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Mitochondria are epicentres of eukaryotic metabolism and bioenergetics. Pioneering efforts in recent decades have established the core protein componentry of these organelles1 and have linked their dysfunction to more than 150 distinct disorders2,3. Still, hundreds of mitochondrial proteins lack clear functions4, and the underlying genetic basis for approximately 40% of mitochondrial disorders remains unresolved5. Here, to establish a more complete functional compendium of human mitochondrial proteins, we profiled more than 200 CRISPR-mediated HAP1 cell knockout lines using mass spectrometry-based multiomics analyses. This effort generated approximately 8.3 million distinct biomolecule measurements, providing a deep survey of the cellular responses to mitochondrial perturbations and laying a foundation for mechanistic investigations into protein function. Guided by these data, we discovered that PIGY upstream open reading frame (PYURF) is an S-adenosylmethionine-dependent methyltransferase chaperone that supports both complex I assembly and coenzyme Q biosynthesis and is disrupted in a previously unresolved multisystemic mitochondrial disorder. We further linked the putative zinc transporter SLC30A9 to mitochondrial ribosomes and OxPhos integrity and established RAB5IF as the second gene harbouring pathogenic variants that cause cerebrofaciothoracic dysplasia. Our data, which can be explored through the interactive online MITOMICS.app resource, suggest biological roles for many other orphan mitochondrial proteins that still lack robust functional characterization and define a rich cell signature of mitochondrial dysfunction that can support the genetic diagnosis of mitochondrial diseases.
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Mitocondrias , Proteínas Mitocondriales , Proteínas de Transporte de Catión , Proteínas de Ciclo Celular , Metabolismo Energético , Humanos , Espectrometría de Masas , Mitocondrias/genética , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Factores de Transcripción , Proteínas de Unión al GTP rab5RESUMEN
Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.
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Proteínas de Unión al Calcio , Enfermedades Mitocondriales , Proteínas de Unión al Calcio/genética , Homeostasis/genética , Humanos , Proteínas de la Membrana/genética , Mitocondrias/genética , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Sistema Nervioso/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMEN
Mitochondrial replacement technology (MRT) aims to reduce the risk of serious disease in children born to women who carry pathogenic mitochondrial DNA (mtDNA) variants. By transplanting nuclear genomes from eggs of an affected woman to enucleated eggs from an unaffected donor, MRT creates new combinations of nuclear and mtDNA. Based on sets of shared sequence variants, mtDNA is classified into ~30 haplogroups. Haplogroup matching between egg donors and women undergoing MRT has been proposed as a means of reducing mtDNA sequence divergence between them. Here we investigate the potential effect of mtDNA haplogroup matching on clinical delivery of MRT and on mtDNA sequence divergence between donor/recipient pairs. Our findings indicate that haplogroup matching would limit the availability of egg donors such that women belonging to rare haplogroups may have to wait > 4 years for treatment. Moreover, we find that intra-haplogroup sequence variation is frequently within the range observed between randomly matched mtDNA pairs. We conclude that haplogroup matching would restrict the availability of MRT, without necessarily reducing mtDNA sequence divergence between donor/recipient pairs.
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ADN Mitocondrial , Mitocondrias , Niño , Humanos , Femenino , Estudios de Factibilidad , Haplotipos , Mitocondrias/genética , ADN Mitocondrial/genéticaRESUMEN
Mitochondria are implicated in the pathogenesis of cardiovascular diseases (CVDs) but the reasons for this are not well understood. Maternally-inherited population variants of mitochondrial DNA (mtDNA) which affect all mtDNA molecules (homoplasmic) are associated with cardiometabolic traits and the risk of developing cardiovascular disease. However, it is not known whether mtDNA mutations only affecting a proportion of mtDNA molecules (heteroplasmic) also play a role. To address this question, we performed a high-depth (~1000-fold) mtDNA sequencing of blood DNA in 1,399 individuals with hypertension (HTN), 1,946 with ischemic heart disease (IHD), 2,146 with ischemic stroke (IS), and 723 healthy controls. We show that the per individual burden of heteroplasmic single nucleotide variants (mtSNVs) increases with age. The age-effect was stronger for low-level heteroplasmies (heteroplasmic fraction, HF, 5-10%), likely reflecting acquired somatic events based on trinucleotide mutational signatures. After correcting for age and other confounders, intermediate heteroplasmies (HF 10-95%) were more common in hypertension, particularly involving non-synonymous variants altering the amino acid sequence of essential respiratory chain proteins. These findings raise the possibility that heteroplasmic mtSNVs play a role in the pathophysiology of hypertension.
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Enfermedades Cardiovasculares , Hipertensión , Enfermedades Mitocondriales , Enfermedades Cardiovasculares/genética , ADN Mitocondrial/genética , Humanos , Hipertensión/genética , Mitocondrias/genética , MutaciónRESUMEN
The SLC25A26 gene encodes a mitochondrial inner membrane carrier that transports S-adenosylmethionine (SAM) into the mitochondrial matrix in exchange for S-adenosylhomocysteine (SAH). SAM is the predominant methyl-group donor for most cellular methylation processes, of which SAH is produced as a by-product. Pathogenic, biallelic SLC25A26 variants are a recognized cause of mitochondrial disease in children, with a severe neonatal onset caused by decreased SAM transport activity. Here, we describe two, unrelated adult cases, one of whom presented with recurrent episodes of severe abdominal pain and metabolic decompensation with lactic acidosis. Both patients had exercise intolerance and mitochondrial myopathy associated with biallelic variants in SLC25A26, which led to marked respiratory chain deficiencies and mitochondrial histopathological abnormalities in skeletal muscle that are comparable to those previously described in early-onset cases. We demonstrate using both mouse and fruit fly models that impairment of SAH, rather than SAM, transport across the mitochondrial membrane is likely the cause of this milder, late-onset phenotype. Our findings associate a novel pathomechanism with a known disease-causing protein and highlight the quests of precision medicine in optimizing diagnosis, therapeutic intervention and prognosis.
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Enfermedades Mitocondriales , S-Adenosilhomocisteína , Animales , Metilación , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6-months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt-tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease.
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Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Miopatías Mitocondriales/genética , Miopatías Mitocondriales/metabolismo , Adolescente , Línea Celular , ADN Mitocondrial/genética , Femenino , Expresión Génica , Humanos , Lactante , Masculino , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación , Linaje , Proteómica , Músculo Cuádriceps/metabolismo , ARNt Metiltransferasas/genética , ARNt Metiltransferasas/metabolismoRESUMEN
BACKGROUND: Autophagy is the major intracellular degradation route in mammalian cells. Systemic ablation of core autophagy-related (ATG) genes in mice leads to embryonic or perinatal lethality, and conditional models show neurodegeneration. Impaired autophagy has been associated with a range of complex human diseases, yet congenital autophagy disorders are rare. METHODS: We performed a genetic, clinical, and neuroimaging analysis involving five families. Mechanistic investigations were conducted with the use of patient-derived fibroblasts, skeletal muscle-biopsy specimens, mouse embryonic fibroblasts, and yeast. RESULTS: We found deleterious, recessive variants in human ATG7, a core autophagy-related gene encoding a protein that is indispensable to classical degradative autophagy. Twelve patients from five families with distinct ATG7 variants had complex neurodevelopmental disorders with brain, muscle, and endocrine involvement. Patients had abnormalities of the cerebellum and corpus callosum and various degrees of facial dysmorphism. These patients have survived with impaired autophagic flux arising from a diminishment or absence of ATG7 protein. Although autophagic sequestration was markedly reduced, evidence of basal autophagy was readily identified in fibroblasts and skeletal muscle with loss of ATG7. Complementation of different model systems by deleterious ATG7 variants resulted in poor or absent autophagic function as compared with the reintroduction of wild-type ATG7. CONCLUSIONS: We identified several patients with a neurodevelopmental disorder who have survived with a severe loss or complete absence of ATG7, an essential effector enzyme for autophagy without a known functional paralogue. (Funded by the Wellcome Centre for Mitochondrial Research and others.).
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Anomalías Múltiples/genética , Ataxia/genética , Proteína 7 Relacionada con la Autofagia/genética , Autofagia/genética , Discapacidades del Desarrollo/genética , Mutación Missense , Adolescente , Adulto , Autofagia/fisiología , Proteína 7 Relacionada con la Autofagia/fisiología , Células Cultivadas , Cerebelo/anomalías , Simulación por Computador , Cara/anomalías , Femenino , Fibroblastos , Genes Recesivos , Humanos , Lactante , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Malformaciones del Sistema Nervioso/genética , Linaje , FenotipoRESUMEN
Genetic processes require the activity of multiple topoisomerases, essential enzymes that remove topological tension and intermolecular linkages in DNA. We have investigated the subcellular localisation and activity of the six human topoisomerases with a view to understanding the topological maintenance of human mitochondrial DNA. Our results indicate that mitochondria contain two topoisomerases, TOP1MT and TOP3A. Using molecular, genomic and biochemical methods we find that both proteins contribute to mtDNA replication, in addition to the decatenation role of TOP3A, and that TOP1MT is stimulated by mtSSB. Loss of TOP3A or TOP1MT also dysregulates mitochondrial gene expression, and both proteins promote transcription elongation in vitro. We find no evidence for TOP2 localisation to mitochondria, and TOP2B knockout does not affect mtDNA maintenance or expression. Our results suggest a division of labour between TOP3A and TOP1MT in mtDNA topology control that is required for the proper maintenance and expression of human mtDNA.
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ADN Mitocondrial , Mitocondrias , Humanos , Mitocondrias/metabolismo , ADN Mitocondrial/metabolismo , ADN-Topoisomerasas de Tipo I/metabolismo , Replicación del ADN/genética , ADN-Topoisomerasas/genéticaRESUMEN
Intracellular heterogeneity contributes significantly to cellular physiology and, in a number of debilitating diseases, cellular pathophysiology. This is greatly influenced by distinct organelle populations and to understand the aetiology of disease, it is important to have tools able to isolate and differentially analyse organelles from precise location within tissues. Here, we report the development of a subcellular biopsy technology that facilitates the isolation of organelles, such as mitochondria, from human tissue. We compared the subcellular biopsy technology to laser capture microdissection (LCM) that is the state-of-the-art technique for the isolation of cells from their surrounding tissues. We demonstrate an operational limit of >20 µm for LCM and then, for the first time in human tissue, show that subcellular biopsy can be used to isolate mitochondria beyond this limit.
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Genómica , Biopsia , Humanos , Captura por Microdisección con Láser/métodosRESUMEN
Policies related to the inclusion of children with disabilities in mainstream classrooms have led to questions regarding how teachers can help cultivate inclusive learning communities where all children are supported and valued. In play-based kindergarten programs, teachers are tasked with ensuring goals for children's learning and development are cultivated in play. However, debates persist regarding the optimal role of the teacher in play and how to meaningfully support the play of children with disabilities. The current multiple case study explored the perspectives and approaches of three kindergarten teachers who highly valued, and strived to enable, participation and inclusion in play-based learning, referred to here as enactors. A minimum of three hours of observation were conducted in each classroom in the fall, and semi-structured teacher interviews were conducted in the fall and spring of the school year. Enactors shared some common themes related to implementing play-based learning to promote inclusion, including a balance of child agency and teacher guidance, involvement that is child-centred and flexible, and the importance of supporting social interactions in play. These views informed both common and unique practices observed in play, including one-on-one conversations, supporting small groups, becoming an active play partner, and collaboratively addressing problems that arose in play. These results illustrate ways enactors gave meaning to the concept of inclusion through their play practices, providing salient examples of play alongside teachers' craft knowledge to help support inclusive play-based learning practices going forward.
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BACKGROUND: Neurocognitive impairment (NCI) remains common in people living with human immunodeficiency virus (PLWH), despite suppressive antiretroviral therapy (ART), but the reasons remain incompletely understood. Mitochondrial dysfunction is a hallmark of aging and of neurodegenerative diseases. We hypothesized that human immunodeficiency virus (HIV) or ART may lead to mitochondrial abnormalities in the brain, thus contributing to NCI. METHODS: We studied postmortem frozen brain samples from 52 PLWH and 40 HIV-negative controls. Cellular mitochondrial DNA (mtDNA) content and levels of large-scale mtDNA deletions were measured by real-time polymerase chain reaction. Heteroplasmic mtDNA point mutations were quantified by deep sequencing (Illumina). Neurocognitive data were taken within 48 months antemortem. RESULTS: We observed a decrease in mtDNA content, an increase in the mtDNA "common deletion," and an increase in mtDNA point mutations with age (all Pâ <â .05). Each of these changes was exacerbated in HIV-positive cases compared with HIV-negative controls (all Pâ <â .05). ART exposures, including nucleoside analogue reverse transcriptase inhibitors, were not associated with changes in mtDNA. The number of mtDNA point mutations was associated with low CD4/CD8 ratio (P = .04) and with NCI (global T-score, P = .007). CONCLUSIONS: In people with predominantly advanced HIV infection, there is exacerbation of age-associated mtDNA damage. This change is driven by HIV per se rather than by ART toxicity and may contribute to NCI. These data suggest that mitochondrial dysfunction may be a mediator of adverse aging phenotypes in PLWH.
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Infecciones por VIH , Envejecimiento/genética , Encéfalo , ADN Mitocondrial/genética , VIH , Infecciones por VIH/complicaciones , Humanos , Mitocondrias/genéticaRESUMEN
Recessively inherited variants in AARS2 (NM_020745.2) encoding mitochondrial alanyl-tRNA synthetase (mt-AlaRS) were first described in patients presenting with fatal infantile cardiomyopathy and multiple oxidative phosphorylation defects. To date, all described patients with AARS2-related fatal infantile cardiomyopathy are united by either a homozygous or compound heterozygous c.1774C>T (p.Arg592Trp) missense founder mutation that is absent in patients with other AARS2-related phenotypes. We describe the clinical, biochemical and molecular investigations of two unrelated boys presenting with fatal infantile cardiomyopathy, lactic acidosis and respiratory failure. Oxidative histochemistry showed cytochrome c oxidase-deficient fibres in skeletal and cardiac muscle. Biochemical studies showed markedly decreased activities of mitochondrial respiratory chain complexes I and IV with a mild decrease of complex III activity in skeletal and cardiac muscle. Using next-generation sequencing, we identified a c.1738C>T (p.Arg580Trp) AARS2 variant shared by both patients that was in trans with a loss-of-function heterozygous AARS2 variant; a c.1008dupT (p.Asp337*) nonsense variant or an intragenic deletion encompassing AARS2 exons 5-7. Interestingly, our patients did not harbour the p.Arg592Trp AARS2 founder mutation. In silico modelling of the p.Arg580Trp substitution suggested a deleterious impact on protein stability and folding. We confirmed markedly decreased mt-AlaRS protein levels in patient fibroblasts, skeletal and cardiac muscle, although mitochondrial protein synthesis defects were confined to skeletal and cardiac muscle. In vitro data showed that the p.Arg580Trp variant had a minimal effect on activation, aminoacylation or misaminoacylation activities relative to wild-type mt-AlaRS, demonstrating that instability of mt-AlaRS is the biological mechanism underlying the fatal cardiomyopathy phenotype in our patients.
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Alanina-ARNt Ligasa/metabolismo , Cardiomiopatías/enzimología , Alanina-ARNt Ligasa/genética , Cardiomiopatías/genética , Enfermedades en Gemelos/genética , Estabilidad de Enzimas , Fibroblastos/metabolismo , Genes Recesivos , Humanos , Lactante , Ácido Láctico , Masculino , Mitocondrias/metabolismo , Proteínas Mitocondriales/biosíntesis , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Linaje , Insuficiencia Respiratoria/enzimologíaRESUMEN
Thyroid hormones are regarded as the major controllers of metabolic rate and oxygen consumption in mammals. Although it has been demonstrated that thyroid hormone supplementation improves bovine embryo development in vitro, the cellular mechanisms underlying these effects are so far unknown. In this study, we investigated the role of thyroid hormone in development of human preimplantation embryos. Embryos were cultured in the presence or absence of 10-7 M triiodothyronine (T3) till blastocyst stage. Inner cell mass (ICM) and trophectoderm (TE) were separated mechanically and subjected to RNAseq or quantification of mitochondrial DNA copy number. Analyses were performed using DESeq (v1.16.0 on R v3.1.3), MeV4.9 and MitoMiner 4.0v2018 JUN platforms. We found that the exposure of human preimplantation embryos to T3 had a profound impact on nuclear gene transcription only in the cells of ICM (1178 regulated genes-10.5% of 11 196 expressed genes) and almost no effect on cells of TE (38 regulated genes-0.3% of expressed genes). The analyses suggest that T3 induces in ICM a shift in ribosome and oxidative phosphorylation activity, as the upregulated genes are contributing to the composition and organization of the respiratory chain and associated cofactors involved in mitoribosome assembly and stability. Furthermore, a number of genes affecting the citric acid cycle energy production have reduced expression. Our findings might explain why thyroid disorders in women have been associated with reduced fertility and adverse pregnancy outcome. Our data also raise a possibility that supplementation of culture media with T3 may improve outcomes for women undergoing in vitro fertilization.
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Blastocisto/metabolismo , Mitocondrias/metabolismo , Hormonas Tiroideas/metabolismo , Femenino , Humanos , Fosforilación Oxidativa , EmbarazoRESUMEN
LonP1 is a mitochondrial matrix protease whose selective substrate specificity is essential for maintaining mitochondrial homeostasis. Recessively inherited, pathogenic defects in LonP1 have been previously reported to underlie cerebral, ocular, dental, auricular and skeletal anomalies (CODAS) syndrome, a complex multisystemic and developmental disorder. Intriguingly, although classical mitochondrial disease presentations are well-known to exhibit marked clinical heterogeneity, the skeletal and dental features associated with CODAS syndrome are pathognomonic. We have applied whole exome sequencing to a patient with congenital lactic acidosis, muscle weakness, profound deficiencies in mitochondrial oxidative phosphorylation associated with loss of mtDNA copy number and MRI abnormalities consistent with Leigh syndrome, identifying biallelic variants in the LONP1 (NM_004793.3) gene; c.1693T > C predicting p.(Tyr565His) and c.2197G > A predicting p.(Glu733Lys); no evidence of the classical skeletal or dental defects observed in CODAS syndrome patients were noted in our patient. In vitro experiments confirmed the p.(Tyr565His) LonP1 mutant alone could not bind or degrade a substrate, consistent with the predicted function of Tyr565, whilst a second missense [p.(Glu733Lys)] variant had minimal effect. Mixtures of p.(Tyr565His) mutant and wild-type LonP1 retained partial protease activity but this was severely depleted when the p.(Tyr565His) mutant was mixed with the p.(Glu733Lys) mutant, data consistent with the compound heterozygosity detected in our patient. In summary, we conclude that pathogenic LONP1 variants can lead to a classical mitochondrial disease presentations associated with severe biochemical defects in oxidative phosphorylation in clinically relevant tissues.
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Proteasas ATP-Dependientes/genética , Anomalías Craneofaciales/genética , Anomalías del Ojo/genética , Trastornos del Crecimiento/genética , Luxación Congénita de la Cadera/genética , Enfermedad de Leigh/genética , Enfermedades Mitocondriales/genética , Proteínas Mitocondriales/genética , Osteocondrodisplasias/genética , Anomalías Dentarias/genética , Biopsia , Línea Celular , Anomalías Craneofaciales/metabolismo , Anomalías Craneofaciales/fisiopatología , Exoma/genética , Anomalías del Ojo/metabolismo , Anomalías del Ojo/fisiopatología , Trastornos del Crecimiento/metabolismo , Trastornos del Crecimiento/fisiopatología , Luxación Congénita de la Cadera/metabolismo , Luxación Congénita de la Cadera/fisiopatología , Humanos , Lactante , Enfermedad de Leigh/metabolismo , Enfermedad de Leigh/fisiopatología , Masculino , Mitocondrias/genética , Mitocondrias/patología , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/fisiopatología , Músculo Esquelético/fisiopatología , Mutación , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/fisiopatología , Fosforilación Oxidativa , Anomalías Dentarias/metabolismo , Anomalías Dentarias/fisiopatología , Secuenciación del ExomaRESUMEN
Mitochondrial dynamics play an important role in cellular homeostasis and a variety of human diseases are linked to its dysregulated function. Here, we describe a 15-year-old boy with a novel disease caused by altered mitochondrial dynamics. The patient was the second child of consanguineous Jewish parents. He developed progressive muscle weakness and exercise intolerance at 6 years of age. His muscle biopsy revealed mitochondrial myopathy with numerous ragged red and cytochrome c oxidase (COX) negative fibers and combined respiratory chain complex I and IV deficiency. MtDNA copy number was elevated and no deletions of the mtDNA were detected in muscle DNA. Whole exome sequencing identified a homozygous nonsense mutation (p.Q92*) in the MIEF2 gene encoding the mitochondrial dynamics protein of 49 kDa (MID49). Immunoblotting revealed increased levels of proteins promoting mitochondrial fusion (MFN2, OPA1) and decreased levels of the fission protein DRP1. Fibroblasts of the patient showed elongated mitochondria, and significantly higher frequency of fusion events, mtDNA abundance and aberrant mitochondrial cristae ultrastructure, compared with controls. Thus, our data suggest that mutations in MIEF2 result in imbalanced mitochondrial dynamics and a combined respiratory chain enzyme defect in skeletal muscle, leading to mitochondrial myopathy.
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Fibroblastos/metabolismo , Dinámicas Mitocondriales/genética , Proteínas Mitocondriales , Enfermedades Musculares , Mutación Missense , Factores de Elongación de Péptidos , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Femenino , Fibroblastos/patología , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Humanos , Masculino , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Enfermedades Musculares/genética , Enfermedades Musculares/metabolismo , Enfermedades Musculares/patología , Factores de Elongación de Péptidos/genética , Factores de Elongación de Péptidos/metabolismo , Cultivo Primario de CélulasRESUMEN
TATA-box binding protein associated factor, RNA polymerase I subunit C (TAF1C) is a component of selectivity factor 1 belonging to RNA polymerase I (Pol I) transcription machinery. We report two unrelated patients with homozygous TAF1C missense variants and an early onset neurological phenotype with severe global developmental delay. Clinical features included lack of speech and ambulation and epilepsy. MRI of the brain demonstrated widespread cerebral atrophy and frontal periventricular white matter hyperintensity. The phenotype resembled that of a previously described variant of UBTF, which encodes another transcription factor of Pol I. TAF1C variants were located in two conserved amino acid positions and were predicted to be deleterious. In patient-derived fibroblasts, TAF1C mRNA and protein expression levels were substantially reduced compared with healthy controls. We propose that the variants impairing TAF1C expression are likely pathogenic and relate to a novel neurological disease. This study expands the disease spectrum related to Pol I transcription machinery, associating the TAF1C missense variants with a severe neurological phenotype for the first time.
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Epilepsia/genética , ARN Polimerasa I/genética , Espasmos Infantiles/genética , Factores Asociados con la Proteína de Unión a TATA/genética , Factor de Transcripción TFIID/genética , Preescolar , Epilepsia/diagnóstico por imagen , Epilepsia/patología , Femenino , Fibroblastos/metabolismo , Homocigoto , Humanos , Lactante , Recién Nacido , Imagen por Resonancia Magnética , Masculino , Mutación Missense/genética , Fenotipo , Espasmos Infantiles/diagnóstico por imagen , Espasmos Infantiles/patologíaRESUMEN
BACKGROUND: Mitochondrial DNA copy number (mtDNA CN) exhibits interindividual and intercellular variation, but few genome-wide association studies (GWAS) of directly assayed mtDNA CN exist. We undertook a GWAS of qPCR-assayed mtDNA CN in the Avon Longitudinal Study of Parents and Children (ALSPAC) and the UK Blood Service (UKBS) cohort. After validating and harmonising data, 5461 ALSPAC mothers (16-43 years at mtDNA CN assay) and 1338 UKBS females (17-69 years) were included in a meta-analysis. Sensitivity analyses restricted to females with white cell-extracted DNA and adjusted for estimated or assayed cell proportions. Associations were also explored in ALSPAC children and UKBS males. RESULTS: A neutrophil-associated locus approached genome-wide significance (rs709591 [MED24], ß (change in SD units of mtDNA CN per allele) [SE] - 0.084 [0.016], p = 1.54e-07) in the main meta-analysis of adult females. This association was concordant in magnitude and direction in UKBS males and ALSPAC neonates. SNPs in and around ABHD8 were associated with mtDNA CN in ALSPAC neonates (rs10424198, ß [SE] 0.262 [0.034], p = 1.40e-14), but not other study groups. In a meta-analysis of unrelated individuals (N = 11,253), we replicated a published association in TFAM (ß [SE] 0.046 [0.017], p = 0.006), with an effect size much smaller than that observed in the replication analysis of a previous in silico GWAS. CONCLUSIONS: In a hypothesis-generating GWAS, we confirm an association between TFAM and mtDNA CN and present putative loci requiring replication in much larger samples. We discuss the limitations of our work, in terms of measurement error and cellular heterogeneity, and highlight the need for larger studies to better understand nuclear genomic control of mtDNA copy number.
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Variaciones en el Número de Copia de ADN , ADN Mitocondrial/genética , Estudio de Asociación del Genoma Completo/métodos , Adolescente , Adulto , Niño , Estudios de Cohortes , Femenino , Humanos , Recién Nacido , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido SimpleRESUMEN
Mitochondrial disease is hugely diverse with respect to associated clinical presentations and underlying genetic causes, with pathogenic variants in over 300 disease genes currently described. Approximately half of these have been discovered in the last decade due to the increasingly widespread application of next generation sequencing technologies, in particular unbiased, whole exome-and latterly, whole genome sequencing. These technologies allow more genetic data to be collected from patients with mitochondrial disorders, continually improving the diagnostic success rate in a clinical setting. Despite these significant advances, some patients still remain without a definitive genetic diagnosis. Large datasets containing many variants of unknown significance have become a major challenge with next generation sequencing strategies and these require significant functional validation to confirm pathogenicity. This interface between diagnostics and research is critical in continuing to expand the list of known pathogenic variants and concomitantly enhance our knowledge of mitochondrial biology. The increasing use of whole exome sequencing, whole genome sequencing and other "omics" techniques such as transcriptomics and proteomics will generate even more data and allow further interrogation and validation of genetic causes, including those outside of coding regions. This will improve diagnostic yields still further and emphasizes the integral role that functional assessment of variant causality plays in this process-the overarching focus of this review.
Asunto(s)
Secuenciación del Exoma/métodos , Genoma Mitocondrial , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Enfermedades Mitocondriales/diagnóstico , Técnicas de Diagnóstico Molecular , Humanos , Enfermedades Mitocondriales/genética , Análisis de Secuencia de ARN , TranscriptomaRESUMEN
Transport And Golgi Organization protein 2 (TANGO2) deficiency has recently been identified as a rare metabolic disorder with a distinct clinical and biochemical phenotype of recurrent metabolic crises, hypoglycemia, lactic acidosis, rhabdomyolysis, arrhythmias, and encephalopathy with cognitive decline. We report nine subjects from seven independent families, and we studied muscle histology, respiratory chain enzyme activities in skeletal muscle and proteomic signature of fibroblasts. All nine subjects carried autosomal recessive TANGO2 mutations. Two carried the reported deletion of exons 3 to 9, one homozygous, one heterozygous with a 22q11.21 microdeletion inherited in trans. The other subjects carried three novel homozygous (c.262C>T/p.Arg88*; c.220A>C/p.Thr74Pro; c.380+1G>A), and two further novel heterozygous (c.6_9del/p.Phe6del); c.11-13delTCT/p.Phe5del mutations. Immunoblot analysis detected a significant decrease of TANGO2 protein. Muscle histology showed mild variation of fiber diameter, no ragged-red/cytochrome c oxidase-negative fibers and a defect of multiple respiratory chain enzymes and coenzyme Q10 (CoQ10 ) in two cases, suggesting a possible secondary defect of oxidative phosphorylation. Proteomic analysis in fibroblasts revealed significant changes in components of the mitochondrial fatty acid oxidation, plasma membrane, endoplasmic reticulum-Golgi network and secretory pathways. Clinical presentation of TANGO2 mutations is homogeneous and clinically recognizable. The hemizygous mutations in two patients suggest that some mutations leading to allele loss are difficult to detect. A combined defect of the respiratory chain enzymes and CoQ10 with altered levels of several membrane proteins provides molecular insights into the underlying pathophysiology and may guide rational new therapeutic interventions.
Asunto(s)
Encefalopatías Metabólicas/genética , Enfermedades Mitocondriales/genética , Debilidad Muscular/genética , Mutación , Proteómica/métodos , Rabdomiólisis/genética , Encefalopatías Metabólicas/diagnóstico , Ácidos Grasos/metabolismo , Femenino , Aparato de Golgi/genética , Aparato de Golgi/metabolismo , Homocigoto , Humanos , Lactante , Masculino , Enfermedades Mitocondriales/diagnóstico , Fosforilación Oxidativa , Fenotipo , Rabdomiólisis/diagnóstico , Secuenciación Completa del GenomaRESUMEN
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.