RESUMEN
OBJECTIVE: This observational cohort study aims to quantify disease burden over time, establish disease progression rates, and identify factors that may determine the disease course of Leigh syndrome. METHODS: Seventy-two Leigh syndrome children who completed the Newcastle Paediatric Mitochondrial Disease Scale (NPMDS) at baseline at 3.7 years (interquartile range [IQR] = 2.0-7.6) and follow-up assessments at 7.5 years (IQR = 3.7-11.0) in clinics were enrolled. Eighty-two percent of this cohort had a confirmed genetic diagnosis, with pathogenic variants in the MT-ATP6 and SURF1 genes being the most common cause. The total NPMDS scores denoted mild (0-14), moderate (15-25), and severe (>25) disease burden. Detailed clinical, neuroradiological, and molecular genetic findings were also analyzed. RESULTS: The median total NPMDS scores rose significantly (Z = -6.9, p < 0.001), and the percentage of children with severe disease burden doubled (22% â 42%) over 2.6 years of follow-up. Poor function (especially mobility, self-care, communication, feeding, and education) and extrapyramidal features contributed significantly to the disease burden (τb ≈ 0.45-0.68, p < 0.001). These children also deteriorated to wheelchair dependence (31% â 57%), exclusive enteral feeding (22% â 46%), and one-to-one assistance for self-care (25% â 43%) during the study period. Twelve children (17%) died after their last NPMDS scores were recorded. These children had higher follow-up NPMDS scores (disease burden; p < 0.001) and steeper increase in NPMDS score per annum (disease progression; p < 0.001). Other predictors of poor outcomes include SURF1 gene variants (p < 0.001) and bilateral caudate changes on neuroimaging (p < 0.01). INTERPRETATION: This study has objectively defined the disease burden and progression of Leigh syndrome. Our analysis has also uncovered potential influences on the trajectory of this neurodegenerative condition. ANN NEUROL 2022;91:117-130.
Asunto(s)
Enfermedad de Leigh , Niño , Preescolar , Estudios de Cohortes , Costo de Enfermedad , Progresión de la Enfermedad , Femenino , Humanos , Lactante , Estudios Longitudinales , MasculinoRESUMEN
In this retrospective, multicentre, observational cohort study, we sought to determine the clinical, radiological, EEG, genetics and neuropathological characteristics of mitochondrial stroke-like episodes and to identify associated risk predictors. Between January 1998 and June 2018, we identified 111 patients with genetically determined mitochondrial disease who developed stroke-like episodes. Post-mortem cases of mitochondrial disease (n = 26) were identified from Newcastle Brain Tissue Resource. The primary outcome was to interrogate the clinico-radiopathological correlates and prognostic indicators of stroke-like episode in patients with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (MELAS). The secondary objective was to develop a multivariable prediction model to forecast stroke-like episode risk. The most common genetic cause of stroke-like episodes was the m.3243A>G variant in MT-TL1 (n = 66), followed by recessive pathogenic POLG variants (n = 22), and 11 other rarer pathogenic mitochondrial DNA variants (n = 23). The age of first stroke-like episode was available for 105 patients [mean (SD) age: 31.8 (16.1)]; a total of 35 patients (32%) presented with their first stroke-like episode ≥40 years of age. The median interval (interquartile range) between first and second stroke-like episodes was 1.33 (2.86) years; 43% of patients developed recurrent stroke-like episodes within 12 months. Clinico-radiological, electrophysiological and neuropathological findings of stroke-like episodes were consistent with the hallmarks of medically refractory epilepsy. Patients with POLG-related stroke-like episodes demonstrated more fulminant disease trajectories than cases of m.3243A>G and other mitochondrial DNA pathogenic variants, in terms of the frequency of refractory status epilepticus, rapidity of progression and overall mortality. In multivariate analysis, baseline factors of body mass index, age-adjusted blood m.3243A>G heteroplasmy, sensorineural hearing loss and serum lactate were significantly associated with risk of stroke-like episodes in patients with the m.3243A>G variant. These factors informed the development of a prediction model to assess the risk of developing stroke-like episodes that demonstrated good overall discrimination (area under the curve = 0.87, 95% CI 0.82-0.93; c-statistic = 0.89). Significant radiological and pathological features of neurodegeneration were more evident in patients harbouring pathogenic mtDNA variants compared with POLG: brain atrophy on cranial MRI (90% versus 44%, P < 0.001) and reduced mean brain weight (SD) [1044 g (148) versus 1304 g (142), P = 0.005]. Our findings highlight the often idiosyncratic clinical, radiological and EEG characteristics of mitochondrial stroke-like episodes. Early recognition of seizures and aggressive instigation of treatment may help circumvent or slow neuronal loss and abate increasing disease burden. The risk-prediction model for the m.3243A>G variant can help inform more tailored genetic counselling and prognostication in routine clinical practice.
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Síndrome MELAS , Enfermedades Mitocondriales , Accidente Cerebrovascular , Adulto , ADN Mitocondrial/genética , Humanos , Síndrome MELAS/genética , Enfermedades Mitocondriales/complicaciones , Enfermedades Mitocondriales/genética , Mutación , Estudios Retrospectivos , Accidente Cerebrovascular/diagnóstico por imagen , Accidente Cerebrovascular/genéticaRESUMEN
Mutations in nuclear-encoded protein subunits of the mitochondrial ribosome are an increasingly recognised cause of oxidative phosphorylation system (OXPHOS) disorders. Among them, mutations in the MRPL44 gene, encoding a structural protein of the large subunit of the mitochondrial ribosome, have been identified in four patients with OXPHOS defects and early-onset hypertrophic cardiomyopathy with or without additional clinical features. A 23-year-old individual with cardiac and skeletal myopathy, neurological involvement, and combined deficiency of OXPHOS complexes in skeletal muscle was clinically and genetically investigated. Analysis of whole-exome sequencing data revealed a homozygous mutation in MRPL44 (c.467 T > G), which was not present in the biological father, and a region of homozygosity involving most of chromosome 2, raising the possibility of uniparental disomy. Short-tandem repeat and genome-wide SNP microarray analyses of the family trio confirmed complete maternal uniparental isodisomy of chromosome 2. Mitochondrial ribosome assembly and mitochondrial translation were assessed in patient derived-fibroblasts. These studies confirmed that c.467 T > G affects the stability or assembly of the large subunit of the mitochondrial ribosome, leading to impaired mitochondrial protein synthesis and decreased levels of multiple OXPHOS components. This study provides evidence of complete maternal uniparental isodisomy of chromosome 2 in a patient with MRPL44-related disease, and confirms that MRLP44 mutations cause a mitochondrial translation defect that may present as a multisystem disorder with neurological involvement.
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Cromosomas Humanos Par 2/genética , Enfermedades Mitocondriales/genética , Proteínas Mitocondriales/genética , Proteínas Ribosómicas/genética , Disomía Uniparental/genética , Adolescente , Secuencia de Bases , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Preescolar , Femenino , Fibroblastos/patología , Homocigoto , Humanos , Lactante , Recién Nacido , Imagen por Resonancia Magnética , Enfermedades Mitocondriales/patología , Músculo Esquelético/metabolismo , Mutación/genética , Fosforilación Oxidativa , Biosíntesis de Proteínas , Adulto JovenRESUMEN
Functioning mitochondria are crucial for cancer metabolism, but aerobic glycolysis is still considered to be an important pathway for energy production in many tumor cells. Here we show that two well established, classic Hodgkin lymphoma (cHL) cell lines harbor deleterious variants within mitochondrial DNA (mtDNA) and thus exhibit reduced steady-state levels of respiratory chain complexes. However, instead of resulting in the expected bioenergetic defect, these mtDNA variants evoke a retrograde signaling response that induces mitochondrial biogenesis and ultimately results in increased mitochondrial mass as well as function and enhances proliferation in vitro as well as tumor growth in mice in vivo. When complex I assembly was impaired by knockdown of one of its subunits, this led to further increased mitochondrial mass and function and, consequently, further accelerated tumor growth in vivo. In contrast, inhibition of mitochondrial respiration in vivo by the mitochondrial complex I inhibitor metformin efficiently slowed down growth. We conclude that, as a new mechanism, mildly deleterious mtDNA variants in cHL cancer cells cause an increase of mitochondrial mass and enhanced function as a compensatory effect using a retrograde signaling pathway, which provides an obvious advantage for tumor growth.
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Carcinogénesis/patología , ADN Mitocondrial/genética , Enfermedad de Hodgkin/patología , Mutación , Biogénesis de Organelos , Animales , Apoptosis , Carcinogénesis/genética , Carcinogénesis/metabolismo , Proliferación Celular , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/metabolismo , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Fosforilación Oxidativa , Células de Reed-Sternberg , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Autosomal dominant progressive external ophthalmoplegia (adPEO) is a late-onset, Mendelian mitochondrial disorder characterised by paresis of the extraocular muscles, ptosis, and skeletal-muscle restricted multiple mitochondrial DNA (mtDNA) deletions. Although dominantly inherited, pathogenic variants in POLG, TWNK and RRM2B are among the most common genetic defects of adPEO, identification of novel candidate genes and the underlying pathomechanisms remains challenging. We report the clinical, genetic and molecular investigations of a patient who presented in the seventh decade of life with PEO. Oxidative histochemistry revealed cytochrome c oxidase-deficient fibres and occasional ragged red fibres showing subsarcolemmal mitochondrial accumulation in skeletal muscle, while molecular studies identified the presence of multiple mtDNA deletions. Negative candidate screening of known nuclear genes associated with PEO prompted diagnostic exome sequencing, leading to the prioritisation of a novel heterozygous c.547G>C variant in GMPR (NM_006877.3) encoding guanosine monophosphate reductase, a cytosolic enzyme required for maintaining the cellular balance of adenine and guanine nucleotides. We show that the novel c.547G>C variant causes aberrant splicing, decreased GMPR protein levels in patient skeletal muscle, proliferating and quiescent cells, and is associated with subtle changes in nucleotide homeostasis protein levels and evidence of disturbed mtDNA maintenance in skeletal muscle. Despite confirmation of GMPR deficiency, demonstrating marked defects of mtDNA replication or nucleotide homeostasis in patient cells proved challenging. Our study proposes that GMPR is the 19th locus for PEO and highlights the complexities of uncovering disease mechanisms in late-onset PEO phenotypes.
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ADN Mitocondrial/genética , GMP-Reductasa/genética , Enfermedades de Inicio Tardío/genética , Músculo Esquelético/enzimología , Oftalmoplejía/genética , Adenina/metabolismo , Anciano , Células Cultivadas , Deficiencia de Citocromo-c Oxidasa/metabolismo , Replicación del ADN , ADN Mitocondrial/metabolismo , Femenino , Fibroblastos/enzimología , GMP-Reductasa/deficiencia , GMP-Reductasa/metabolismo , Guanina/metabolismo , Células HEK293 , Células HeLa , Heterocigoto , Humanos , Enfermedades de Inicio Tardío/metabolismo , Enfermedades de Inicio Tardío/patología , Músculo Esquelético/patología , Oftalmoplejía/enzimología , Oftalmoplejía/fisiopatología , Fosforilación Oxidativa , Empalme del ARN , Eliminación de Secuencia , Secuenciación del ExomaRESUMEN
Distinct clinical syndromes have been associated with pathogenic MT-ATP6 variants. In this cohort study, we identified 125 individuals (60 families) including 88 clinically affected individuals and 37 asymptomatic carriers. Thirty-one individuals presented with Leigh syndrome and 7 with neuropathy ataxia retinitis pigmentosa. The remaining 50 patients presented with variable nonsyndromic features including ataxia, neuropathy, and learning disability. We confirmed maternal inheritance in 39 families and demonstrated that tissue segregation patterns and phenotypic threshold are variant dependent. Our findings suggest that MT-ATP6-related mitochondrial DNA disease is best conceptualized as a mitochondrial disease spectrum disorder and should be routinely included in genetic ataxia and neuropathy gene panels. ANN NEUROL 2019;86:310-315.
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Variación Genética/genética , Enfermedades Mitocondriales/epidemiología , Enfermedades Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Enfermedades Mitocondriales/diagnóstico , Reino Unido/epidemiología , Adulto JovenRESUMEN
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.
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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
OBJECTIVE: Single, large-scale deletions in mitochondrial DNA (mtDNA) are a common cause of mitochondrial disease. This study aimed to investigate the relationship between the genetic defect and molecular phenotype to improve understanding of pathogenic mechanisms associated with single, large-scale mtDNA deletions in skeletal muscle. METHODS: We investigated 23 muscle biopsies taken from adult patients (6 males/17 females with a mean age of 43 years) with characterized single, large-scale mtDNA deletions. Mitochondrial respiratory chain deficiency in skeletal muscle biopsies was quantified by immunoreactivity levels for complex I and complex IV proteins. Single muscle fibers with varying degrees of deficiency were selected from 6 patient biopsies for determination of mtDNA deletion level and copy number by quantitative polymerase chain reaction. RESULTS: We have defined 3 "classes" of single, large-scale deletion with distinct patterns of mitochondrial deficiency, determined by the size and location of the deletion. Single fiber analyses showed that fibers with greater respiratory chain deficiency harbored higher levels of mtDNA deletion with an increase in total mtDNA copy number. For the first time, we have demonstrated that threshold levels for complex I and complex IV deficiency differ based on deletion class. INTERPRETATION: Combining genetic and immunofluorescent assays, we conclude that thresholds for complex I and complex IV deficiency are modulated by the deletion of complex-specific protein-encoding genes. Furthermore, removal of mt-tRNA genes impacts specific complexes only at high deletion levels, when complex-specific protein-encoding genes remain. These novel findings provide valuable insight into the pathogenic mechanisms associated with these mutations. Ann Neurol 2018;83:115-130.
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ADN Mitocondrial/genética , Enfermedades Mitocondriales/genética , Eliminación de Secuencia/genética , Adulto , Anciano , Biopsia , Estudios de Cohortes , Complejo I de Transporte de Electrón/genética , Complejo IV de Transporte de Electrones/genética , Femenino , Eliminación de Gen , Dosificación de Gen , Humanos , Masculino , Persona de Mediana Edad , Enfermedades Mitocondriales/patología , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/patología , Fosforilación Oxidativa , Adulto JovenRESUMEN
PURPOSE: Biallelic mutations in SCYL1 were recently identified as causing a syndromal disorder characterized by peripheral neuropathy, cerebellar atrophy, ataxia, and recurrent episodes of liver failure. The occurrence of SCYL1 deficiency among patients with previously undetermined infantile cholestasis or acute liver failure has not been studied; furthermore, little is known regarding the hepatic phenotype. METHODS: We aimed to identify patients with SCYL1 variants within an exome-sequencing study of individuals with infantile cholestasis or acute liver failure of unknown etiology. Deep clinical and biochemical phenotyping plus analysis of liver biopsies and functional studies on fibroblasts were performed. RESULTS: Seven patients from five families with biallelic SCYL1 variants were identified. The main clinical phenotype was recurrent low γ-glutamyl-transferase (GGT) cholestasis or acute liver failure with onset in infancy and a variable neurological phenotype of later onset (CALFAN syndrome). Liver crises were triggered by febrile infections and were transient, but fibrosis developed. Functional studies emphasize that SCYL1 deficiency is linked to impaired intracellular trafficking. CONCLUSION: SCYL1 deficiency can cause recurrent low-GGT cholestatic liver dysfunction in conjunction with a variable neurological phenotype. Like NBAS deficiency, it is a member of the emerging group of congenital disorders of intracellular trafficking causing hepatopathy.
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Colestasis/genética , Fallo Hepático Agudo/genética , Degeneración Nerviosa/genética , Factores de Transcripción/genética , Proteínas Adaptadoras del Transporte Vesicular , Alelos , Niño , Preescolar , Colestasis/complicaciones , Colestasis/diagnóstico , Colestasis/patología , Proteínas de Unión al ADN , Exoma/genética , Femenino , Humanos , Lactante , Fallo Hepático Agudo/complicaciones , Fallo Hepático Agudo/diagnóstico , Fallo Hepático Agudo/patología , Masculino , Mutación , Degeneración Nerviosa/complicaciones , Degeneración Nerviosa/diagnóstico , Degeneración Nerviosa/patología , gamma-Glutamiltransferasa/genéticaRESUMEN
Mitochondrial DNA (mtDNA) rearrangements are an important cause of mitochondrial disease and age related mitochondrial dysfunction in tissues including brain and skeletal muscle. It is known that different mtDNA deletions accumulate in single cells, but the detailed nature of these rearrangements is still unknown. To evaluate this we used a complementary set of sensitive assays to explore the mtDNA rearrangements in individual cells from patients with sporadic inclusion body myositis, a late-onset inflammatory myopathy with prominent mitochondrial changes. We identified large-scale mtDNA deletions in individual muscle fibres with 20% of cytochrome c oxidase-deficient myofibres accumulating two or more mtDNA deletions. The majority of deletions removed only the major arc but â¼10% of all deletions extended into the minor arc removing the origin of light strand replication (OL) and a variable number of genes. Some mtDNA molecules contained two deletion sites. Additionally, we found evidence of mitochondrial genome duplications allowing replication and clonal expansion of these complex rearranged molecules. The extended spectrum of mtDNA rearrangements in single cells provides insight into the process of clonal expansion which is fundamental to our understanding of the role of mtDNA mutations in ageing and disease.
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ADN Mitocondrial , Reordenamiento Génico , Miositis por Cuerpos de Inclusión/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores , Biopsia , Niño , Femenino , Genoma Mitocondrial , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Miositis por Cuerpos de Inclusión/patología , Eliminación de Secuencia , Adulto JovenRESUMEN
Identifying the genetic basis for mitochondrial diseases is technically challenging given the size of the mitochondrial proteome and the heterogeneity of disease presentations. Using next-generation exome sequencing, we identified in a patient with severe combined mitochondrial respiratory chain defects and corresponding perturbation in mitochondrial protein synthesis, a homozygous p.Arg323Gln mutation in TRIT1. This gene encodes human tRNA isopentenyltransferase, which is responsible for i6A37 modification of the anticodon loops of a small subset of cytosolic and mitochondrial tRNAs. Deficiency of i6A37 was previously shown in yeast to decrease translational efficiency and fidelity in a codon-specific manner. Modelling of the p.Arg323Gln mutation on the co-crystal structure of the homologous yeast isopentenyltransferase bound to a substrate tRNA, indicates that it is one of a series of adjacent basic side chains that interact with the tRNA backbone of the anticodon stem, somewhat removed from the catalytic center. We show that patient cells bearing the p.Arg323Gln TRIT1 mutation are severely deficient in i6A37 in both cytosolic and mitochondrial tRNAs. Complete complementation of the i6A37 deficiency of both cytosolic and mitochondrial tRNAs was achieved by transduction of patient fibroblasts with wild-type TRIT1. Moreover, we show that a previously-reported pathogenic m.7480A>G mt-tRNASer(UCN) mutation in the anticodon loop sequence A36A37A38 recognised by TRIT1 causes a loss of i6A37 modification. These data demonstrate that deficiencies of i6A37 tRNA modification should be considered a potential mechanism of human disease caused by both nuclear gene and mitochondrial DNA mutations while providing insight into the structure and function of TRIT1 in the modification of cytosolic and mitochondrial tRNAs.
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Transferasas Alquil y Aril/genética , Enfermedades Mitocondriales/genética , Sulfurtransferasas/genética , Células Cultivadas , Deficiencia de Citocromo-c Oxidasa/genética , Citosol , ADN Mitocondrial/genética , Transporte de Electrón/genética , Complejo IV de Transporte de Electrones/genética , Femenino , Humanos , Masculino , Mitocondrias/genética , Biosíntesis de Proteínas/genética , ARN/genética , ARN Mitocondrial , ARN de Transferencia/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
OBJECTIVE: The prevalence of mitochondrial disease has proven difficult to establish, predominantly as a result of clinical and genetic heterogeneity. The phenotypic spectrum of mitochondrial disease has expanded significantly since the original reports that associated classic clinical syndromes with mitochondrial DNA (mtDNA) rearrangements and point mutations. The revolution in genetic technologies has allowed interrogation of the nuclear genome in a manner that has dramatically improved the diagnosis of mitochondrial disorders. We comprehensively assessed the prevalence of all forms of adult mitochondrial disease to include pathogenic mutations in both nuclear and mtDNA. METHODS: Adults with suspected mitochondrial disease in the North East of England were referred to a single neurology center from 1990 to 2014. For the midyear period of 2011, we evaluated the minimum prevalence of symptomatic nuclear DNA mutations and symptomatic and asymptomatic mtDNA mutations causing mitochondrial diseases. RESULTS: The minimum prevalence rate for mtDNA mutations was 1 in 5,000 (20 per 100,000), comparable with our previously published prevalence rates. In this population, nuclear mutations were responsible for clinically overt adult mitochondrial disease in 2.9 per 100,000 adults. INTERPRETATION: Combined, our data confirm that the total prevalence of adult mitochondrial disease, including pathogenic mutations of both the mitochondrial and nuclear genomes (≈1 in 4,300), is among the commonest adult forms of inherited neurological disorders. These figures hold important implications for the evaluation of interventions, provision of evidence-based health policies, and planning of future services.
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Núcleo Celular/genética , ADN Mitocondrial/genética , Enfermedades Mitocondriales/epidemiología , Enfermedades Mitocondriales/genética , Mutación/genética , Adulto , Estudios Transversales , Inglaterra/epidemiología , Humanos , Enfermedades Mitocondriales/diagnóstico , Prevalencia , Adulto JovenRESUMEN
Progressive myopathy is a major clinical feature of patients with mitochondrial DNA (mtDNA) disease. There is limited treatment available for these patients although exercise and other approaches to activate muscle stem cells (satellite cells) have been proposed. The majority of mtDNA defects are heteroplasmic (a mixture of mutated and wild-type mtDNA present within the muscle) with high levels of mutated mtDNA and low levels of wild-type mtDNA associated with more severe disease. The culture of satellite cell-derived myoblasts often reveals no evidence of the original mtDNA mutation although it is not known if this is lost by selection or simply not present in these cells. We have explored if the mtDNA mutation is present in the satellite cells in one of the commonest genotypes associated with mitochondrial myopathies (patients with single, large-scale mtDNA deletions). Analysis of satellite cells from eight patients showed that the level of mtDNA mutation in the satellite cells is the same as in the mature muscle but is most often subsequently lost during culture. We show that there are two periods of selection against the mutated form, one early on possibly during satellite cell activation and the other during the rapid replication phase of myoblast culture. Our data suggest that the mutations are also lost during rapid replication in vivo, implying that strategies to activate satellite cells remain a viable treatment for mitochondrial myopathies in specific patient groups.
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ADN Mitocondrial/genética , Mitocondrias/genética , Miopatías Mitocondriales/genética , Células Satélite del Músculo Esquelético/metabolismo , Adulto , Variaciones en el Número de Copia de ADN , Femenino , Eliminación de Gen , Variación Genética , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/patología , Miopatías Mitocondriales/terapia , Fibras Musculares Esqueléticas/metabolismo , Mutación , NADH Deshidrogenasa/genética , ARN Ribosómico 18S/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Succinate dehydrogenase (SDH) is a crucial metabolic enzyme complex that is involved in ATP production, playing roles in both the tricarboxylic cycle and the mitochondrial respiratory chain (complex II). Isolated complex II deficiency is one of the rarest oxidative phosphorylation disorders with mutations described in three structural subunits and one of the assembly factors; just one case is attributed to recessively inherited SDHD mutations. We report the pathological, biochemical, histochemical and molecular genetic investigations of a male neonate who had left ventricular hypertrophy detected on antenatal scan and died on day one of life. Subsequent postmortem examination confirmed hypertrophic cardiomyopathy with left ventricular non-compaction. Biochemical analysis of his skeletal muscle biopsy revealed evidence of a severe isolated complex II deficiency and candidate gene sequencing revealed a novel homozygous c.275A>G, p.(Asp92Gly) SDHD mutation which was shown to be recessively inherited through segregation studies. The affected amino acid has been reported as a Dutch founder mutation p.(Asp92Tyr) in families with hereditary head and neck paraganglioma. By introducing both mutations into Saccharomyces cerevisiae, we were able to confirm that the p.(Asp92Gly) mutation causes a more severe oxidative growth phenotype than the p.(Asp92Tyr) mutant, and provides functional evidence to support the pathogenicity of the patient's SDHD mutation. This is only the second case of mitochondrial complex II deficiency due to inherited SDHD mutations and highlights the importance of sequencing all SDH genes in patients with biochemical and histochemical evidence of isolated mitochondrial complex II deficiency.
Asunto(s)
Cardiomiopatía Hipertrófica Familiar/genética , Genes Recesivos , Cardiopatías Congénitas/genética , Homocigoto , Proteínas Mitocondriales/genética , Mutación Missense , Succinato Deshidrogenasa/genética , Sustitución de Aminoácidos , Cardiomiopatía Hipertrófica Familiar/enzimología , Ciclo del Ácido Cítrico/genética , Cardiopatías Congénitas/enzimología , Humanos , Recién Nacido , MasculinoRESUMEN
Complex I (CI) is the largest of the five multi-subunit complexes constituting the human oxidative phosphorylation (OXPHOS) system. Seven of its catalytic core subunits are encoded by mitochondrial DNA (ND (NADH dehydrogenase)1-6, ND4L (NADH dehydrogenase subunit 4L)), with mutations in all seven having been reported in association with isolated CI deficiency. We investigated two unrelated adult patients presenting with marked exercise intolerance, persistent lactic acidaemia and severe muscle-restricted isolated CI deficiency associated with sub-sarcolemmal mitochondrial accumulation. Screening of the mitochondrial genome detected novel mutations in the MTND1 (NADH dehydrogenase subunit 1) gene, encoding subunit of CI [Patient 1, m.3365T>C predicting p.(Leu20Pro); Patient 2, m.4175G>A predicting p.(Trp290*)] at high levels of mitochondrial DNA heteroplasmy in skeletal muscle. We evaluated the effect of these novel MTND1 mutations on complex assembly showing that CI assembly, although markedly reduced, was viable in the absence of detectable ND1 signal. Real-time PCR and Western blotting showed overexpression of different CI assembly factor transcripts and proteins in patient tissue. Together, our data indicate that the mechanism underlying the expression of the biochemical defect may involve a compensatory response to the novel MTND1 gene mutations, promoting assembly factor up-regulation and stabilization of respiratory chain super-complexes, resulting in partial rescue of the clinical phenotype.
Asunto(s)
Complejo I de Transporte de Electrón/deficiencia , Tolerancia al Ejercicio/genética , Miopatías Mitocondriales/genética , Mutación , NADH Deshidrogenasa/genética , Adolescente , ADN Mitocondrial/genética , Prueba de Esfuerzo/métodos , Femenino , Humanos , Miopatías Mitocondriales/enzimología , Músculo Esquelético/enzimología , Linaje , Adulto JovenRESUMEN
Single, large-scale deletions of mitochondrial DNA are a common cause of mitochondrial disease and cause a broad phenotypic spectrum ranging from mild myopathy to devastating multi-system syndromes such as Kearns-Sayre syndrome. Studies to date have been inconsistent on the value of putative predictors of clinical phenotype and disease progression such as mutation load and the size or location of the deletion. Using a cohort of 87 patients with single, large-scale mitochondrial DNA deletions we demonstrate that a variety of outcome measures such as COX-deficient fibre density, age-at-onset of symptoms and progression of disease burden, as measured by the Newcastle Mitochondrial Disease Adult Scale, are significantly (P < 0.05) correlated with the size of the deletion, the deletion heteroplasmy level in skeletal muscle, and the location of the deletion within the genome. We validate these findings with re-analysis of 256 cases from published data and clarify the previously conflicting information of the value of these predictors, identifying that multiple regression analysis is necessary to understand the effect of these interrelated predictors. Furthermore, we have used mixed modelling techniques to model the progression of disease according to these predictors, allowing a better understanding of the progression over time of this strikingly variable disease. In this way we have developed a new paradigm in clinical mitochondrial disease assessment and management that sidesteps the perennial difficulty of ascribing a discrete clinical phenotype to a broad multi-dimensional and progressive spectrum of disease, establishing a framework to allow better understanding of disease progression.
Asunto(s)
ADN Mitocondrial/genética , Progresión de la Enfermedad , Eliminación de Gen , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/genética , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , ADN Mitocondrial/antagonistas & inhibidores , Femenino , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana EdadRESUMEN
Despite being a canonical presenting feature of mitochondrial disease, the genetic basis of progressive external ophthalmoplegia remains unknown in a large proportion of patients. Here we show that mutations in SPG7 are a novel cause of progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions. After excluding known causes, whole exome sequencing, targeted Sanger sequencing and multiplex ligation-dependent probe amplification analysis were used to study 68 adult patients with progressive external ophthalmoplegia either with or without multiple mitochondrial DNA deletions in skeletal muscle. Nine patients (eight probands) were found to carry compound heterozygous SPG7 mutations, including three novel mutations: two missense mutations c.2221G>A; p.(Glu741Lys), c.2224G>A; p.(Asp742Asn), a truncating mutation c.861dupT; p.Asn288*, and seven previously reported mutations. We identified a further six patients with single heterozygous mutations in SPG7, including two further novel mutations: c.184-3C>T (predicted to remove a splice site before exon 2) and c.1067C>T; p.(Thr356Met). The clinical phenotype typically developed in mid-adult life with either progressive external ophthalmoplegia/ptosis and spastic ataxia, or a progressive ataxic disorder. Dysphagia and proximal myopathy were common, but urinary symptoms were rare, despite the spasticity. Functional studies included transcript analysis, proteomics, mitochondrial network analysis, single fibre mitochondrial DNA analysis and deep re-sequencing of mitochondrial DNA. SPG7 mutations caused increased mitochondrial biogenesis in patient muscle, and mitochondrial fusion in patient fibroblasts associated with the clonal expansion of mitochondrial DNA mutations. In conclusion, the SPG7 gene should be screened in patients in whom a disorder of mitochondrial DNA maintenance is suspected when spastic ataxia is prominent. The complex neurological phenotype is likely a result of the clonal expansion of secondary mitochondrial DNA mutations modulating the phenotype, driven by compensatory mitochondrial biogenesis.
Asunto(s)
ADN Mitocondrial/metabolismo , Metaloendopeptidasas/metabolismo , Enfermedades Mitocondriales/complicaciones , Enfermedades Mitocondriales/genética , Mutación/genética , Oftalmoplejía Externa Progresiva Crónica/complicaciones , Oftalmoplejía Externa Progresiva Crónica/genética , ATPasas Asociadas con Actividades Celulares Diversas , Anciano , Enfermedad Crónica , Análisis Mutacional de ADN , ADN Mitocondrial/genética , Estimulación Eléctrica , Complejo IV de Transporte de Electrones/metabolismo , Potenciales Evocados Motores/genética , Femenino , Estudios de Asociación Genética , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Oftalmoplejía Externa Progresiva Crónica/patología , Fenotipo , Tiempo de ReacciónRESUMEN
PURPOSE: Mitochondrial disorders are a common cause of inherited metabolic disease and can be due to mutations affecting mitochondrial DNA or nuclear DNA. The current diagnostic approach involves the targeted resequencing of mitochondrial DNA and candidate nuclear genes, usually proceeds step by step, and is time consuming and costly. Recent evidence suggests that variations in mitochondrial DNA sequence can be obtained from whole-exome sequence data, raising the possibility of a comprehensive single diagnostic test to detect pathogenic point mutations. METHODS: We compared the mitochondrial DNA sequence derived from off-target exome reads with conventional mitochondrial DNA Sanger sequencing in 46 subjects. RESULTS: Mitochondrial DNA sequences can be reliably obtained using three different whole-exome sequence capture kits. Coverage correlates with the relative amount of mitochondrial DNA in the original genomic DNA sample, heteroplasmy levels can be determined using variant and total read depths, and-providing there is a minimum read depth of 20-fold-rare sequencing errors occur at a rate similar to that observed with conventional Sanger sequencing. CONCLUSION: This offers the prospect of using whole-exome sequence in a diagnostic setting to screen not only all protein coding nuclear genes but also all mitochondrial DNA genes for pathogenic mutations. Off-target mitochondrial DNA reads can also be used to assess quality control and maternal ancestry, inform on ethnic origin, and allow genetic disease association studies not previously anticipated with existing whole-exome data sets.
Asunto(s)
Análisis Mutacional de ADN/métodos , ADN Mitocondrial/genética , Enfermedades Mitocondriales/genética , Mutación Puntual , Polimorfismo de Nucleótido Simple , Núcleo Celular/metabolismo , Mapeo Cromosómico , Biología Computacional , ADN/genética , Exoma , Reacciones Falso Positivas , Genoma , Humanos , Mitocondrias/metabolismo , Fenotipo , Reacción en Cadena de la PolimerasaRESUMEN
IMPORTANCE: Mitochondrial disorders have emerged as a common cause of inherited disease, but their diagnosis remains challenging. Multiple respiratory chain complex defects are particularly difficult to diagnose at the molecular level because of the massive number of nuclear genes potentially involved in intramitochondrial protein synthesis, with many not yet linked to human disease. OBJECTIVE: To determine the molecular basis of multiple respiratory chain complex deficiencies. DESIGN, SETTING, AND PARTICIPANTS: We studied 53 patients referred to 2 national centers in the United Kingdom and Germany between 2005 and 2012. All had biochemical evidence of multiple respiratory chain complex defects but no primary pathogenic mitochondrial DNA mutation. Whole-exome sequencing was performed using 62-Mb exome enrichment, followed by variant prioritization using bioinformatic prediction tools, variant validation by Sanger sequencing, and segregation of the variant with the disease phenotype in the family. RESULTS: Presumptive causal variants were identified in 28 patients (53%; 95% CI, 39%-67%) and possible causal variants were identified in 4 (8%; 95% CI, 2%-18%). Together these accounted for 32 patients (60% 95% CI, 46%-74%) and involved 18 different genes. These included recurrent mutations in RMND1, AARS2, and MTO1, each on a haplotype background consistent with a shared founder allele, and potential novel mutations in 4 possible mitochondrial disease genes (VARS2, GARS, FLAD1, and PTCD1). Distinguishing clinical features included deafness and renal involvement associated with RMND1 and cardiomyopathy with AARS2 and MTO1. However, atypical clinical features were present in some patients, including normal liver function and Leigh syndrome (subacute necrotizing encephalomyelopathy) seen in association with TRMU mutations and no cardiomyopathy with founder SCO2 mutations. It was not possible to confidently identify the underlying genetic basis in 21 patients (40%; 95% CI, 26%-54%). CONCLUSIONS AND RELEVANCE: Exome sequencing enhances the ability to identify potential nuclear gene mutations in patients with biochemically defined defects affecting multiple mitochondrial respiratory chain complexes. Additional study is required in independent patient populations to determine the utility of this approach in comparison with traditional diagnostic methods.
Asunto(s)
Análisis Mutacional de ADN , Exoma , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/genética , Análisis de Secuencia de ADN/métodos , Adolescente , Adulto , Niño , Preescolar , Biología Computacional , Femenino , Haplotipos , Humanos , Lactante , MasculinoRESUMEN
We report three families presenting with hypertrophic cardiomyopathy, lactic acidosis, and multiple defects of mitochondrial respiratory chain (MRC) activities. By direct sequencing of the candidate gene MTO1, encoding the mitochondrial-tRNA modifier 1, or whole exome sequencing analysis, we identified novel missense mutations. All MTO1 mutations were predicted to be deleterious on MTO1 function. Their pathogenic role was experimentally validated in a recombinant yeast model, by assessing oxidative growth, respiratory activity, mitochondrial protein synthesis, and complex IV activity. In one case, we also demonstrated that expression of wt MTO1 could rescue the respiratory defect in mutant fibroblasts. The severity of the yeast respiratory phenotypes partly correlated with the different clinical presentations observed in MTO1 mutant patients, although the clinical outcome was highly variable in patients with the same mutation and seemed also to depend on timely start of pharmacological treatment, centered on the control of lactic acidosis by dichloroacetate. Our results indicate that MTO1 mutations are commonly associated with a presentation of hypertrophic cardiomyopathy, lactic acidosis, and MRC deficiency, and that ad hoc recombinant yeast models represent a useful system to test the pathogenic potential of uncommon variants, and provide insight into their effects on the expression of a biochemical phenotype.