RESUMEN
Electron transfer flavoprotein dehydrogenase, also called ETF-ubiquinone oxidoreductase (ETF-QO), is a protein localized in the inner membrane of mitochondria, playing a central role in the electron-transfer system. Indeed, ETF-QO mediates electron transport from flavoprotein dehydrogenases to the ubiquinone pool. ETF-QO mutations are often associated with riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (RR-MADD, OMIM#231680), a multisystem genetic disease characterized by various clinical manifestations with different degrees of severity. In this review, we outline the clinical features correlated with ETF-QO deficiency and the benefits obtained from different treatments, such as riboflavin, L-carnitine and/or coenzyme Q10 supplementation, and a diet poor in fat and protein. Moreover, we provide a detailed summary of molecular and bioinformatic investigations, describing the mutations identified in ETFDH gene and highlighting their predicted impact on enzymatic structure and activity. In addition, we report biochemical and functional analysis, performed in HEK293 cells and patient fibroblasts and muscle cells, to show the relationship between the nature of ETFDH mutations, the variable impairment of enzyme function, and the different degrees of RR-MADD severity. Finally, we describe in detail 5 RR-MADD patients carrying different ETFDH mutations and presenting variable degrees of clinical symptom severity.
Asunto(s)
Flavoproteínas Transportadoras de Electrones , Proteínas Hierro-Azufre , Mitocondrias , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH , Animales , Carnitina/genética , Carnitina/metabolismo , Flavoproteínas Transportadoras de Electrones/genética , Flavoproteínas Transportadoras de Electrones/metabolismo , Humanos , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Mitocondrias/enzimología , Mitocondrias/genética , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/enzimología , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/genética , Ubiquinona/metabolismoRESUMEN
BACKGROUND: Deficiency of electron transfer flavoprotein dehydrogenase (ETFDH) is associated with multiple acyl-CoA dehydrogenase deficiency (MADD). This disorder is an autosomal recessive lipid storage myopathy (LSM) that exhibits a wide range of clinical features, including myopathy, weakness and multisystem dysfunctions. Many patients with late onset of MADD improve when treated with riboflavin and are also referred to as RR-MADD (riboflavin-responsive multiple Acyl-CoA dehydrogenase disorder). METHODS: In this study, we report the clinical and genetic characterization of a novel RR-MADD patient. Biochemical data were obtained from analysis of muscle and plasma samples. DNA and RNA were extracted from peripheral blood, and sequence analysis and expression study of ETFDH gene were performed. Finally, the impact of mutations on ETFDH folding was evaluated using bioinformatic tools. RESULTS: Patient initially presented with vomiting, muscle weakness, and acidosis. Muscle biopsy revealed typical myopathological patterns of lipid storage myopathy and blood acylcarnitine profiles showed a combined elevation of long and medium chain acylcarnitines, supporting the diagnosis of RR-MADD. Molecular analysis of ETFDH gene revealed two heterozygous mutations, a novel splice variation in intron 10, c.1285 + 1G > A, and the previously reported c.560C > T missense mutation. RT-PCR analysis showed an alteration of ETFDH RNA splicing which in turn should lead to the production of a truncated protein. The in silico prediction analysis of ETFDH tridimensional structure demonstrated that the missense mutation resulted in instability and loss of protein activation, while the splice site variation induced a dramatic conformational change of the truncated protein. After MCT diet supplemented with carnitine and riboflavin, the patient showed significant biochemical and clinical improvement, in spite of severe molecular defect. CONCLUSION: This case report extends the spectrum of ETFDH mutations in MADD, providing further evidence that patients presenting at least one missense mutation in the FAD-binding domain may respond to either carnitine or riboflavin treatment, due to the recovery of some enzymatic activity.
Asunto(s)
Flavoproteínas Transportadoras de Electrones/genética , Proteínas Hierro-Azufre/genética , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/genética , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Carnitina/uso terapéutico , Simulación por Computador , Análisis Mutacional de ADN , Quimioterapia Combinada , Flavoproteínas Transportadoras de Electrones/metabolismo , Femenino , Humanos , Proteínas Hierro-Azufre/metabolismo , Persona de Mediana Edad , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/tratamiento farmacológico , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/enzimología , Músculo Esquelético/enzimología , Mutación Missense , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Conformación Proteica , Riboflavina/uso terapéuticoRESUMEN
This article reports the results of tandem mass spectrometry and the mutation features of the ETFDH gene for an infant with multiple acyl-CoA dehydrogenase deficiency. The results of tandem mass spectrometry showed that C14 : 1, C8, C6, C10, and C12 increased. Exon sequencing was performed on this infant and his parents and revealed double heterozygous mutations in the ETFDH gene of the infant: c.992A>T and c.1450T>C. The former was inherited from his mother, and the latter was inherited from his father. c.1450T>C was shown to be the pathogenic mutation in the HGMD database. PolyPhen2, SIFT, and PROVEAN all predicted that the novel mutation c.992A>T might be pathogenic, and the mutant amino acids were highly conserved across various species. The findings expand the mutation spectrum of the ETFDH gene, and provide molecular evidence for the etiological diagnosis of the patient with multiple acyl-CoA dehydrogenase deficiency as well as for the genetic counseling and prenatal diagnosis in the family.
Asunto(s)
Flavoproteínas Transportadoras de Electrones/genética , Proteínas Hierro-Azufre/genética , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Secuencia de Bases , Exones , Humanos , Recién Nacido , Masculino , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/enzimología , MutaciónRESUMEN
The heterodimeric human (h) electron-transferring flavoprotein (ETF) transfers electrons from at least 13 different flavin dehydrogenases to the mitochondrial respiratory chain through a non-covalently bound FAD cofactor. Here, we describe the discovery of an irreversible and pH-dependent oxidation of the 8α-methyl group to 8-formyl-FAD (8f-FAD), which represents a unique chemical modification of a flavin cofactor in the human flavoproteome. Furthermore, a set of hETF variants revealed that several conserved amino acid residues in the FAD-binding pocket of electron-transferring flavoproteins are required for the conversion to the formyl group. Two of the variants generated in our study, namely αR249C and αT266M, cause glutaric aciduria type II, a severe inherited disease. Both of the variants showed impaired formation of 8f-FAD shedding new light on the potential molecular cause of disease development. Interestingly, the conversion of FAD to 8f-FAD yields a very stable flavin semiquinone that exhibited slightly lower rates of electron transfer in an artificial assay system than hETF containing FAD. In contrast, the formation of 8f-FAD enhanced the affinity to human dimethylglycine dehydrogenase 5-fold, indicating that formation of 8f-FAD modulates the interaction of hETF with client enzymes in the mitochondrial matrix. Thus, we hypothesize that the FAD cofactor bound to hETF is subject to oxidation in the alkaline (pH 8) environment of the mitochondrial matrix, which may modulate electron transport between client dehydrogenases and the respiratory chain. This discovery challenges the current concepts of electron transfer processes in mitochondria.
Asunto(s)
Flavoproteínas Transportadoras de Electrones/metabolismo , Flavina-Adenina Dinucleótido/análogos & derivados , Flavina-Adenina Dinucleótido/metabolismo , Modelos Moleculares , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Biocatálisis , Dominio Catalítico , Secuencia Conservada , Transporte de Electrón , Flavoproteínas Transportadoras de Electrones/química , Flavoproteínas Transportadoras de Electrones/genética , Flavina-Adenina Dinucleótido/química , Humanos , Concentración de Iones de Hidrógeno , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/enzimología , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/genética , Mutagénesis Sitio-Dirigida , Mutación , Oxidación-Reducción , Ingeniería de Proteínas , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
OBJECTIVE: To analyze the clinical features and gene mutations in an adolescent patient affected with late-onset multiple aeyl-CoA dehydrogenase deficiency (MADD) with severe fatty liver. METHODS: Potential mutations of the ETFDH gene were detected with polymerase chain reaction (PCR) and DNA sequencing. RESULTS: The 13-year-and-10-month girl has presented with weakness without any other special manifestation. Laboratory tests demonstrated an elevation of myocardial enzyme levels, total cholesterol, lactic acid and abnormal serum free fatty acids. H magnetic resonance spectroscopy revealed severe fatty liver. An increase in multiple plasma acyl-carnitines was detected by gas chromatography/mass spectrometry and isobutyrylglycine in urine by screening with tandem mass spectrometry. Genetic analysis demonstrated 2 heterozygous missense mutations c.250G>A (p.Ala84Thr) and c.353G>T (p.Cys118Phe) in the ETFDH gene. The diagnosis of MADD was confirmed. The patient was given large dose of vitamin B2, which resulted in rapid clinical and biochemical improvement. CONCLUSION: A common mutation c.250G>A and a novel mutation c.353G>T in the ETFDH gene were identified in the patient. The pathogenic role of c.353G>T (p.Cys118Phe) deserves further study. Early diagnosis of MADD and appropriate therapy is crucial for the prognosis.
Asunto(s)
Flavoproteínas Transportadoras de Electrones/genética , Hígado Graso/genética , Proteínas Hierro-Azufre/genética , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Adolescente , Adulto , Secuencia de Bases , Ácidos Grasos no Esterificados/sangre , Hígado Graso/sangre , Hígado Graso/enzimología , Femenino , Humanos , Lactante , Masculino , Datos de Secuencia Molecular , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/sangre , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/enzimología , Mutación , LinajeRESUMEN
OBJECTIVE: To identify pathogenic mutation in a boy affected with riboflavin responsive-multiple acyl-CoA dehydrogenase deficiency (RR-MADD). METHODS: The patient was initially diagnosed as primary carnitine deficiency (PCD) and has been treated with carnitine supplementation for 7 years. Clinical manifestations and characteristics of fibula muscle specimen were analyzed. Potential mutation in electron transfer flavoprotein dehydrogenase (ETFDH) gene (for the patient and his parents) and carnitine transfer protein gene (SLC22A5) (for the patient) was screened. RESULTS: Electronic microscopy of the muscle specimen has suggested lipid storage myopathy. Mutation analysis has found that the patient carried compound heterozygous mutations, c.250G>A and c.380T>C, in exon 3 of the ETFDH gene, whilst his father and mother were heterozygous for the c.380T>C and c.250G>A mutations, respectively. Screening of the SLC22A5 gene has yielded no clinically meaningful result. After the establishment of diagnosis of RR-MADD, the condition of the patient has improved greatly with supplementation of high doses of riboflavin along with continuous carnitine supplement. CONCLUSION: The c.250G>A (p.Ala84Thr) mutation of exon 3 of the ETFDH gene has been a hot spot in Southern Chinese population, whilst the c.380T>C (p.Leu127Pro) is rarely reported. Our case has suggested that therapeutic diagnosis cannot substitute genetic testing. The mechanism for having stabilized the patient with only carnitine supplementation for 7 years needs further investigation.
Asunto(s)
Flavoproteínas Transportadoras de Electrones/genética , Proteínas Hierro-Azufre/genética , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/genética , Proteínas de Transporte de Catión Orgánico/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Riboflavina/metabolismo , Adolescente , Adulto , Secuencia de Bases , Niño , Análisis Mutacional de ADN , Flavoproteínas Transportadoras de Electrones/metabolismo , Femenino , Humanos , Proteínas Hierro-Azufre/metabolismo , Masculino , Datos de Secuencia Molecular , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/enzimología , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/metabolismo , Músculo Esquelético/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Miembro 5 de la Familia 22 de Transportadores de SolutosRESUMEN
We describe the pregnancy of a patient of French-Canadian descent with multiple Acyl-CoA dehydrogenation deficiency (MADD). The proband was found to harbor a previously reported homozygous missense mutation on EFTDH gene (p.Pro534Leu:c.1601C>T) confirming the biochemical diagnosis of MADD. This mutation was not found in 50 controls from the same ethnic background. The clinical and molecular information of all patients with ETFDH mutations reported in the literature up-to-date are summarized.
Asunto(s)
Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/complicaciones , Complicaciones del Embarazo/enzimología , Flavoproteínas Transportadoras de Electrones/genética , Femenino , Humanos , Proteínas Hierro-Azufre/genética , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/enzimología , Mutación/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Embarazo , Adulto JovenRESUMEN
We report a case of a 37 year-old male with multiple acyl-CoA dehydrogenation deficiency (MADD). The patient had suffered from exercise intolerance in his hip and thigh muscles for one year. Then, restriction of carbohydrates for a diet made his symptoms rapidly deteriorate. Blood test revealed compound heterozygosity for two novel missense mutations in the electron transfer flavoprotein dehydrogenase gene (ETFDH), and an abnormal LDH isoenzyme pattern: LDH-1 (60.0%) and LDH-2 (26.0%) predominated with abnormally elevated LDH-1/LDH-2 ratio (2.3), compared with muscle-derived LDH-5 (4.0%). Oral riboflavin treatment significantly improved his exercise intolerance and the LDH profile: LDH-1 (34.4%), LDH-2 (34.9%), LDH-5 (6.9%) and LDH-1/LDH-2 ratio (1.0). The abnormal LDH isoenzyme pattern may be one feature of adult-onset MADD selectively affecting type I muscle fibers with relatively high LDH-1 content.
Asunto(s)
L-Lactato Deshidrogenasa/sangre , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/diagnóstico , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/enzimología , Adulto , Humanos , Isoenzimas/sangre , Lactato Deshidrogenasa 5 , Masculino , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/genética , Riboflavina/uso terapéuticoRESUMEN
This is the case of a 41 year old man, suffering general weakness and elevated liver enzymes, sensitive to a treatment with riboflavin and coenzyme Q(10). Tandem mass spectroscopy and molecular analysis reveal a multiple acyl-CoA-dehydrogenase deficiency (MADD) with two novel heterozygote missense mutations of the EFTDH gene.
Asunto(s)
Transporte de Electrón/genética , Flavoproteínas Transportadoras de Electrones/deficiencia , Flavoproteínas Transportadoras de Electrones/genética , Proteínas Hierro-Azufre/deficiencia , Proteínas Hierro-Azufre/genética , Enfermedades Mitocondriales/enzimología , Enfermedades Mitocondriales/genética , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/enzimología , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/genética , Mutación Missense/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/deficiencia , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Adulto , Tamización de Portadores Genéticos , Humanos , Trastornos del Metabolismo de los Lípidos/diagnóstico , Trastornos del Metabolismo de los Lípidos/enzimología , Trastornos del Metabolismo de los Lípidos/genética , Masculino , Enfermedades Mitocondriales/diagnóstico , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/diagnósticoRESUMEN
We describe a 22-year-old male who developed severe hypoglycemia and lethargy during an acute illness at 4 months of age and subsequently grew and developed normally. At age 4 years he developed recurrent vomiting with mild hyperammonemia and dehydration requiring frequent hospitalizations. Glutaric aciduria Type II was suspected based upon biochemical findings and managed with cornstarch, carnitine and riboflavin supplements. He did not experience metabolic crises between ages 4-12 years. He experienced recurrent vomiting, mild hyperammonemia, and generalized weakness associated with acute illnesses and growth spurts. At age 18 years, he developed exercise intolerance and proximal muscle weakness leading to the identification of multiple acyl-CoA dehydrogenase and complex II/III deficiencies in both skeletal muscle and liver. Subsequent molecular characterization of the ETFDH gene revealed novel heterozygous mutations, p.G274X:c.820 G > T (exon 7) and p.P534L: c.1601 C > T (exon 12), the latter within the iron sulfur-cluster and predicted to affect ubiquinone reductase activity of ETFDH and the docking of ETF to ETFDH. Our case supports the concept of a structural interaction between ETFDH and other enzyme partners, and suggests that the conformational change upon ETF binding to ETFDH may play a key role in linking ETFDH to II/III super-complex formation.
Asunto(s)
Complejo III de Transporte de Electrones/deficiencia , Complejo II de Transporte de Electrones/deficiencia , Flavoproteínas Transportadoras de Electrones/genética , Proteínas Hierro-Azufre/genética , Hígado/enzimología , Errores Innatos del Metabolismo/genética , Enfermedades Mitocondriales/genética , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/genética , Músculo Esquelético/enzimología , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Biomarcadores/sangre , Biomarcadores/orina , Análisis Mutacional de ADN , Complejo II de Transporte de Electrones/química , Complejo II de Transporte de Electrones/genética , Complejo II de Transporte de Electrones/metabolismo , Complejo III de Transporte de Electrones/química , Complejo III de Transporte de Electrones/genética , Complejo III de Transporte de Electrones/metabolismo , Flavoproteínas Transportadoras de Electrones/química , Flavoproteínas Transportadoras de Electrones/deficiencia , Exones , Predisposición Genética a la Enfermedad , Heterocigoto , Humanos , Proteínas Hierro-Azufre/química , Proteínas Hierro-Azufre/deficiencia , Masculino , Errores Innatos del Metabolismo/diagnóstico , Errores Innatos del Metabolismo/enzimología , Enfermedades Mitocondriales/diagnóstico , Enfermedades Mitocondriales/enzimología , Simulación del Acoplamiento Molecular , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/diagnóstico , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/deficiencia , Fenotipo , Unión Proteica , Conformación Proteica , Adulto JovenRESUMEN
We report an adolescent case of late-onset riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency (MADD) characterized by intermittent nausea and depressive state as early symptoms. At the age of 12 years and 11 months, the patient experienced intermittent nausea and vomiting, and depressive state. She was on medication for depression for 5 months but it was ineffective. Brain magnetic resonance imaging showed disseminated high-intensity areas in the periventricular white matter and in the splenium of the corpus callosum on T2-weighted images and fluid-attenuated inversion-recovery images. Progressive muscle weakness occurred and blood creatine kinase level was found to be elevated. The muscle biopsy revealed lipid storage myopathy. Urine organic acid analysis and mutation analysis of the ETFDH gene confirmed the diagnosis of MADD. With oral supplements of riboflavin and l-carnitine, in addition to a high-calorie and reduced-fat diet, her clinical symptoms improved dramatically. Early diagnosis is important because riboflavin treatment has been effective in a significant number of patients with MADD.