RESUMEN
Leigh syndrome with cytochrome oxidase (COX) deficiency has been associated with SURF1 mutations. For patient diagnosis, distinction between neutral polymorphisms and pathogenic missense SURF1 mutations in Leigh syndrome is essential. We show that several missense SURF1 mutations did not allow a stable protein to be expressed. Absence of immunologically reactive SURF1 is, therefore, helpful to demonstrate their pathogenicity. In addition, we show that out of two previously described missense mutations housed by the same allele, only one, the T737 C was pathogenic. Indeed, transfection of T737 C mutated SURF1 in SURF1-deficient cells did not restore normal SURF1 stability and COX activity. On the contrary, the G604 C-mutated SURF1 did it and, hence, is a neutral variant.
RESUMEN
The transcript levels of nuclear and mitochondrial genes involved in oxidative phosphorylation were quantified in human myoblasts and myotubes cultured from biopsies of patients harboring either heteroplasmic point mutation or deletion of mitochondrial DNA. The transcript patterns were determined by two different methodologies, competitive reverse-transcription polymerase chain reaction and classical Northern blot analysis, both referred to the mitochondrial to nuclear DNA ratio. In myoblasts from the patients with MELAS (myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) and KSS (Kearns-Sayre) syndromes, both methodologies revealed an increase of mtDNA transcript levels. A higher level of the nuclear ATP synthase beta transcript was observed in the MELAS patient cells and could be the consequence of a feedback effect of the mitochondrial DNA mutation. Moreover, the nuclear and mitochondrial transcript accumulation is more pronounced after myoblast differentiation. Thus, the OXPHOS expression is specifically altered in patients with mitochondrial diseases. The competitive RT-PCR, a rapid and sensitive technique, could be applied to investigation of mitochondrial myopathies.
Asunto(s)
Expresión Génica/genética , Síndrome de Kearns-Sayre/metabolismo , Síndrome MELAS/metabolismo , Músculo Esquelético/metabolismo , Fosforilación Oxidativa , Complejos de ATP Sintetasa , Northern Blotting , Diferenciación Celular , Núcleo Celular/metabolismo , Células Cultivadas , ADN/metabolismo , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Humanos , Síndrome de Kearns-Sayre/genética , Síndrome de Kearns-Sayre/patología , Síndrome MELAS/genética , Síndrome MELAS/patología , Complejos Multienzimáticos/genética , Músculo Esquelético/patología , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Mutación Puntual/genética , ARN Mensajero/metabolismo , ARN Ribosómico 18S/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia/genética , Activación TranscripcionalRESUMEN
The expression of several mitochondrial and nuclear genes involved in ATP production was examined in cells cultured from muscle biopsies of patients harboring mitochondrial pathologies. The transcript patterns in muscle cells from the patients affected by carnitine palmitoyl transferase II or 2-ketoglutarate dehydrogenase deficiencies were almost similar to control patterns. In the opposite, patterns were strikingly abnormal in all the other cell cultures from patients with defects in enzymatic complexes involved in oxidative phosphorylation: mitochondrial complex II and III deficiencies, two MELAS syndromes (myopathy, encephalopathy, lactic acidosis and stroke like episodes), a case of Kearns-Sayre syndrome and a case of chronic progressive external ophthalmoplegia. In cultured muscle cells from patients with mtDNA mutations, the percentage of mutated mtDNA was low as compared with those determined in the corresponding skeletal muscle biopsy. Moreover, the complex II defect resulting of a nuclear mutation was not expressed in the cell cultures. Thus, an undetermined transcriptional event, transmitted from muscle biopsies to cultured muscle cells, should be involved to account for such abnormal transcript patterns.
Asunto(s)
Miopatías Mitocondriales/genética , Músculo Esquelético/metabolismo , Fosforilación Oxidativa , Adolescente , Adulto , Biopsia , Carnitina O-Palmitoiltransferasa/genética , Núcleo Celular/metabolismo , Células Cultivadas , Preescolar , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Femenino , Humanos , Lactante , Complejo Cetoglutarato Deshidrogenasa/genética , Masculino , Persona de Mediana Edad , Mitocondrias Musculares/enzimología , Mitocondrias Musculares/metabolismo , Miopatías Mitocondriales/metabolismo , Miopatías Mitocondriales/patología , Músculo Esquelético/patologíaRESUMEN
Myoblast cultures were established from muscle biopsies of two patients harboring heteroplasmic mitochondrial (mt) DNA deletions. The accumulation kinetics of the deleted mtDNA was followed during myoblast to myotube differentiation. The percentage of deleted mtDNA was determined by quantitative PCR in myoblasts, myotubes, and muscle biopsies. The deleted form accounted for 65% of the mtDNA present in a muscle biopsy from a patient harboring a 5.6-kb deletion. The percentage of deleted mtDNA was 1.2% in myoblasts and increased progressively after differentiation, up to 12% at 21 days after the commitment time. In a second patient harboring a 2.8-kb deletion, the percentage of deleted mtDNA increased much more slowly: from 0.07% in myoblasts to 0.21% after 22 days of differentiation, as compared with 45% in the muscle biopsy. Thus, a three- and ten-fold increase, respectively, in the fraction of deleted mtDNA occurred during the differentiation of myoblasts to myotubes from the two patients. The faster accumulation of deleted mtDNA int he first patient's cells was linked to an earlier myoblast to myotube differentiation, suggesting that the level of deleted mtDNA is inversely related to the rate of cell proliferation.
Asunto(s)
ADN Mitocondrial/genética , Síndrome de Kearns-Sayre/genética , Fibras Musculares Esqueléticas/química , Oftalmoplejía Externa Progresiva Crónica/genética , Eliminación de Secuencia , Adolescente , Diferenciación Celular , División Celular , Células Cultivadas , Femenino , Humanos , Síndrome de Kearns-Sayre/patología , Masculino , Persona de Mediana Edad , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/patología , Reacción en Cadena de la PolimerasaRESUMEN
Seventy amniotic fluids (AF) were sampled because of abnormal ultrasound findings (mainly non-immune hydrops fetalis (54 cases) or of the presence of vacuolated lymphocytes in fetal blood (3 cases)). They were analysed by a procedure involving AF supernatant analysis (glycosaminoglycans, oligosaccharides, free sialic acid and acid hydrolase activities) and biochemical study of cultured AF cells. Ten cases of lysosomal storage diseases (LSD) were diagnosed. The reported procedure allows an orientating screening within 3 days by analysis of 15 ml of third trimester AF supernatant (except for Gaucher and Niemann-Pick diseases). In some cases, the results allow an LSD diagnosis and a medical abortion without waiting for the formal diagnosis (in cultured AF cells that needs 3 more weeks), considering the poor prognosis of these LSD presenting in utero. Furthermore, the formal assessment of the diagnosis in the cultured fetal cells allows accurate genetic counselling for the couple.
Asunto(s)
Líquido Amniótico/química , Hidropesía Fetal/diagnóstico por imagen , Enfermedades por Almacenamiento Lisosomal/diagnóstico , Diagnóstico Prenatal , Células Cultivadas , Femenino , Glucuronidasa/deficiencia , Glicosaminoglicanos/análisis , Humanos , Ácido N-Acetilneuramínico/análisis , Oligosacáridos/análisis , Embarazo , Ultrasonografía PrenatalRESUMEN
Carnitine palmitoyltransferase II deficiency (CPT II) is an autosomal recessive disorder and the most frequent cause of hereditary myoglobinuria. We report the case of a young man who presented a severe fever-induced episode of rhabdomyolysis and myoglobinuria resulting in acute renal failure. Cultured skeletal muscle cells have been used for the biochemical and molecular characterization of the defect in this patient. Immunoblot analysis revealed reduced steady-state level of CPT II protein. A PCR-based method detected the common Ser113Leu substitution only in one allele, suggesting that the patient is a compound heterozygote for this common mutation and a different as yet unidentified mutation.
Asunto(s)
Carnitina O-Palmitoiltransferasa/deficiencia , Carnitina O-Palmitoiltransferasa/genética , Músculo Esquelético/enzimología , Mioglobinuria/enzimología , Mioglobinuria/genética , Fenotipo , Adulto , Secuencia de Bases , Western Blotting , Células Cultivadas , Humanos , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , RecurrenciaRESUMEN
Amniocytes isolated from two pregnancies at risk for fatty acid oxidation defects were incubated with stable isotopically labelled palmitate, in the presence of L-carnitine, to probe that pathway. The labelled acylcarnitines were then quantitated using tandem mass spectrometry. Amniocytes from a pregnancy at risk for medium-chain acyl-CoA dehydrogenase (MCAD) deficiency produced a characteristic acylcarnitine profile with increased levels of octanoylcarnitine and decanoylcarnitine, indicative of MCAD deficiency. DNA analysis confirmed that the fetus was homozygous for the MCAD A985G mutation. Acylcarnitine and DNA analysis of the infant's blood obtained post-partum confirmed MCAD deficiency. Amniocytes from a pregnancy at risk for an unspecified fat oxidation defect produced increased levels of long-chain acylcarnitines consistent with a deficiency in very-long-chain acyl-CoA dehydrogenase (VLCAD). Measurements of the enzymatic activity confirmed VLCAD deficiency in amniocytes. Acylcarnitine profiles of the infant's blood obtained post-partum in addition to enzyme activities measured in fibroblasts confirmed VLCAD deficiency. The successful prenatal diagnosis of VLCAD and MCAD deficiencies using in vitro probes of fatty acid oxidation in fibroblasts suggests that this approach can potentially recognize many mitochondrial fatty acid oxidation defects even if no prior diagnosis is determined in the family at risk.
Asunto(s)
Acil-CoA Deshidrogenasa de Cadena Larga/deficiencia , Amniocentesis , Errores Innatos del Metabolismo Lipídico/diagnóstico , Mitocondrias/enzimología , Acil-CoA Deshidrogenasa , Acil-CoA Deshidrogenasa de Cadena Larga/metabolismo , Hidrolasas de Éster Carboxílico/metabolismo , Femenino , Edad Gestacional , Humanos , Recién Nacido , Errores Innatos del Metabolismo Lipídico/enzimología , Masculino , Espectrometría de Masas , Oxidación-Reducción , EmbarazoAsunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Pruebas Enzimáticas Clínicas , Glicina/sangre , Transferasas de Hidroximetilo y Formilo , Diagnóstico Prenatal/métodos , Transferasas/sangre , Aminometiltransferasa , Femenino , Humanos , Embarazo , Primer Trimestre del EmbarazoAsunto(s)
Miopatías Mitocondriales/diagnóstico , Músculo Esquelético/patología , Adolescente , Adulto , Carnitina O-Palmitoiltransferasa/deficiencia , Células Cultivadas , Niño , ADN Mitocondrial/metabolismo , Femenino , Humanos , Complejo Cetoglutarato Deshidrogenasa/deficiencia , Masculino , Miopatías Mitocondriales/genética , Miopatías Mitocondriales/patología , Mutación PuntualRESUMEN
Thirty-seven pregnancies at risk for Niemann-Pick type C disease were monitored by study of cultured amniotic fluid cells (8 cases) or chorionic villus cells (29 cases) in 23 couples over the period 1984-91. An early protocol combined determination of sphingomyelinase activity with electron microscopy. The current strategy, based on the demonstration of specific abnormalities in intracellular processing of exogenous cholesterol, combines the study of the early phase (first 6 h) of LDL-induced cholesteryl ester formation and the histochemical evaluation (filipin staining after 24 h of LDL uptake) of the LDL-induced accumulation of unesterified cholesterol. Thirteen fetuses were predicted to be affected. Confirmation of the diagnosis was made by study of cholesterol processing in fetal skin fibroblast cultures and/or by demonstration of a characteristic lipid storage in fetal liver, already present at 14 w gestation. Definition of the biochemical phenotype (classical, variant, or intermediate) of the index case, with regard to cholesterol-processing abnormalities, is an absolute prerequisite to adequate genetic counseling in a given family. Prenatal diagnosis has now proved a safe procedure in the predominant (approximately 85%) group of families with the classical phenotype.