hastened by the infection and were possibly worsened by the pharmacological treatment. This study underscores the benefit of early genetic diagnosis of the patients with mitochondrial diseases, since they may be a target group of patients especially vulnerable to environmental factors.
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Infecções por Borrelia/complicações , DNA Polimerase gama/genética , Epilepsia/genética , Doenças Mitocondriais/genética , Mutação , Antibacterianos/efeitos adversos , Infecções por Borrelia/tratamento farmacológico , Ceftriaxona/efeitos adversos , Células Cultivadas , Pré-Escolar , DNA Mitocondrial/genética , Epilepsia/etiologia , Feminino , Humanos , Doenças Mitocondriais/etiologiaRESUMO
Protein import into mitochondria is facilitated by translocases within the outer and the inner mitochondrial membranes that are dedicated to a highly specific subset of client proteins. The mitochondrial carrier translocase (TIM22 complex) inserts multispanning proteins, such as mitochondrial metabolite carriers and translocase subunits (TIM23, TIM17A/B and TIM22), into the inner mitochondrial membrane. Both types of substrates are essential for mitochondrial metabolic function and biogenesis. Here, we report on a subject, diagnosed at 1.5 years, with a neuromuscular presentation, comprising hypotonia, gastroesophageal reflux disease and persistently elevated serum and Cerebrospinal fluid lactate (CSF). Patient fibroblasts displayed reduced oxidative capacity and altered mitochondrial morphology. Using trans-mitochondrial cybrid cell lines, we excluded a candidate variant in mitochondrial DNA as causative of these effects. Whole-exome sequencing identified compound heterozygous variants in the TIM22 gene (NM_013337), resulting in premature truncation in one allele (p.Tyr25Ter) and a point mutation in a conserved residue (p.Val33Leu), within the intermembrane space region, of the TIM22 protein in the second allele. Although mRNA transcripts of TIM22 were elevated, biochemical analyses revealed lower levels of TIM22 protein and an even greater deficiency of TIM22 complex formation. In agreement with a defect in carrier translocase function, carrier protein amounts in the inner membrane were found to be reduced. This is the first report of pathogenic variants in the TIM22 pore-forming subunit of the carrier translocase affecting the biogenesis of inner mitochondrial membrane proteins critical for metabolite exchange.
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Proteínas de Transporte/genética , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Miopatias Mitocondriais/genética , Criança , DNA Mitocondrial/genética , Feminino , Fibroblastos/metabolismo , Predisposição Genética para Doença , Humanos , Ácido Láctico/líquido cefalorraquidiano , Proteínas de Membrana Transportadoras/genética , Mitocôndrias/patologia , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/patologia , Miopatias Mitocondriais/líquido cefalorraquidiano , Miopatias Mitocondriais/patologia , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Sequenciamento do ExomaRESUMO
The frequency of dermatological manifestations in diseases due to mitochondrial DNA mutations is not well known, although multiple symmetric lipomatosis has been repeatedly associated to mitochondrial DNA mutations. Here, we present a patient suffering from multiple symmetric lipomatosis and other skin signs. We found a new mitochondrial DNA mutation, m.8357T>C, in the tRNALys -coding gene and, using a cybrid approach, confirmed its pathogenicity. A meta-analysis of the dermatological signs of the patient shows that they are not common in patients with confirmed mitochondrial DNA mutations and suggests that, in these cases, lipomatosis is not related to the oxidative phosphorylation dysfunction, but to an alteration of an additional function associated to particular mitochondrial tRNAs.
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DNA Mitocondrial/genética , Lipomatose Simétrica Múltipla/genética , RNA de Transferência/genética , Adulto , Humanos , Lipomatose Simétrica Múltipla/diagnóstico por imagem , Lipomatose Simétrica Múltipla/fisiopatologia , Masculino , Pessoa de Meia-Idade , Mitocôndrias/genética , Mitocôndrias/patologia , Mutação/genéticaRESUMO
Physical stress, including high temperatures, may damage the central metabolic nicotinamide nucleotide cofactors [NAD(P)H], generating toxic derivatives [NAD(P)HX]. The highly conserved enzyme NAD(P)HX dehydratase (NAXD) is essential for intracellular repair of NAD(P)HX. Here we present a series of infants and children who suffered episodes of febrile illness-induced neurodegeneration or cardiac failure and early death. Whole-exome or whole-genome sequencing identified recessive NAXD variants in each case. Variants were predicted to be potentially deleterious through in silico analysis. Reverse-transcription PCR confirmed altered splicing in one case. Subject fibroblasts showed highly elevated concentrations of the damaged cofactors S-NADHX, R-NADHX and cyclic NADHX. NADHX accumulation was abrogated by lentiviral transduction of subject cells with wild-type NAXD. Subject fibroblasts and muscle biopsies showed impaired mitochondrial function, higher sensitivity to metabolic stress in media containing galactose and azide, but not glucose, and decreased mitochondrial reactive oxygen species production. Recombinant NAXD protein harbouring two missense variants leading to the amino acid changes p.(Gly63Ser) and p.(Arg608Cys) were thermolabile and showed a decrease in Vmax and increase in KM for the ATP-dependent NADHX dehydratase activity. This is the first study to identify pathogenic variants in NAXD and to link deficient NADHX repair with mitochondrial dysfunction. The results show that NAXD deficiency can be classified as a metabolite repair disorder in which accumulation of damaged metabolites likely triggers devastating effects in tissues such as the brain and the heart, eventually leading to early childhood death.
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Hidroliases/deficiência , Doenças Neurodegenerativas/genética , Pré-Escolar , Simulação por Computador , Feminino , Febre/complicações , Febre/metabolismo , Fibroblastos/metabolismo , Vetores Genéticos , Humanos , Hidroliases/genética , Lactente , Cinética , Lentivirus , Masculino , Mitocôndrias/metabolismo , Mutação , NAD/análogos & derivados , NAD/metabolismo , Doenças Neurodegenerativas/complicações , Doenças Neurodegenerativas/metabolismo , Cultura Primária de Células , Sequenciamento Completo do GenomaRESUMO
Mitochondrial oxidative phosphorylation disorders are extremely heterogeneous conditions. Their clinical and genetic variability makes the identification of reliable and specific biomarkers very challenging. Until now, only a few studies have focused on the effect of a defective oxidative phosphorylation functioning on the cell's secretome, although it could be a promising approach for the identification and pre-selection of potential circulating biomarkers for mitochondrial diseases. Here, we review the insights obtained from secretome studies with regard to oxidative phosphorylation dysfunction, and the biomarkers that appear, so far, to be promising to identify mitochondrial diseases. We propose two new biomarkers to be taken into account in future diagnostic trials.
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DNA Mitocondrial/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Fator 15 de Diferenciação de Crescimento/metabolismo , Interleucina-6/metabolismo , Doenças Mitocondriais/metabolismo , Fosforilação Oxidativa , Fator A de Crescimento do Endotélio Vascular/metabolismo , Biomarcadores/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fator 15 de Diferenciação de Crescimento/genética , Humanos , Doenças Mitocondriais/genética , Via Secretória/efeitos dos fármacos , Via Secretória/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
AIM: To perform a deep phenotype characterisation in a pedigree of 3 siblings with Leigh syndrome and compound heterozygous NDUFAF6 mutations. METHOD: A multi-gene panel of childhood-onset basal ganglia neurodegeneration inherited conditions was analysed followed by functional studies in fibroblasts. RESULTS: Three siblings developed gait dystonia in infancy followed by rapid progression to generalised dystonia and psychomotor regression. Brain magnetic resonance showed symmetric and bilateral cytotoxic lesions in the putamen and proliferation of the lenticular-striate arteries, latter spreading to the caudate and progressing to cavitation and volume loss. We identified a frameshift novel change (c.554_558delTTCTT; p.Tyr187AsnfsTer65) and a pathogenic missense change (c.371T>C; p.Ile124Thr) in the NDUFAF6 gene, which segregated with an autosomal recessive inheritance within the family. Patient mutations were associated with the absence of the NDUFAF6 protein and reduced activity and assembly of mature complex I in fibroblasts. By functional complementation assay, the mutant phenotype was rescued by the canonical version of the NDUFAF6. A literature review of 14 NDUFAF6 patients showed a consistent phenotype of an early childhood insidious onset neurological regression with prominent dystonia associated with basal ganglia degeneration and long survival. INTERPRETATION: NDUFAF6-related Leigh syndrome is a relevant cause of childhood onset dystonia and isolated bilateral striatal necrosis. By genetic complementation, we could demonstrate the pathogenicity of novel genetic variants in NDUFAF6.
Assuntos
Distúrbios Distônicos/genética , Complexo I de Transporte de Elétrons/genética , Doença de Leigh/genética , Proteínas Mitocondriais/genética , Degeneração Estriatonigral/congênito , Biópsia , Criança , Estudos de Coortes , Feminino , Fibroblastos , Expressão Gênica , Variação Genética , Humanos , Doença de Leigh/complicações , Masculino , Músculos/patologia , Mutação , Linhagem , Irmãos , Degeneração Estriatonigral/genéticaRESUMO
The m.1555A>G mtDNA variant causes maternally inherited deafness, but the reasons for the highly variable clinical penetrance are not known. Exome sequencing identified a heterozygous start loss mutation in SSBP1, encoding the single stranded binding protein 1 (SSBP1), segregating with hearing loss in a multi-generational family transmitting m.1555A>G, associated with mtDNA depletion and multiple deletions in skeletal muscle. The SSBP1 mutation reduced steady state SSBP1 levels leading to a perturbation of mtDNA metabolism, likely compounding the intra-mitochondrial translation defect due to m.1555A>G in a tissue-specific manner. This family demonstrates the importance of rare trans-acting genetic nuclear modifiers in the clinical expression of mtDNA disease.
Assuntos
Proteínas de Ligação a DNA/genética , Saúde da Família , Perda Auditiva/genética , Proteínas Mitocondriais/genética , Mutação/genética , Adolescente , Criança , Pré-Escolar , Análise Mutacional de DNA , Complexo II de Transporte de Elétrons/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Perda Auditiva/complicações , Heterozigoto , Humanos , Lactente , Masculino , Doenças Mitocondriais/complicações , Doenças Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Músculo Esquelético/ultraestrutura , Adulto JovemRESUMO
BACKGROUND: Thymine kinase 2 (TK2) is a mitochondrial matrix protein encoded in nuclear DNA and phosphorylates the pyrimidine nucleosides: thymidine and deoxycytidine. Autosomal recessive TK2 mutations cause a spectrum of disease from infantile onset to adult onset manifesting primarily as myopathy. OBJECTIVE: To perform a retrospective natural history study of a large cohort of patients with TK2 deficiency. METHODS: The study was conducted by 42 investigators across 31 academic medical centres. RESULTS: We identified 92 patients with genetically confirmed diagnoses of TK2 deficiency: 67 from literature review and 25 unreported cases. Based on clinical and molecular genetics findings, we recognised three phenotypes with divergent survival: (1) infantile-onset myopathy (42.4%) with severe mitochondrial DNA (mtDNA) depletion, frequent neurological involvement and rapid progression to early mortality (median post-onset survival (POS) 1.00, CI 0.58 to 2.33 years); (2) childhood-onset myopathy (40.2%) with mtDNA depletion, moderate-to-severe progression of generalised weakness and median POS at least 13 years; and (3) late-onset myopathy (17.4%) with mild limb weakness at onset and slow progression to respiratory insufficiency with median POS of 23 years. Ophthalmoparesis and facial weakness are frequent in adults. Muscle biopsies show multiple mtDNA deletions often with mtDNA depletion. CONCLUSIONS: In TK2 deficiency, age at onset, rate of weakness progression and POS are important variables that define three clinical subtypes. Nervous system involvement often complicates the clinical course of the infantile-onset form while extraocular muscle and facial involvement are characteristic of the late-onset form. Our observations provide essential information for planning future clinical trials in this disorder.
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Estudos de Associação Genética , Predisposição Genética para Doença , Proteínas Mitocondriais/deficiência , Doenças Musculares/diagnóstico , Doenças Musculares/genética , Timidina Quinase/deficiência , Adolescente , Adulto , Idade de Início , Idoso , Criança , Pré-Escolar , Feminino , Genes Recessivos , Testes Genéticos , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Doenças Musculares/mortalidade , Mutação , Fenótipo , Estudos Retrospectivos , Análise de Sobrevida , Adulto JovemRESUMO
Mitochondrial diseases are a group of genetic disorders leading to the dysfunction of mitochondrial energy metabolism pathways. We aimed to assess the clinical phenotype and the biochemical cerebrospinal fluid (CSF) biogenic amine profiles of patients with different diagnoses of genetic mitochondrial diseases. We recruited 29 patients with genetically confirmed mitochondrial diseases harboring mutations in either nuclear or mitochondrial DNA (mtDNA) genes. Signs and symptoms of impaired neurotransmission and neuroradiological data were recorded. CSF monoamines, pterins, and 5-methyltetrahydrofolate (5MTHF) concentrations were analyzed using high-performance liquid chromatography with electrochemical and fluorescence detection procedures. The mtDNA mutations were studied by Sanger sequencing, Southern blot, and real-time PCR, and nuclear DNA was assessed either by Sanger or next-generation sequencing. Five out of 29 cases showed predominant dopaminergic signs not attributable to basal ganglia involvement, harboring mutations in different nuclear genes. A chi-square test showed a statistically significant association between high homovanillic acid (HVA) values and low CSF 5-MTHF values (chi-square = 10.916; p = 0.001). Seven out of the eight patients with high CSF HVA values showed cerebral folate deficiency. Five of them harbored mtDNA deletions associated with Kearns-Sayre syndrome (KSS), one had a mitochondrial point mutation at the mtDNA ATPase6 gene, and one had a POLG mutation. In conclusion, dopamine deficiency clinical signs were present in some patients with mitochondrial diseases with different genetic backgrounds. High CSF HVA values, together with a severe cerebral folate deficiency, were observed in KSS patients and in other mtDNA mutation syndromes.
Assuntos
Aminas Biogênicas/líquido cefalorraquidiano , Ácido Homovanílico/líquido cefalorraquidiano , Doenças Mitocondriais/líquido cefalorraquidiano , Doenças Mitocondriais/diagnóstico , Pterinas/líquido cefalorraquidiano , Tetra-Hidrofolatos/líquido cefalorraquidiano , DNA Mitocondrial/genética , Humanos , Doenças Mitocondriais/genética , Mutação Puntual , Deleção de Sequência , Tetra-Hidrofolatos/deficiênciaRESUMO
AIM: Gamma-aminobutyric acid (GABA) is a major modulator in brain maturation and its role in many different neurodevelopmental disorders has been widely reported. Although the involvement of GABA in different disorders has been related to its regulatory function as an inhibitory neurotransmitter in the mature brain, co-transmitter, and signalling molecule, little is known about its role as a clinical biomarker in neuropaediatric disorders. The aim of this study is to report the cerebrospinal fluid (CSF) free-GABA concentrations in a large cohort of patients (n=85) with different neurological disorders. METHOD: GABA was measured in the CSF of neuropaediatric patients using capillary electrophoresis with laser-induced fluorescence detection. Other neurotransmitters (amino acids and monoamines) were also analysed. RESULTS: GABA concentrations in CSF were abnormal, with a greater frequency (44%) than monoamines (20%) in neuropaediatric patients compared with our reference values. Although we included a few patients with inborn errors of metabolism, GABA levels in CSF were more frequently abnormal in metabolic disorders than in other nosological groups. INTERPRETATION: Our work suggests further research into brain GABAergic status in neuropaediatric disorders, which could also lead to new therapeutic strategies. WHAT THIS PAPER ADDS: Homeostasis of GABA seems more vulnerable than that of monoamines in the developing brain. The highest GABA levels are found in the primary GABA neurotransmitter disorder SSADH deficiency. GABA alterations are not specific for any clinical or neuroimaging presentation.
Assuntos
Epilepsia/líquido cefalorraquidiano , Deficiência Intelectual/líquido cefalorraquidiano , Erros Inatos do Metabolismo/líquido cefalorraquidiano , Doenças Mitocondriais/líquido cefalorraquidiano , Transtornos dos Movimentos/líquido cefalorraquidiano , Doenças do Sistema Nervoso/líquido cefalorraquidiano , Ácido gama-Aminobutírico/líquido cefalorraquidiano , Adolescente , Adulto , Biomarcadores/líquido cefalorraquidiano , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Adulto JovemRESUMO
BACKGROUND: Several methods have been developed to predict the pathogenicity of missense mutations but none has been specifically designed for classification of variants in mtDNA-encoded polypeptides. Moreover, there is not available curated dataset of neutral and damaging mtDNA missense variants to test the accuracy of predictors. Because mtDNA sequencing of patients suffering mitochondrial diseases is revealing many missense mutations, it is needed to prioritize candidate substitutions for further confirmation. Predictors can be useful as screening tools but their performance must be improved. RESULTS: We have developed a SVM classifier (Mitoclass.1) specific for mtDNA missense variants. Training and validation of the model was executed with 2,835 mtDNA damaging and neutral amino acid substitutions, previously curated by a set of rigorous pathogenicity criteria with high specificity. Each instance is described by a set of three attributes based on evolutionary conservation in Eukaryota of wildtype and mutant amino acids as well as coevolution and a novel evolutionary analysis of specific substitutions belonging to the same domain of mitochondrial polypeptides. Our classifier has performed better than other web-available tested predictors. We checked performance of three broadly used predictors with the total mutations of our curated dataset. PolyPhen-2 showed the best results for a screening proposal with a good sensitivity. Nevertheless, the number of false positive predictions was too high. Our method has an improved sensitivity and better specificity in relation to PolyPhen-2. We also publish predictions for the complete set of 24,201 possible missense variants in the 13 human mtDNA-encoded polypeptides. CONCLUSIONS: Mitoclass.1 allows a better selection of candidate damaging missense variants from mtDNA. A careful search of discriminatory attributes and a training step based on a curated dataset of amino acid substitutions belonging exclusively to human mtDNA genes allows an improved performance. Mitoclass.1 accuracy could be improved in the future when more mtDNA missense substitutions will be available for updating the attributes and retraining the model.
Assuntos
Análise Mutacional de DNA/métodos , DNA Mitocondrial , Aprendizado de Máquina , Mitocôndrias/metabolismo , Mutação de Sentido Incorreto , Peptídeos/genética , Biologia Computacional/métodos , Humanos , Mitocôndrias/genética , Sensibilidade e EspecificidadeRESUMO
Mitochondrial DNA mutations at MT-ATP6 gene are relatively common in individuals suffering from striatal necrosis syndromes. These patients usually do not show apparent histochemical and/or biochemical signs of oxidative phosphorylation dysfunction. Because of this, MT-ATP6 is not typically analyzed in many other mitochondrial disorders that have not been previously associated to mutations in this gene. To correct this bias, we have performed a screening of the MT-ATP6 gene in a large collection of patients suspected of suffering different mitochondrial DNA (mtDNA) disorders. In three cases, biochemical, molecular-genetics and other analyses in patient tissues and cybrids were also carried out. We found three new pathologic mutations. Two of them in patients showing phenotypes that have not been commonly associated to mutations in the MT-ATP6 gene. These results remark the importance of sequencing the MT-ATP6 gene in patients with striatal necrosis syndromes, but also within other mitochondrial pathologies. This gene should be sequenced at least in all those patients suspected of suffering an mtDNA disorder disclosing normal results for histochemical and biochemical analyses of respiratory chain.