RESUMEN
The goal of the study was to retrospectively evaluate a cohort of children and adults with mitochondrial diseases (MDs) in a single-center experience. Neurological clinical examination, brain magnetic resonance imaging (MRI) and spectroscopy, muscle biopsy, metabolic and molecular-genetic analysis were evaluated in 26 children and 36 adult patients with MD in Slovenia from 2004 to 2018. Nijmegen MD criteria (MDC) were applied to all patients and the need for a muscle biopsy was estimated. Exome-sequencing was used in half of the patients. Twenty children (77.0%) and 12 adults (35.0%) scored a total of ≥8 on MDC, a result that is compatible with the diagnosis of definite MD. Yield of exome-sequencing was 7/22 (31.0%), but the method was not applied systematically in all patients from the beginning of diagnostics. Brain MRI morphological changes, which can be an imaging clue for the diagnosis of MD, were found in 17/24 children (71.0%). In 7/26 (29.0%) children, and in 20/30 (67.0%) adults, abnormal mitochondria were found on electron microscopy (EM) and ragged-red fibers were found in 16/30 (53.0%) adults. Respiratory chain enzymes (RCEs) and/or pyruvate dehydrogenase complex (PDHc) activities were abnormal in all the children and six adult cases. First, our data revealed that MDC was useful in the clinical diagnosis of MD, and second, until the use of NGS methods, extensive, laborious and invasive diagnostic procedures were performed to reach a final diagnosis. In patients with suspected MD, there is a need to prioritize molecular diagnosis with the more modern next-generation sequencing (NGS) method.
RESUMEN
We report a novel pathogenic variant (c.223G > C; p.Gly75Arg) in the gene encoding the small mitoribosomal subunit protein mS34 in a long-surviving patient with Leigh Syndrome who was genetically diagnosed at age 34 years. The patient presented with delayed motor milestones and a stepwise motor deterioration during life, along with brain MRI alterations involving the subcortical white matter, deep grey nuclei and in particular the internal globi pallidi, that appeared calcified on CT scan. The novel variant is associated with a reduction of mS34 protein levels and of the OXPHOS complex I and IV subunits in peripheral blood mononuclear cells of the case. This study expands the number of variants that, by affecting the stability of the mitoribosome, may cause an OXPHOS deficiency in Leigh Syndrome and reports, for the first time, an unusual long survival in a patient with a homozygous MRPS34 pathogenic variant.
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Mitochondrial DNA depletion syndromes (MDSs) form a group of autosomal recessive disorders characterized by profoundly decreased mitochondrial DNA copy numbers in affected tissues. Three main clinical presentations are known: myopathic, encephalomyopathic and hepatocerebral. The first is associated with mutations in thymidine kinase 2 (TK2) and p53-induced ribonucleotide reductase B subunit (RRM2B); the second with mutations in succinate synthase A (SUCLA2) and B (SUCLG1); the third with mutations in Twinkle (PEO1), pol-gammaA (POLG1), deoxyguanosine kinase (DGUOK) and MPV17 (MPV17). In this work, we review the MDS-associated phenotypes and present our own experience of 32 MDS patients, with the aim of defining the mutation frequency of the known genes, the clinical spectrum of the diseases, and the genotype-phenotype correlations. Five of our patients carried previously unreported mutations in one of the eight MDS genes.
Asunto(s)
ADN Mitocondrial/genética , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/patología , Acidosis Láctica/etiología , Edad de Inicio , Encéfalo/patología , Niño , Preescolar , Estudios de Cohortes , Electroencefalografía , Electromiografía , Femenino , Humanos , Lactante , Recién Nacido , Hígado/patología , Imagen por Resonancia Magnética , Masculino , Encefalomiopatías Mitocondriales/genética , Encefalomiopatías Mitocondriales/patología , Miopatías Mitocondriales/genética , Miopatías Mitocondriales/patología , Músculo Esquelético/patología , Mutación/fisiología , Timidina Quinasa/genéticaRESUMEN
Mutations in ETHE1, a gene located at chromosome 19q13, have recently been identified in patients affected by ethylmalonic encephalopathy (EE). EE is a devastating infantile metabolic disorder, characterised by widespread lesions in the brain, hyperlactic acidaemia, petechiae, orthostatic acrocyanosis, and high levels of ethylmalonic acid in body fluids. To investigate to what extent ETHE1 is responsible for EE, we analysed this gene in 29 patients with typical EE and in 11 patients presenting with early onset progressive encephalopathy with ethylmalonic aciduria (non-EE EMA). Frameshift, stop, splice site, and missense mutations of ETHE1 were detected in all the typical EE patients analysed. Western blot analysis of the ETHE1 protein indicated that some of the missense mutations are associated with the presence of the protein, suggesting that the corresponding wild type amino acid residues have a catalytic function. No ETHE1 mutations were identified in non-EE EMA patients. Experiments based on two dimensional blue native electrophoresis indicated that ETHE1 protein works as a supramolecular, presumably homodimeric, complex, and a three dimensional model of the protein suggests that it is likely to be a mitochondrial matrix thioesterase acting on a still unknown substrate. Finally, the 625G-->A single nucleotide polymorphism in the gene encoding the short chain acyl-coenzyme A dehydrogenase (SCAD) was previously proposed as a co-factor in the aetiology of EE and other EMA syndromes. SNP analysis in our patients ruled out a pathogenic role of SCAD variants in EE, but did show a highly significant prevalence of the 625A alleles in non-EE EMA patients.
Asunto(s)
Encefalopatías Metabólicas/genética , Proteínas Mitocondriales/genética , Mutación , Proteínas de Transporte Nucleocitoplasmático/genética , Alelos , Western Blotting , Encefalopatías Metabólicas/diagnóstico , Butiril-CoA Deshidrogenasa/genética , Butiril-CoA Deshidrogenasa/fisiología , Análisis Mutacional de ADN , Electroforesis en Gel Bidimensional , Humanos , Malonatos/análisis , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Modelos Moleculares , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Filogenia , Polimorfismo de Nucleótido SimpleRESUMEN
Isolated complex I deficiency, the most frequent OXPHOS disorder in infants and children, is genetically heterogeneous. Mutations have been found in seven mitochondrial DNA (mtDNA) and eight nuclear DNA encoded subunits, respectively, but in most of the cases the genetic basis of the biochemical defect is unknown. We analyzed the entire mtDNA and 11 nuclear encoded complex I subunits in 23 isolated complex I-deficient children, classified into five clinical groups: Leigh syndrome, progressive leukoencephalopathy, neonatal cardiomyopathy, severe infantile lactic acidosis, and a miscellaneous group of unspecified encephalomyopathies. A genetic definition was reached in eight patients (35%). Mutations in mtDNA were found in six out of eight children with Leigh syndrome, indicating a prevalent association between this phenotype and abnormalities in ND genes. In two patients with leukoencephalopathy, homozygous mutations were detected in two different nuclear-encoded complex I genes, including a novel transition in NDUFS1 subunit. In addition to these, a child affected by mitochondrial encephalomyopathy had heterozygous mutations in NDUFA8 and NDUFS2 genes, while another child with neonatal cardiomyopathy had a complex rearrangement in a single NDUFS7 allele. The latter cases suggest the possibility of unconventional patterns of inheritance in complex I defects.
Asunto(s)
Complejo I de Transporte de Electrón/deficiencia , Errores Innatos del Metabolismo/etiología , Mutación , Acidosis Láctica/etiología , Acidosis Láctica/genética , Cardiomiopatías/etiología , Cardiomiopatías/genética , Niño , ADN Mitocondrial , Complejo I de Transporte de Electrón/genética , Humanos , Lactante , Proteínas Hierro-Azufre/genética , Enfermedad de Leigh/etiología , Enfermedad de Leigh/genética , Leucoencefalopatía Multifocal Progresiva/etiología , Leucoencefalopatía Multifocal Progresiva/genética , Errores Innatos del Metabolismo/genética , Proteínas Mitocondriales/genética , NAD(P)H Deshidrogenasa (Quinona)/genética , NADH Deshidrogenasa/genética , Proteínas/genéticaRESUMEN
OBJECTIVE: To define the clinical and EEG features of the epileptic syndromes occurring in adult and infantile mitochondrial encephalopathies (ME). METHODS: Thirty-one patients with recurrent and apparently unprovoked seizures associated with primary ME were included in the study. Diagnosis of ME was based on the recognition of a morphologic, biochemical, or molecular defect. RESULTS: Epileptic seizures were the first recognized symptom in 53% of the patients. Many adults (43%) and most infants (70%) had nontypical ME phenotypes. Partial seizures, mainly with elementary motor symptoms, and focal or multifocal EEG epileptiform activities characterized the epileptic presentation in 71% of the patients. Generalized myoclonic seizures were an early and consistent symptom only in the five patients with an A8344G mitochondrial DNA point mutation with classic myoclonus epilepsy with ragged red fibers (MERRF) syndrome or "overlapping" characteristics. Photoparoxysmal EEG responses were observed not only in patients with typical MERRF, but also in adult patients with ME with lactic acidosis and strokelike episodes (MELAS), or overlapping phenotypes, and in one child with Leigh syndrome. CONCLUSIONS: Epilepsy is an important sign in the early presentation of ME and may be the most apparent neurologic sign of nontypical ME, often leading to the diagnostic workup. Except for those with an A8344G mitochondrial DNA point mutation, most of our patients had partial seizures or EEG signs indicating a focal origin.
Asunto(s)
Encéfalo/fisiopatología , Epilepsia/etiología , Epilepsia/fisiopatología , Encefalomiopatías Mitocondriales/complicaciones , Encefalomiopatías Mitocondriales/fisiopatología , Adolescente , Adulto , Edad de Inicio , Encéfalo/metabolismo , Encéfalo/patología , Niño , Preescolar , Electroencefalografía , Epilepsia/patología , Femenino , Humanos , Lactante , Recién Nacido , Enfermedad de Leigh/complicaciones , Enfermedad de Leigh/patología , Enfermedad de Leigh/fisiopatología , Síndrome MELAS/complicaciones , Síndrome MELAS/patología , Síndrome MELAS/fisiopatología , Síndrome MERRF/complicaciones , Síndrome MERRF/patología , Síndrome MERRF/fisiopatología , Imagen por Resonancia Magnética , Masculino , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Encefalomiopatías Mitocondriales/patología , FenotipoRESUMEN
A mutation was found in an Italian child affecting the gene encoding the mitochondrial transfer RNA for leucine (codon UUR). This mutation (3291T-->C) had previously been reported in a single Japanese patient. In contrast with the original patient, who suffered from early-onset mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), our patient presented an apparently isolated mild myopathy. Mutational analysis in the proband and her family showed that the mutation was heteroplasmic, and that its relative amount was positively correlated with the severity of the phenotype. These findings lead to the definitive confirmation that the 3291T-->C is indeed pathogenic. As commonly found in mitochondrial-DNA related disorders, also for this mutation different clinical manifestations can be associated with the same genetic abnormality.
Asunto(s)
ADN Mitocondrial/genética , Miopatías Mitocondriales/genética , Miopatías Mitocondriales/patología , Enfermedades Neuromusculares/diagnóstico , Mutación Puntual , Biopsia con Aguja , Enfermedad Celíaca/complicaciones , Enfermedad Celíaca/diagnóstico , Niño , Análisis Mutacional de ADN , Diagnóstico Diferencial , Transporte de Electrón , Femenino , Humanos , Síndrome MELAS/genética , Miopatías Mitocondriales/complicaciones , Fibras Musculares de Contracción Rápida/patología , Músculo Esquelético/diagnóstico por imagen , Músculo Esquelético/enzimología , Músculo Esquelético/patología , Enfermedades Neuromusculares/complicaciones , Enfermedades Neuromusculares/genética , Linaje , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Polimorfismo Conformacional Retorcido-Simple , Tomografía Computarizada por Rayos XRESUMEN
We describe a patient who suffered from impaired ocular motility from age 10 years and at 16 years developed ptosis, proximal weakness and progressive fatigability. At 35 years she developed massive myoclonic jerks, and head and distal tremor. A muscle biopsy showed a high percentage of cytochrome c oxidase negative fibers but no ragged-red fibers. A novel heteroplasmic mutation (8342G-->A) was found in the mitochondrial transfer RNA(Lys) gene by single-strand conformation polymorphism screening, followed by sequence and restriction fragment length polymorphism analysis. Approximately 80% of muscle mitochondrial DNA (mtDNA) harbored the mutation, while the mutation was absent in lymphocyte DNA of the proband, as well as of her mother, daughter and a maternal aunt. However, the pathogenicity of the mutation was confirmed by restriction fragment length polymorphism analysis of single muscle fibers, which revealed a significantly greater level of mutant mtDNA in cytochrome c oxidase negative over cytochrome c oxidase positive fibers.
Asunto(s)
Epilepsias Mioclónicas/genética , Oftalmoplejía Externa Progresiva Crónica/genética , Mutación Puntual , ARN de Transferencia de Lisina/genética , ARN/genética , Adenina/química , Adulto , Autoanálisis , Secuencia de Bases , Epilepsias Mioclónicas/metabolismo , Epilepsias Mioclónicas/patología , Femenino , Guanina/química , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oftalmoplejía Externa Progresiva Crónica/metabolismo , Oftalmoplejía Externa Progresiva Crónica/patología , Linaje , ARN MitocondrialRESUMEN
Very-long-chain acyl-coenzyme A dehydrogenase (VLCAD) deficiency is an increasingly recognized defect of mitochondrial fatty acid beta-oxidation manifesting with episodes of metabolic decompensation or isolated recurrent myoglobinuria. In this report the clinical, biochemical, and molecular studies in a series of five patients (four Italian and one Spanish) with this disorder are discussed. Biochemical studies included the determination of fibroblast substrate oxidation rates and enzyme activity and Western blot analysis of VLCAD protein. Molecular analysis was performed by sequencing the VLCAD gene from the genomic DNA. Clinical features were within the spectrum previously reported. Four patients presented in infancy or childhood with episodes of severe metabolic decompensation and dicarboxylic aciduria. Two exhibited cardiomyopathy. The fifth patient presented with isolated recurrent rhabdomyolysis, with no cardiomyopathy or dicarboxylic aciduria. In all patients a significant loss of VLCAD activity associated with a marked reduction of VLCAD protein levels occurred. Molecular analysis disclosed one novel missense mutation (Cys437Tyr) and four previously reported mutations, including two missense substitutions (Phe418Leu and Arg419Trp), a single amino acid deletion (Lys258del), and one splice site mutation (IVS8-C(-2)), which was present in all four Italian patients. All patients exhibited compound heterozygosity. The phenotypic variability and the high genotypic heterogeneity of this hereditary metabolic disorder is reported.
Asunto(s)
Ácido Graso Desaturasas/deficiencia , Ácido Graso Desaturasas/genética , Miopatías Mitocondriales/diagnóstico , Miopatías Mitocondriales/genética , Mutación , Acil-CoA Deshidrogenasa , Adulto , Western Blotting , Preescolar , Diagnóstico Diferencial , Dieta con Restricción de Grasas , Resultado Fatal , Femenino , Humanos , Lactante , Masculino , Miopatías Mitocondriales/dietoterapia , Miopatías Mitocondriales/etiología , Análisis de Secuencia de ADN , Resultado del TratamientoRESUMEN
Mitochondrial disorders are a group of heterogeneous diseases associated with abnormalities of the oxidative phosphorylation (OXPHOS), the most important source of energy for the cell. The number of mitochondrial syndromes and of identified causative genes is constantly increasing. Taken as a whole they are among the most frequent genetic diseases in humans at any age. The respiratory chain is the only metabolic pathway under double genome control and molecular genetics of these disorders is complicated by the existence of strict interactions between mitochondrial DNA and nuclear DNA. In childhood and infancy, clinical presentation differs from mitochondrial disorders with adult onset. The phenotypes are much more severe, often involving brain, frequently presenting as multisystemic disorders and seldom as isolated myopathy. Mutations in nDNA are more frequent than in adulthood. The major phenotypes presenting in infancy are here correlated with genetic defects and biochemical data with the aim to facilitate diagnosis work-up.
RESUMEN
BACKGROUND: Pelizaeus-Merzbacher-like disease (PMLD) is an inherited hypomyelinating leukoencephalopathy with onset in early infancy. Like Pelizaeus-Merzbacher disease (PMD), PMLD is characterized clinically by nystagmus, cerebellar ataxia, and spasticity, due to a permanent lack of myelin deposition in the brain. Mutations in the GJA12 gene, encoding connexin 47 (Cx47), were recently reported in five children with autosomal recessive PMLD. OBJECTIVES: To evaluate the impact of mutations in the GJA12 gene in, and define the clinical and neuroimaging features of, autosomal recessive PMLD. RESULTS: The authors screened for GJA12 mutations in 10 additional PMLD families originating from Italy, Pakistan, and Saudi Arabia. Three novel homozygous GJA12 mutations were identified in 12 mutant cases distributed in 3 of 10 families. The mutations segregated with the disease according to an autosomal recessive trait and included one missense (G236S) and two nonsense (L281fs285X and P131fs144X) changes. CONCLUSIONS: The identification of homozygous mutations predicting the synthesis of aberrant and truncated polypeptides, and their tight segregation with the disease in very large families, clearly demonstrate that the loss of Cx47 function is the cause of the disease. The phenotype of GJA12-related Pelizaeus-Merzbacher-like disease is fairly homogeneous and similar to that of Pelizaeus-Merzbacher disease. However, slower progression of symptoms, greater preservation of cognitive functions, and partial myelination of corticospinal tracts at MRI were distinctive features, which could help in the differential diagnosis.
Asunto(s)
Demencia Vascular/diagnóstico , Demencia Vascular/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/diagnóstico , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Medición de Riesgo/métodos , Adolescente , Niño , Preescolar , Comorbilidad , Demencia Vascular/epidemiología , Femenino , Predisposición Genética a la Enfermedad/epidemiología , Predisposición Genética a la Enfermedad/genética , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/epidemiología , Humanos , Internacionalidad , Italia/epidemiología , Masculino , Pakistán/epidemiología , Linaje , Enfermedad de Pelizaeus-Merzbacher/diagnóstico , Enfermedad de Pelizaeus-Merzbacher/epidemiología , Enfermedad de Pelizaeus-Merzbacher/genética , Factores de Riesgo , Arabia Saudita/epidemiologíaRESUMEN
In childhood mitochondrial encephalopathies the common MRI features are bilateral symmetric abnormalities in basal nuclei and brainstem. The presence of diffuse white matter abnormality has been described only in a few cases. Among a series of 110 children with mitochondrial encephalopathies, 8 patients with MR imaging consistent with a leukoencephalopathy were retrospectively evaluated. Diagnosis was based on the recognition of the biochemical defect in muscle homogenate. H-MR spectroscopic imaging was performed in six of them. Biochemical analysis demonstrated a defect of respiratory chain complexes in six patients: complex I in two cases, complex II in two, complex IV in one, multiple complexes defect in one. Pyruvate dehydrogenase deficiency was demonstrated in two patients. MRI showed severe involvement of the brain white matter without significant basal nuclei or brainstem abnormalities. Two patients developed large cystic areas since onset; in two others progressive vacuolisation of affected white matter was seen later in the course of the disease. One patient with pyruvate dehydrogenase deficiency also presented with a diffuse cortical polymicrogyria. H-MR spectroscopic imaging showed a decrease of N-acetylaspartate, choline and creatine with lactate accumulation in five patients, and was normal in one. These findings suggest that mitochondrial disorders should be included in the differential diagnosis of white matter disorders.
Asunto(s)
Encefalopatías Metabólicas/complicaciones , Encefalopatías Metabólicas/diagnóstico , Encéfalo/patología , Enfermedades Mitocondriales/complicaciones , Atrofia/patología , Tronco Encefálico/anomalías , Diagnóstico Diferencial , Progresión de la Enfermedad , Femenino , Humanos , Lactante , Imagen por Resonancia Magnética , Masculino , Enfermedades Mitocondriales/metabolismo , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Trastornos Psicomotores/diagnóstico , Piruvato Deshidrogenasa (Lipoamida)/análisis , Piruvato Deshidrogenasa (Lipoamida)/deficiencia , Estudios RetrospectivosRESUMEN
Sequence analysis of mitochondrial and nuclear candidate genes of complex I in children with deficiency of this complex and exhibiting Leigh-like syndrome has revealed, in one of them, a novel mutation in the NDUFS4 gene encoding the 18 kDa subunit. Phosphorylation of this subunit by cAMP-dependent protein kinase has previously been found to activate the complex. The present mutation consists of a homozygous G-->A transition at nucleotide position +44 of the coding sequence of the gene, resulting in the change of a tryptophan codon to a stop codon. Such mutation causes premature termination of the protein after only 14 amino acids of the putative mitochondrial targeting peptide. Fibroblast cultures from the patient exhibited severe reduction of the rotenone-sensitive NADH-->UQ oxidoreductase activity of complex I, which was insensitive to cAMP stimulation. Two-dimensional electrophoresis showed the absence of detectable normally assembled complex I in the inner mitochondrial membrane. These findings show that the expression of the NDUFS4 gene is essential for the assembly of a functional complex I.
Asunto(s)
Codón sin Sentido , Enfermedad de Leigh/genética , NADH NADPH Oxidorreductasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , ADN Complementario , Complejo I de Transporte de Electrón , Electroforesis en Gel Bidimensional , Femenino , Humanos , Recién Nacido , Datos de Secuencia Molecular , NADH Deshidrogenasa , NADH NADPH Oxidorreductasas/químicaRESUMEN
The neuronal ceroid lipofuscinoses (NCL) are progressive neurodegenerative diseases occurring in infancy and adulthood. Atypical forms of these diseases have been described and are particularly represented in the late-infantile and juvenile onset groups. Recent progress in biochemistry and molecular genetics has identified some of these variants as separate disease entities while disclosing the phenotypic variability of some classic forms. We report the results of a retrospective analysis performed on a series of 27 NCL patients, 15 of which were atypical as to clinical and/or pathological findings. Most of such patients, belonging to the late-infantile onset group and displaying homogeneous clinical-pathological features, were suggestive for CLN6. The two atypical juvenile NCL patients had features which resembled the "protracted form" of the disease. Given their relative frequency, strict clinical and pathological criteria are still the most useful tools for identifying and characterizing atypical forms and for defining phenotype-genotype correlations.
Asunto(s)
Glicoproteínas de Membrana , Chaperonas Moleculares , Lipofuscinosis Ceroideas Neuronales/clasificación , Lipofuscinosis Ceroideas Neuronales/diagnóstico , Adulto , Edad de Inicio , Niño , Preescolar , Electroencefalografía , Epilepsias Mioclónicas/diagnóstico , Epilepsias Mioclónicas/etiología , Potenciales Evocados Somatosensoriales , Potenciales Evocados Visuales , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Lipofuscinosis Ceroideas Neuronales/genética , Fenotipo , Proteínas/genética , Estudios RetrospectivosRESUMEN
A generalized defect of complex IV (cytochrome C oxidase, COX) is frequently found in subacute necrotizing encephalomyelopathy (Leigh's syndrome), the most common mitochondrial disorder in infancy. We previously demonstrated the nuclear origin of the COX defect in one case, by fusing nuclear DNA-less cytoplasts derived from normal fibroblasts with mitochondrial DNA (mtDNA)-less transformant fibroblasts derived from a patient with COX-defective [COX(-)] Leigh's syndrome. The resulting cybrid line showed a specific and serve COX(-) phenotype. Conversely, in the present study, we demonstrated that a COX(+) phenotype could be restored in hybrids obtained by fusing COX(-) transformant fibroblasts of seven additional Leigh's syndrome patients with mtDNA-less, COX(-) tumor-derived rho degree cells. Both these results are explained by the presence of a mutation in a nuclear gene. In a second set of experiments, in order to demonstrate whether COX(-) Leigh's syndrome is due to a defect in the same gene, or in different genes, we tested several hybrids derived by fusing our original COX(-) cell line with each of the remaining seven cell lines. COX activity was evaluated in situ by histochemical techniques and in cell extracts by a spectrophotometric assay. No COX complementers were found among the resulting hybrid lines. This result demonstrates that all our cases were genetically homogeneous, and suggests that a major nuclear disease locus is associated with several, perhaps most, of the cases of infantile COX(-) Leigh's syndrome. This information should make it easier to identify the gene responsible.
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Complejo IV de Transporte de Electrones/metabolismo , Enfermedad de Leigh/enzimología , Fusión Celular , Línea Celular , Niño , Preescolar , Transporte de Electrón , Complejo IV de Transporte de Electrones/genética , Femenino , Prueba de Complementación Genética , Genotipo , Humanos , Enfermedad de Leigh/genética , Masculino , Succinato Deshidrogenasa/metabolismo , SíndromeRESUMEN
We identified a novel heteroplasmic mutation in the mitochodrial DNA gene encoding the ND5 subunit of complex I. This mutation (13514A-->G) hits the same codon affected by a previously reported mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS)-associated mutation (13513G-->A), but the amino acid replacement is different (D393G vs D393N). The 13514A-->G mutation was found in two unrelated MELAS-like patients. However, in contrast to typical MELAS, lactic acidosis was absent or mild and the muscle biopsy was morphologically normal. Strongly positive correlation between the percentage of heteroplasmy and defective activity of complex I was found in cybrids. We found an additional 13513G-->A-positive case, affected by a progressive mitochondrial encephalomyopathy. Our results clearly demonstrate that the amino acid position D393 is crucial for the function of complex I. Search for D393 mutations should be part of the routine screening for mitochondrial disorders.
Asunto(s)
ADN Mitocondrial/genética , Síndrome MELAS/genética , NADH NADPH Oxidorreductasas/genética , Adolescente , Adulto , Encéfalo/patología , Complejo I de Transporte de Electrón , Femenino , Humanos , Síndrome MELAS/patología , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Mutación/genética , Polimorfismo Genético/genéticaRESUMEN
We show that the blue native gel polyacrylamide electrophoresis system (BN-PAGE) can be applied to pyruvate dehydrogenase complex (PDC). BN-PAGE has been used extensively to study the multisubunit enzymes of oxidative phosphorylation, as nondenaturing separation in the first dimension maintains holoenzyme integrity. However, the standard protocol was inappropriate for PDC as, at 10 MDa, it is approximately ten times larger than the largest respiratory chain enzyme complex. Therefore, agarose was substituted for polyacrylamide. Moreover, a substantial decrease in salt concentration was necessary to prevent dissociation of PDC. As with standard BN-PAGE, immunoblots of second-dimensional sodium dodecyl sulfate-PAGE (SDS-PAGE) provided more detailed information on specific subunits and subcomplexes. The method was applied to human heart mitochondrial fragments, control cultured human cells, rho0 cells that lack mitochondrial DNA, and two cell lines derived from patients with PDC deficiency. The PDC deficient cell lines showed a clear correlation between amount of PDC holoenzyme and disease severity. In cells lacking mitochondrial DNA, synthesis and assembly of all PDC subunits (all nuclearly encoded) appeared normal, suggesting that respiratory function has no regulatory role in PDC biogenesis. Blue native agarose gel electrophoresis coupled with standard second-dimensional SDS-PAGE provides a new tool to be used in conjunction with biochemical assays and immunoblots of one-dimensional SDS-PAGE to further elucidate the nature of PDC in normal and disease states. Furthermore, other cellular protein complexes of 1 MDa or more can be analysed by this method.
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Electroforesis en Gel de Agar/métodos , Complejo Piruvato Deshidrogenasa/aislamiento & purificación , Humanos , Complejo Piruvato Deshidrogenasa/químicaRESUMEN
Deficiency of carnitine palmitoyltransferase II (CPTase II; palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21) is a clinically heterogeneous autosomal recessive disorder of energy metabolism. We studied the molecular basis of CPTase II deficiency in an early-onset patient presenting with hypoketotic hypoglycemia and cardiomyopathy. cDNA and genomic DNA analysis demonstrated that the patient was homozygous for a mutant CPTase II allele (termed ICV), which carried three missense mutations: a G-1203----A transition, predicting a Val-368----Ile substitution (V368I); a C-1992----T transition, predicting an Arg-631----Cys substitution (R631C); and an A-2040----G transition, predicting a Met-647----Val substitution (M647V). Genomic DNA analysis of family members showed that the mutations cosegregated with the disease in the family. However, screening of 59 healthy controls demonstrated that both the V368I and M647V mutations are sequence polymorphisms with allele frequencies of 0.5 and 0.25, respectively. By contrast, the R631C substitution was not detected in 22 normal individuals or in 12 of 14 CPTase II-deficient patients with the adult muscular form. Notably, 2 adult CPTase II-deficient patients were heterozygous for the ICV allele, thus suggesting compound heterozygosity for this and a different mutant allele. The consequences of the three mutations on enzyme activity were investigated by expressing normal and mutated CPTase II cDNAs in COS cells. The R631C substitution drastically depressed the catalytic activity of CPTase II, thus confirming that this is the crucial mutation. Interestingly, the V368I and M647V substitutions, which did not affect enzyme activity alone, exacerbated the effects of the R631C substitution. Biochemical characterization of mutant CPTase II in patient's cells showed that the mutations are associated with (i) severe reduction of Vmax (approximately 90%), (ii) normal apparent Km values, and (iii) decreased protein stability.
Asunto(s)
Carnitina O-Palmitoiltransferasa/deficiencia , Errores Innatos del Metabolismo/genética , Secuencia de Bases , Carnitina O-Palmitoiltransferasa/genética , Clonación Molecular , ADN/genética , Análisis Mutacional de ADN , Metabolismo Energético , Humanos , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Linaje , Reacción en Cadena de la Polimerasa , Polimorfismo GenéticoRESUMEN
AIM: To contribute to the establishment of a rational clinical, neuroradiological, and molecular approach to neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) and maternally inherited Leigh's syndrome (MILS). METHODS AND RESULTS: The T8993G mutation in the mitochondrial genome was found in several maternal members of six pedigrees, whose clinical status ranged from no symptoms to severe infantile subacute necrotising encephalomyelopathy (Leigh's disease). In one case a MELAS-like syndrome was documented both clinically and neuroradiologically. Relevant genetic features of the series were anticipation of symptoms through subsequent generations, and the presence of several cases in whom the mutation apparently occurred recently or was new. A uniform distribution of the mutation in many tissues was shown in one patient subjected to necropsy. In general, a good correlation was found between clinical severity and mutation heteroplasmy in readily accessible tissues, such as lymphocytes or fibroblasts. By contrast, a consistent reduction of the mitochondrial ATPase activity, to about half of the normal values, was found in most of the clinically affected cases, irrespective of the amount of mutant mitochondrial DNA. CONCLUSIONS: Although the measurement of ATP hydrolysis in cultured fibroblasts was a reliable, and sometimes instrumental, means to identify T8993G positive patients, the relation between the mutation and the oxidative phosphorylation defect is probably very complex, and its understanding requires more complex biochemical analysis.
Asunto(s)
Adenosina Trifosfatasas/genética , ADN Mitocondrial/genética , Enfermedad de Leigh/diagnóstico , Mitocondrias Musculares/enzimología , Encefalomiopatías Mitocondriales/diagnóstico , Mutación , Adenosina Trifosfatasas/metabolismo , Adulto , Edad de Inicio , Encéfalo/patología , Niño , Preescolar , Análisis Mutacional de ADN , Femenino , Genotipo , Humanos , Lactante , Enfermedad de Leigh/genética , Enfermedad de Leigh/metabolismo , Síndrome MELAS/diagnóstico , Síndrome MELAS/genética , Imagen por Resonancia Magnética , Masculino , Encefalomiopatías Mitocondriales/genética , Encefalomiopatías Mitocondriales/metabolismo , Fosforilación Oxidativa , Linaje , Fenotipo , Retinitis Pigmentosa/diagnóstico , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
Carnitine palmitoyltransferase (CPT) II deficiency is the most common inherited disorder of lipid metabolism affecting skeletal muscle. To facilitate the identification of disease-causing mutations in the CPT II gene (CPT1), we have established the genomic organization of this gene. CPT1 spans approximately 20 kb of 1p32 and is composed of five exons ranging from 81 to 1305 bp. The sequences of the exon--intron boundaries were determined for each exon and conformed to the consensus splice junction sequences. The 5' and 3' untranslated regions in exon 1 and 5, respectively, were also determined, including the polyadenylation signal and the polyadenylation site. The mature transcript is predicted to be 3090 nt in length. CPT1 exons from CPT II-deficient patients were amplified and directly sequenced. Two novel disease-causing mutations were identified and characterized. The first mutation was a C-665-to-A transversion in exon 1 resulting in a proline-to-histidine substitution at residue 50 of the protein (P50H). This amino acid substitution occurs within a leucine-proline motif that is highly conserved in acyltransferases from different species. The mutation was detected in both alleles of patient 05SB of Italian ancestry, and in one allele of patients 11EG, 38PG, and 26FD of Italian, Dutch, and French ancestry, respectively. The second mutation was a rare G-2173-to-A transition in exon 5 causing an aspartic-acid-to-asparagine substitution at amino acid 553 (D553N) and the generation of a new MseI site. The mutation was detected only in one allele of patient 15MB, of Italian ancestry, who was also heterozygous for the common S113L substitution. Transfection experiments in COS cells demonstrated that both mutations drastically depressed the catalytic activity of CPT II. Biochemical characterization of P50H mutant CPT II in cultured cells from patient 05SB showed that the mutation does not affect substrate binding sites. Finally, immunoblot analysis demonstrated that both mutations were associated with markedly reduced steady-state level of the protein, thus indicating decreased stability of the mutant CPT II.