RESUMEN
l-2-hydroxyglutarate dehydrogenase (L2HGDH) is a mitochondrial membrane-associated metabolic enzyme, which catalyzes the oxidation of l-2-hydroxyglutarate (l-2-HG) to 2-oxoglutarate (2-OG). Mutations in human L2HGDH lead to abnormal accumulation of l-2-HG, which causes a neurometabolic disorder named l-2-hydroxyglutaric aciduria (l-2-HGA). Here, we report the crystal structures of Drosophila melanogaster L2HGDH (dmL2HGDH) in FAD-bound form and in complex with FAD and 2-OG and show that dmL2HGDH exhibits high activity and substrate specificity for l-2-HG. dmL2HGDH consists of an FAD-binding domain and a substrate-binding domain, and the active site is located at the interface of the two domains with 2-OG binding to the re-face of the isoalloxazine moiety of FAD. Mutagenesis and activity assay confirmed the functional roles of key residues involved in the substrate binding and catalytic reaction and showed that most of the mutations of dmL2HGDH equivalent to l-2-HGA-associated mutations of human L2HGDH led to complete loss of the activity. The structural and biochemical data together reveal the molecular basis for the substrate specificity and catalytic mechanism of L2HGDH and provide insights into the functional roles of human L2HGDH mutations in the pathogeneses of l-2-HGA.
Asunto(s)
Oxidorreductasas de Alcohol , Encefalopatías Metabólicas Innatas , Drosophila melanogaster , Modelos Moleculares , Animales , Humanos , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Encefalopatías Metabólicas Innatas/enzimología , Encefalopatías Metabólicas Innatas/genética , Encefalopatías Metabólicas Innatas/fisiopatología , Drosophila melanogaster/enzimología , Glutaratos/metabolismo , Mutación , Dominio Catalítico/genética , Especificidad por Sustrato/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Succinyl-CoA:3-oxoacid coenzyme A transferase deficiency (SCOTD) is a rare autosomal recessive disorder of ketone body utilization caused by mutations in OXCT1. We performed a systematic literature search and evaluated clinical, biochemical and genetic data on 34 previously published and 10 novel patients with SCOTD. Structural mapping and in silico analysis of protein variants is also presented. All patients presented with severe ketoacidotic episodes. Age at first symptoms ranged from 36 h to 3 years (median 7 months). About 70% of patients manifested in the first year of life, approximately one quarter already within the neonatal period. Two patients died, while the remainder (95%) were alive at the time of the report. Almost all the surviving patients (92%) showed normal psychomotor development and no neurologic abnormalities. A total of 29 missense mutations are reported. Analysis of the published crystal structure of the human SCOT enzyme, paired with both sequence-based and structure-based methods to predict variant pathogenicity, provides insight into the biochemical consequences of the reported variants. Pathogenic variants cluster in SCOT protein regions that affect certain structures of the protein. The described pathogenic variants can be viewed in an interactive map of the SCOT protein at https://michelanglo.sgc.ox.ac.uk/r/oxct. This comprehensive data analysis provides a systematic overview of all cases of SCOTD published to date. Although SCOTD is a rather benign disorder with often favourable outcome, metabolic crises can be life-threatening or even fatal. As the diagnosis can only be made by enzyme studies or mutation analyses, SCOTD may be underdiagnosed.
Asunto(s)
Acidosis , Encefalopatías Metabólicas Innatas , Coenzima A Transferasas/deficiencia , Mutación Missense , Trastornos del Neurodesarrollo , Acidosis/enzimología , Acidosis/genética , Encefalopatías Metabólicas Innatas/enzimología , Encefalopatías Metabólicas Innatas/genética , Coenzima A Transferasas/química , Coenzima A Transferasas/genética , Coenzima A Transferasas/metabolismo , Cristalografía por Rayos X , Humanos , Cuerpos Cetónicos/química , Cuerpos Cetónicos/genética , Cuerpos Cetónicos/metabolismo , Trastornos del Neurodesarrollo/enzimología , Trastornos del Neurodesarrollo/genética , Dominios ProteicosAsunto(s)
Aspartato Carbamoiltransferasa/deficiencia , Encefalopatías Metabólicas Innatas , Carbamoil-Fosfato Sintasa (Glutamina-Hidrolizante)/deficiencia , Dihidroorotasa/deficiencia , Epilepsia Refractaria , Anticonvulsivantes/administración & dosificación , Encefalopatías Metabólicas Innatas/complicaciones , Encefalopatías Metabólicas Innatas/diagnóstico , Encefalopatías Metabólicas Innatas/tratamiento farmacológico , Encefalopatías Metabólicas Innatas/enzimología , Consanguinidad , Epilepsia Refractaria/diagnóstico , Epilepsia Refractaria/tratamiento farmacológico , Epilepsia Refractaria/enzimología , Epilepsia Refractaria/etiología , Humanos , Lactante , Fenitoína/administración & dosificación , Uridina/administración & dosificaciónRESUMEN
D-2-hydroxyglutaric aciduria is a neurometabolic disorder, characterized by the accumulation of D-2-hydroxyglutarate (D-2HG) in human mitochondria. Increased levels of D-2HG are detected in humans exhibiting point mutations in the genes encoding isocitrate dehydrogenase, citrate carrier, the electron transferring flavoprotein (ETF) and its downstream electron acceptor ETF-ubiquinone oxidoreductase or D-2-hydroxyglutarate dehydrogenase (hD2HGDH). However, while the pathogenicity of several amino acid replacements in the former four proteins has been studied extensively, not much is known about the effect of certain point mutations on the biochemical properties of hD2HGDH. Therefore, we recombinantly produced wild type hD2HGDH as well as two recently identified disease-related variants (hD2HGDH-I147S and -V444A) and performed their detailed biochemical characterization. We could show that hD2HGDH is a FAD dependent protein, which is able to catalyze the oxidation of D-2HG and D-lactate to α-ketoglutarate and pyruvate, respectively. The two variants were obtained as apo-proteins and were thus catalytically inactive. The addition of FAD failed to restore enzymatic activity of the variants, indicating that the cofactor binding site is compromised by the single amino acid replacements. Further analyses revealed that both variants form aggregates that are apparently unable to bind the FAD cofactor. Since, D-2-hydroxyglutaric aciduria may also result from a loss of function of either the ETF or its downstream electron acceptor ETF-ubiquinone oxidoreductase, ETF may serve as the cognate electron acceptor of reduced hD2HGDH. Here, we show that hD2HGDH directly reduces recombinant human ETF, thus establishing a metabolic link between the oxidation of D-2-hydroxyglutarate and the mitochondrial electron transport chain.
Asunto(s)
Oxidorreductasas de Alcohol/química , Encefalopatías Metabólicas Innatas/enzimología , Mutación Missense , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Sustitución de Aminoácidos , Encefalopatías Metabólicas Innatas/genética , Catálisis , Flavoproteínas Transportadoras de Electrones/química , Flavoproteínas Transportadoras de Electrones/metabolismo , Glutaratos/química , Glutaratos/metabolismo , Humanos , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Ácido Láctico/química , Ácido Láctico/metabolismoRESUMEN
Missense mutations of the human mitochondrial citrate carrier, encoded by the SLC25A1 gene, lead to an autosomal recessive neurometabolic disorder characterised by neonatal-onset encephalopathy with severe muscular weakness, intractable seizures, respiratory distress, and lack of psychomotor development, often resulting in early death. Here, we have measured the effect of all twelve known pathogenic mutations on the transport activity. The results show that nine mutations abolish transport of citrate completely, whereas the other three reduce the transport rate by >70%, indicating that impaired citrate transport is the most likely primary cause of the disease. Some mutations may be detrimental to the structure of the carrier, whereas others may impair key functional elements, such as the substrate binding site and the salt bridge network on the matrix side of the carrier. To understand the consequences of impaired citrate transport on metabolism, the substrate specificity was also determined, showing that the human citrate carrier predominantly transports citrate, isocitrate, cis-aconitate, phosphoenolpyruvate and malate. Although D-2- and L-2 hydroxyglutaric aciduria is a metabolic hallmark of the disease, it is unlikely that the citrate carrier plays a significant role in the removal of hydroxyglutarate from the cytosol for oxidation to oxoglutarate in the mitochondrial matrix. In contrast, computer simulations of central metabolism predict that the export of citrate from the mitochondrion cannot be fully compensated by other pathways, restricting the cytosolic production of acetyl-CoA that is required for the synthesis of lipids, sterols, dolichols and ubiquinone, which in turn explains the severe disease phenotypes.
Asunto(s)
Proteínas de Transporte de Anión , Ácido Cítrico/metabolismo , Simulación por Computador , Dolicoles , Proteínas Mitocondriales , Modelos Biológicos , Mutación Missense , Esteroles , Ubiquinona , Proteínas de Transporte de Anión/química , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Transporte Biológico Activo/genética , Encefalopatías Metabólicas Innatas/enzimología , Encefalopatías Metabólicas Innatas/genética , Dominio Catalítico , Dolicoles/biosíntesis , Dolicoles/química , Dolicoles/genética , Humanos , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Transportadores de Anión Orgánico , Esteroles/biosíntesis , Esteroles/química , Esteroles/metabolismo , Ubiquinona/biosíntesis , Ubiquinona/química , Ubiquinona/genéticaRESUMEN
Carnosinase (CN1) is a dipeptidase, encoded by the CNDP1 gene, that degrades histidine-containing dipeptides, such as carnosine, anserine and homocarnosine. Loss of CN1 function (also called carnosinase deficiency or aminoacyl-histidine dipeptidase deficiency) has been reported in a small number of patients with highly elevated blood carnosine concentrations, denoted carnosinaemia; it is unclear whether the variety of clinical symptoms in these individuals is causally related to carnosinase deficiency. Reduced CN1 function should increase serum carnosine concentrations but the genetic basis of carnosinaemia has not been formally confirmed to be due to CNDP1 mutations. A CNDP1 polymorphism associated with low CN1 activity correlates with significantly reduced risk for diabetic nephropathy, especially in women with type 2 diabetes, and may slow progression of chronic kidney disease in children with glomerulonephritis. Studies in rodents demonstrate antiproteinuric and vasculoprotective effects of carnosine, the precise molecular mechanisms, however, are still incompletely understood. Thus, carnosinemia due to CN1 deficiency may be a non-disease; in contrast, carnosine may potentially protect against long-term sequelae of reactive metabolites accumulating, e.g. in diabetes and chronic renal failure.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Encefalopatías Metabólicas Innatas/enzimología , Diabetes Mellitus Tipo 2/enzimología , Nefropatías Diabéticas/enzimología , Dipeptidasas/deficiencia , Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Errores Innatos del Metabolismo de los Aminoácidos/epidemiología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Animales , Encefalopatías Metabólicas Innatas/diagnóstico , Encefalopatías Metabólicas Innatas/epidemiología , Encefalopatías Metabólicas Innatas/genética , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/epidemiología , Diabetes Mellitus Tipo 2/genética , Nefropatías Diabéticas/diagnóstico , Nefropatías Diabéticas/epidemiología , Nefropatías Diabéticas/genética , Dipeptidasas/genética , Humanos , Mutación , Polimorfismo Genético , Pronóstico , Factores Protectores , Factores de RiesgoRESUMEN
OBJECTIVE: To explore pathogenic mutation in a family affected with 2-hydroxyglutaric aciduria. METHODS: Exons of 3 candidate genes, including L2HGDH, D2HGDH and SLC25A1, were amplified with polymerase chain reaction and subjected to direct sequencing. RESULTS: DNA sequencing has found that the proband and his affected younger brother have both carried a heterozygous mutation c.845G>A (p.R282Q) in the exon 7 of the L2HGDH gene. The same mutation was not detected in the his sister who was healthy. Pedigree analysis has confirmed that the above mutation was inherited from the mother. No mutation was detected in exons and flanking sequences of the D2HGDH and SLC25A1 genes. CONCLUSION: Mutation of the L2HGDH gene probably underlies the 2-hydroxyglutaric aciduria in this family.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Encefalopatías Metabólicas Innatas/enzimología , Encefalopatías Metabólicas Innatas/genética , Mutación , Secuencia de Bases , Encéfalo/diagnóstico por imagen , Encefalopatías Metabólicas Innatas/diagnóstico por imagen , Niño , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Radiografía , Adulto JovenRESUMEN
Mitochondrial energy metabolism disorder is one of the important reasons of leukodystrophy. Mutations of mitochondrial complex I genes have been implicated in more common neurological disorders such as Leigh syndrome. We describe a case of a child manifested as regression of mental and motor development, aggravated obviously after suffering infection. Physical and auxiliary examinations demonstrated that a series of changes including white matter lesions of magnetic resonance imaging, peripheral neuropathy with high muscle tension and hyperreflexia of limbs pointed to the diagnosis of leukodystrophy, with what can't explain the high levels of lactate and creatine kinase. Spontaneously, genetic analysis covered known leukodystrophy and mitochondrial genes were adapted for this child and his parents. Results showed the child was compound heterozygous mutation (c.278A > G; c.247G > A) within exon 2 in the NDUFAF1 gene, his parents carried a heterozygous mutation each. The authors report a case of leukodystrophy associated with mitochondrial complex I deficiency due to a novel mutation in the NDUFAF1 gene. This is the first report that NDUFAF1 mutations cause leukodystrophy.
Asunto(s)
Encefalopatías Metabólicas Innatas/genética , Exones , Heterocigoto , Proteínas Mitocondriales/deficiencia , NADH Deshidrogenasa/deficiencia , Mutación Puntual , Encefalopatías Metabólicas Innatas/diagnóstico , Encefalopatías Metabólicas Innatas/enzimología , Encefalopatías Metabólicas Innatas/patología , Humanos , Lactante , MasculinoRESUMEN
Creatine is physiologically provided equally by diet and by endogenous synthesis from arginine and glycine with successive involvements of arginine glycine amidinotransferase [AGAT] and guanidinoacetate methyl transferase [GAMT]. A specific plasma membrane transporter, creatine transporter [CRTR] (SLC6A8), further enables cells to incorporate creatine and through uptake of its precursor, guanidinoacetate, also directly contributes to creatine biosynthesis. Breakthrough in the role of creatine has arisen from studies on creatine deficiency disorders. Primary creatine disorders are inherited as autosomal recessive (mutations affecting GATM [for glycine-amidinotransferase, mitochondrial]) and GAMT genes) or X-linked (SLC6A8 gene) traits. They have highlighted the role of creatine in brain functions altered in patients (global developmental delay, intellectual disability, behavioral disorders). Creatine modulates GABAergic and glutamatergic cerebral pathways, presynaptic CRTR (SLC6A8) ensuring re-uptake of synaptic creatine. Secondary creatine disorders, addressing other genes, have stressed the extraordinary imbrication of creatine metabolism with many other cellular pathways. This high dependence on multiple pathways supports creatine as a cellular sensor, to cell methylation and energy status. Creatine biosynthesis consumes 40% of methyl groups produced as S-adenosylmethionine, and creatine uptake is controlled by AMP activated protein kinase, a ubiquitous sensor of energy depletion. Today, creatine is considered as a potential sensor of cell methylation and energy status, a neurotransmitter influencing key (GABAergic and glutamatergic) CNS neurotransmission, therapeutic agent with anaplerotic properties (towards creatine kinases [creatine-creatine phosphate cycle] and creatine neurotransmission), energetic and antioxidant compound (benefits in degenerative diseases through protection against energy depletion and oxidant species) with osmolyte behavior (retention of water by muscle). This review encompasses all these aspects by providing an illustrated metabolic account for brain and body creatine in health and disease, an algorithm to diagnose metabolic and gene bases of primary and secondary creatine deficiencies, and a metabolic exploration by (1)H-MRS assessment of cerebral creatine levels and response to therapeutic measures.
Asunto(s)
Amidinotransferasas/metabolismo , Creatina/metabolismo , Guanidinoacetato N-Metiltransferasa/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Transporte de Neurotransmisores en la Membrana Plasmática/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Amidinotransferasas/deficiencia , Amidinotransferasas/genética , Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/deficiencia , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Animales , Transporte Biológico Activo , Encefalopatías Metabólicas Innatas/diagnóstico , Encefalopatías Metabólicas Innatas/enzimología , Encefalopatías Metabólicas Innatas/genética , Encefalopatías Metabólicas Innatas/metabolismo , Creatina/biosíntesis , Creatina/deficiencia , Creatina/genética , Discapacidades del Desarrollo/diagnóstico , Discapacidades del Desarrollo/enzimología , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/metabolismo , Metabolismo Energético , Guanidinoacetato N-Metiltransferasa/deficiencia , Guanidinoacetato N-Metiltransferasa/genética , Atrofia Girata/diagnóstico , Atrofia Girata/enzimología , Atrofia Girata/genética , Atrofia Girata/metabolismo , Humanos , Hiperamonemia/diagnóstico , Hiperamonemia/enzimología , Hiperamonemia/genética , Hiperamonemia/metabolismo , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/enzimología , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Trastornos del Desarrollo del Lenguaje/diagnóstico , Trastornos del Desarrollo del Lenguaje/enzimología , Trastornos del Desarrollo del Lenguaje/genética , Trastornos del Desarrollo del Lenguaje/metabolismo , Discapacidad Intelectual Ligada al Cromosoma X/diagnóstico , Discapacidad Intelectual Ligada al Cromosoma X/enzimología , Discapacidad Intelectual Ligada al Cromosoma X/genética , Discapacidad Intelectual Ligada al Cromosoma X/metabolismo , Metilación , Proteínas de Transporte de Membrana Mitocondrial , Trastornos del Movimiento/congénitoRESUMEN
OBJECTIVE: Acyl-CoA oxidase (ACOX1) deficiency is a rare disorder of peroxisomal very-long chain fatty acid oxidation. No reports detailing attempted treatment, longitudinal imaging, or neuropathology exist. We describe the natural history of clinical symptoms and brain imaging in two siblings with ACOX1 deficiency, including the younger sibling's response to allogeneic unrelated donor hematopoietic stem cell transplantation (HSCT). METHODS: We conducted retrospective chart review to obtain clinical history, neuro-imaging, and neuropathology data. ACOX1 genotyping were performed to confirm the disease. In vitro fibroblast and neural stem cell fatty acid oxidation assays were also performed. RESULTS: Both patients experienced a fatal neurodegenerative course, with late-stage cerebellar and cerebral gray matter atrophy. Serial brain magnetic resonance imaging in the younger sibling indicated demyelination began in the medulla and progressed rostrally to include the white matter of the cerebellum, pons, midbrain, and eventually subcortical white matter. The successfully engrafted younger sibling had less brain inflammation, cortical atrophy, and neuronal loss on neuro-imaging and neuropathology compared to the untreated older sister. Fibroblasts and stem cells demonstrated deficient very long chain fatty acid oxidation. INTERPRETATION: Although HSCT did not halt the course of ACOX1 deficiency, it reduced the extent of white matter inflammation in the brain. Demyelination continued because of ongoing neuronal loss, which may be due to inability of transplant to prevent progression of gray matter disease, adverse effects of chronic corticosteroid use to control graft-versus-host disease, or intervention occurring beyond a critical point for therapeutic efficacy.
Asunto(s)
Acil-CoA Oxidasa/deficiencia , Encefalopatías Metabólicas Innatas/terapia , Trasplante de Células Madre Hematopoyéticas/métodos , Encéfalo/patología , Encefalopatías Metabólicas Innatas/enzimología , Encefalopatías Metabólicas Innatas/patología , Preescolar , Resultado Fatal , Femenino , Movimientos de la Cabeza/fisiología , Humanos , Lactante , Recién Nacido , Masculino , Hipotonía Muscular/etiología , Células-Madre Neurales/trasplante , Hermanos , Resultado del TratamientoRESUMEN
Complex II deficiency is a rare cause of mitochondrial respiratory chain defects with a prevalence of 2-23%. It is exclusively nuclear encoded and functions in the citric acid cycle by oxidizing succinate to fumarate and in the mitochondrial electron transport chain (ETC) by transferring electrons to ubiquinone. Of the four subunits, SDHA and SDHB are catalytic and SDHC and SDHD are anchoring. Mutations in SDHA and SDHAF1 (assembly factor) have been found in patients with CII deficiency and a mitochondrial phenotype. We present a patient with CII deficiency with a previously undescribed phenotype of dilated cardiomyopathy, left ventricular noncompaction, failure to thrive, hypotonia, and developmental delay. Also, a comprehensive review of 36 cases published in the literature was undertaken. The results show that CII deficiency has a variable phenotype with no correlation with residual complex activity in muscle although the phenotype and enzyme activities are comparable within a family. For some, the condition was fatal in infancy, others had multisystem involvement and some had onset in adulthood with mild symptoms and normal cognition. Neurological involvement is most commonly observed and brain imaging commonly shows leukoencephalopathy, Leigh syndrome, or cerebellar atrophy. Mutations in SDHAF1 are associated with leukoencephalopathy. Other organ systems like heart, muscle, and eyes are only involved in about 50% of the cases but cardiomyopathy is associated with high mortality and morbidity. In some patients, riboflavin has provided clinical improvement.
Asunto(s)
Encefalopatías Metabólicas Innatas/diagnóstico , Succinato Deshidrogenasa/deficiencia , Encefalopatías Metabólicas Innatas/sangre , Encefalopatías Metabólicas Innatas/enzimología , Complejo II de Transporte de Electrones/deficiencia , Complejo II de Transporte de Electrones/genética , Resultado Fatal , Femenino , Humanos , Lactante , Ácido Láctico/sangre , Ácido Láctico/líquido cefalorraquídeo , Succinato Deshidrogenasa/genéticaRESUMEN
Hydrogen sulfide (H(2)S) is a recently described endogenously produced gaseous signaling molecule that influences various cellular processes in the central nervous system, cardiovascular system, and gastrointestinal tract. The biogenesis of H(2)S involves the cytoplasmic transsulfuration enzymes, cystathionine ß-synthase and γ-cystathionase, whereas its catabolism occurs in the mitochondrion and couples to the energy-yielding electron transfer chain. Low steady-state levels of H(2)S appear to be controlled primarily by efficient oxygen-dependent catabolism via sulfide quinone oxidoreductase, persulfide dioxygenase (ETHE1), rhodanese, and sulfite oxidase. Mutations in the persulfide dioxgenase, i.e. ETHE1, result in ethylmalonic encephalopathy, an inborn error of metabolism. In this study, we report the biochemical characterization and kinetic properties of human persulfide dioxygenase and describe the biochemical penalties associated with two patient mutations, T152I and D196N. Steady-state kinetic analysis reveals that the T152I mutation results in a 3-fold lower activity, which is correlated with a 3-fold lower iron content compared with the wild-type enzyme. The D196N mutation results in a 2-fold higher K(m) for the substrate, glutathione persulfide.
Asunto(s)
Encefalopatías Metabólicas Innatas/enzimología , Sulfuro de Hidrógeno/metabolismo , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Mutación Missense , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Púrpura/enzimología , Encefalopatías Metabólicas Innatas/genética , Encefalopatías Metabólicas Innatas/metabolismo , Humanos , Cinética , Proteínas Mitocondriales/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Púrpura/genética , Púrpura/metabolismoRESUMEN
We used exome sequencing of blood DNA in four unrelated patients to identify the genetic basis of metaphyseal chondromatosis with urinary excretion of D-2-hydroxy-glutaric acid (MC-HGA), a rare entity comprising severe chondrodysplasia, organic aciduria, and variable cerebral involvement. No evidence for recessive mutations was found; instead, two patients showed mutations in IDH1 predicting p.R132H and p.R132S as apparent somatic mosaicism. Sanger sequencing confirmed the presence of the mutation in blood DNA in one patient, and in blood and saliva (but not in fibroblast) DNA in the other patient. Mutations at codon 132 of IDH1 change the enzymatic specificity of the cytoplasmic isocitrate dehydrogenase enzyme. They result in increased D-2-hydroxy-glutarate production, α-ketoglutarate depletion, activation of HIF-1α (a key regulator of chondrocyte proliferation at the growth plate), and reduction of N-acetyl-aspartyl-glutamate level in glial cells. Thus, somatic mutations in IDH1 may explain all features of MC-HGA, including sporadic occurrence, metaphyseal disorganization, and chondromatosis, urinary excretion of D-2-hydroxy-glutaric acid, and reduced cerebral myelinization.
Asunto(s)
Encefalopatías Metabólicas Innatas/genética , Condromatosis/genética , Isocitrato Deshidrogenasa/genética , Encefalopatías Metabólicas Innatas/sangre , Encefalopatías Metabólicas Innatas/enzimología , Encefalopatías Metabólicas Innatas/patología , Encefalopatías Metabólicas Innatas/orina , Condromatosis/sangre , Condromatosis/enzimología , Condromatosis/patología , Análisis Mutacional de ADN/métodos , Exoma , Femenino , Estudios de Asociación Genética/métodos , Genoma Humano , Genotipo , Glutaratos/orina , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Lactante , Isocitrato Deshidrogenasa/sangre , Ácidos Cetoglutáricos/metabolismo , Masculino , Mutación , Saliva/química , Especificidad por SustratoRESUMEN
The recent discovery of heterozygous isocitrate dehydrogenase 2 (IDH2) mutations of residue Arg(140) to Gln(140) or Gly(140) (IDH2(wt/R140Q), IDH2(wt/R140G)) in d-2-hydroxyglutaric aciduria (D-2-HGA) has defined the primary genetic lesion in 50% of D-2-HGA patients, denoted type II. Overexpression studies with IDH1(R132H) and IDH2(R172K) mutations demonstrated that the enzymes acquired a new function, converting 2-ketoglutarate (2-KG) to d-2-hydroxyglutarate (D-2-HG), in lieu of the normal IDH reaction which reversibly converts isocitrate to 2-KG. To confirm the IDH2(wt/R140Q) gain-of-function in D-2-HGA type II, and to evaluate potential therapeutic strategies, we developed a specific and sensitive IDH2(wt/R140Q) enzyme assay in lymphoblasts. This assay determines gain-of-function activity which converts 2-KG to D-2-HG in homogenates of D-2-HGA type II lymphoblasts, and uses stable-isotope-labeled 2-keto[3,3,4,4-(2)H(4)]glutarate. The specificity and sensitivity of the assay are enhanced with chiral separation and detection of stable-isotope-labeled D-2-HG by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Eleven potential inhibitors of IDH2(wt/R140Q) enzyme activity were evaluated with this procedure. The mean reaction rate in D-2-HGA type II lymphoblasts was 8-fold higher than that of controls and D-2-HGA type I cells (14.4nmolh(-1)mgprotein(-1) vs. 1.9), with a corresponding 140-fold increase in intracellular D-2-HG level. Optimal inhibition of IDH2(wt/R140Q) activity was obtained with oxaloacetate, which competitively inhibited IDH2(wt/R140Q) activity. Lymphoblast IDH2(wt/R140Q) showed long-term cell culture stability without loss of the heterozygous IDH2(wt/R140Q) mutation, underscoring the utility of the lymphoblast model for future biochemical and therapeutic studies.
Asunto(s)
Encefalopatías Metabólicas Innatas/enzimología , Isocitrato Deshidrogenasa/metabolismo , Linfocitos/enzimología , Encefalopatías Metabólicas Innatas/genética , Encefalopatías Metabólicas Innatas/terapia , Estudios de Casos y Controles , Células Cultivadas , Cromatografía Liquida , Inhibidores Enzimáticos/farmacología , Glutaratos/metabolismo , Humanos , Isocitrato Deshidrogenasa/genética , Ácidos Cetoglutáricos/metabolismo , Linfocitos/efectos de los fármacos , Linfocitos/patología , Mutación/genética , Sensibilidad y Especificidad , Espectrometría de Masas en TándemRESUMEN
Ethylmalonic encephalopathy (EE) is an autosomal recessive, invariably fatal disorder associated with mutations in ETHE1, a gene encoding a mitochondrial sulfur dioxygenase (SDO). The main consequence of the absence of Ethe1-SDO is the accumulation of sulfide (H(2)S) in critical tissues, including colonic mucosa, liver, muscle, and brain. To make progress in the elucidation of the biochemical mechanisms leading to cytochrome c oxidase (COX) deficiency, we (i) generated tissue-specific conditional Ethe1 knockout mice to clarify the different contributions of endogenous and exogenous H(2)S production, and (ii) studied the development of H(2)S-driven COX deficiency in Ethe1(-/-) mouse tissues and human cells. Ethe1(-/-) conditional animals displayed COX deficiency limited to the specific targeted tissue. The accumulation of H(2)S over time causes progressive COX deficiency in animal tissues and human cells, which is associated with reduced amount of COX holoenzyme, and of several COX subunits, including mitochondrially encoded cytochrome c oxidase 1 (MTCO1), MTCO2, COX4, and COX5A. This reduction is not paralleled by consistent downregulation in expression of the corresponding mRNAs. Tissue-specific ablation of Ethe1 causes COX deficiency in targeted organs, suggesting that failure in neutralizing endogenous, tissue-specific production of H(2)S is sufficient to cause the biochemical defect but neither to determine a clinical impact nor to induce the biomarker profile typical of EE. The mechanism by which H(2)S causes COX deficiency consists of rapid heme a inhibition and accelerated long-term degradation of COX subunits. However, the pleiotropic devastating effects of H(2)S accumulation in EE cannot be fully explained by the sole defect of COX in critical tissues, but are likely consequent to several toxic actions on a number of enzymatic activities in different tissues, including endothelial lining of the small vessels, leading to multiorgan failure.
Asunto(s)
Encefalopatías Metabólicas Innatas/enzimología , Complejo IV de Transporte de Electrones/metabolismo , Púrpura/enzimología , Sulfuros/toxicidad , Animales , Western Blotting , Línea Celular , Dioxigenasas/genética , Humanos , Ratones , Ratones Noqueados , Proteínas Mitocondriales/genética , Reacción en Cadena de la PolimerasaRESUMEN
Ethylmalonic encephalopathy (EE) is a rare metabolic disorder caused by dysfunction of ETHE1, a mitochondrial dioxygenase involved in hydrogen sulfide (H2S) detoxification. Patients present in infancy with psychomotor retardation, chronic diarrhea, orthostatic acrocyanosis and relapsing petechiae. High levels of lactic acid, ethymalonic acid (EMA) and methylsuccinic acid (MSA) are detected in body fluids. Several pathways may contribute to the pathophysiology, including isoleucine, methionine and fatty acid metabolism. We report on a 15-month-old male presenting with typical EE associated with a homozygous ETHE1 mutation. We investigated oral isoleucine (150 mg/kg), methionine (100 mg/kg), fatty acid loading tests and isoleucine-restricted diet (200 mg/day) for any effects on several metabolic parameters. Before loading tests or specific dietary interventions, EMA, C4-C5 acylcarnitines and most acylglycines were elevated, indicating functional deficiency of short chain acyl-CoA (SCAD) as well as all branched acyl-CoA dehydrogenases. Excretion of EMA and n-butyrylglycine increased following each of the loads, and isoleucine led to increased levels of derivative metabolites. An isoleucine-restricted diet for 8 days corrected some of the abnormalities but led to no obvious clinical improvement and only partial effects on EMA. A principal component analysis supports the inference that these dietary conditions have consistent effects on the global metabolic profile. Our results suggest that multiple pathways modulate EMA levels in EE. They might all interact with H2S toxicity. Prolonged dietary interventions involving the restriction for branched aminoacids, fatty acids and methionine could be discussed as auxiliary therapeutical strategies in EE.
Asunto(s)
Encefalopatías Metabólicas Innatas/enzimología , Proteínas Mitocondriales/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Púrpura/enzimología , Aminoácidos/uso terapéutico , Biomarcadores/sangre , Biomarcadores/orina , Encefalopatías Metabólicas Innatas/diagnóstico , Encefalopatías Metabólicas Innatas/dietoterapia , Encefalopatías Metabólicas Innatas/genética , Dieta con Restricción de Proteínas , Suplementos Dietéticos , Predisposición Genética a la Enfermedad , Homocigoto , Humanos , Lactante , Masculino , Malonatos/sangre , Malonatos/orina , Proteínas Mitocondriales/genética , Mutación , Proteínas de Transporte Nucleocitoplasmático/genética , Fenotipo , Análisis de Componente Principal , Púrpura/diagnóstico , Púrpura/dietoterapia , Púrpura/genética , Resultado del TratamientoRESUMEN
Human succinic semialdehyde dehydrogenase deficiency, an autosomal recessive disorder of γ-aminobutyric acid (GABA) catabolism, was modeled by a murine model sharing the phenotype of ataxia and seizures. Magnetic resonance imaging (MRI) with volumetry was obtained on 7 patients versus controls, and MRI with stereology was derived in 3 murine genotypes: null, wild-type, and heterozygous mutants. All patients had T1 hypointensity and T2 hyperintensity in globus pallidus, and 5 also had similar changes in subthalamic and cerebellar dentate nuclei. There was a trend for patients to have a smaller cerebellar vermis. Homozygous null mice had significantly lower total brain and cerebellar volumes than wild-types and heterozygotes. Stereology confirmed cerebellar atrophy and was otherwise normal in multiple regions. Cerebellar volume loss is present in the murine disorder with a trend for cerebellar atrophy in patients. Reduced cerebellar volume can reflect neurodegeneration and may be related to the clinical manifestations.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/patología , Encefalopatías Metabólicas Innatas/enzimología , Encefalopatías Metabólicas Innatas/patología , Enfermedades Cerebelosas/enzimología , Enfermedades Cerebelosas/patología , Modelos Animales de Enfermedad , Adolescente , Adulto , Errores Innatos del Metabolismo de los Aminoácidos/genética , Animales , Atrofia , Encefalopatías Metabólicas Innatas/diagnóstico por imagen , Enfermedades Cerebelosas/diagnóstico por imagen , Niño , Discapacidades del Desarrollo , Femenino , Humanos , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Persona de Mediana Edad , Radiografía , Distribución Aleatoria , Succionato-Semialdehído Deshidrogenasa/deficiencia , Succionato-Semialdehído Deshidrogenasa/genética , Adulto JovenRESUMEN
A 25-year-old man with exertional myoglobinuria had no evidence of hemolytic anemia, but he had severe parkinsonism that was responsive to levodopa. Phosphoglycerate kinase (PGK) activity was markedly decreased in muscle, and molecular analysis of the PGK1 gene identified the p.T378P mutation that was recently reported in a patient with isolated myopathy. This case reinforces the concept that PGK deficiency is a clinically heterogeneous disorder and raises the question of a relationship between PGK deficiency and idiopathic juvenile Parkinson disease.
Asunto(s)
Enfermedades Metabólicas/enzimología , Enfermedades Musculares/enzimología , Trastornos Parkinsonianos/enzimología , Fosfoglicerato Quinasa/deficiencia , Adulto , Edad de Inicio , Encefalopatías Metabólicas Innatas/complicaciones , Encefalopatías Metabólicas Innatas/enzimología , Encefalopatías Metabólicas Innatas/genética , Causalidad , Análisis Mutacional de ADN , Regulación Enzimológica de la Expresión Génica/genética , Marcadores Genéticos , Predisposición Genética a la Enfermedad/genética , Genotipo , Humanos , Masculino , Enfermedades Metabólicas/complicaciones , Enfermedades Metabólicas/genética , Músculo Esquelético/embriología , Músculo Esquelético/fisiopatología , Enfermedades Musculares/genética , Enfermedades Musculares/fisiopatología , Mutación/genética , Mioglobinuria/enzimología , Mioglobinuria/etiología , Mioglobinuria/fisiopatología , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/fisiopatología , Fosfoglicerato Quinasa/genéticaRESUMEN
L-2-Hydroxyglutaric aciduria (L2HGA) is a rare, neurometabolic disorder with an autosomal recessive mode of inheritance. Affected individuals only have neurological manifestations, including psychomotor retardation, cerebellar ataxia, and more variably macrocephaly, or epilepsy. The diagnosis of L2HGA can be made based on magnetic resonance imaging (MRI), biochemical analysis, and mutational analysis of L2HGDH. About 200 patients with elevated concentrations of 2-hydroxyglutarate (2HG) in the urine were referred for chiral determination of 2HG and L2HGDH mutational analysis. All patients with increased L2HG (n=106; 83 families) were included. Clinical information on 61 patients was obtained via questionnaires. In 82 families the mutations were detected by direct sequence analysis and/or multiplex ligation dependent probe amplification (MLPA), including one case where MLPA was essential to detect the second allele. In another case RT-PCR followed by deep intronic sequencing was needed to detect the mutation. Thirty-five novel mutations as well as 35 reported mutations and 14 nondisease-related variants are reviewed and included in a novel Leiden Open source Variation Database (LOVD) for L2HGDH variants (http://www.LOVD.nl/L2HGDH). Every user can access the database and submit variants/patients. Furthermore, we report on the phenotype, including neurological manifestations and urinary levels of L2HG, and we evaluate the phenotype-genotype relationship.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Encefalopatías Metabólicas Innatas/enzimología , Encefalopatías Metabólicas Innatas/genética , Estudios de Asociación Genética , Mutación/genética , Animales , Encefalopatías Metabólicas Innatas/patología , Modelos Animales de Enfermedad , HumanosRESUMEN
Mitochondrial fatty acid beta-oxidation (FAO) disorders have become an important group of inherited metabolic disorders causing serious pediatric and maternal morbidity and mortality. More than 20 defects affecting beta-oxidation have been discovered, characterized by distinct enzyme or transporter deficiencies. This growing number of FAO disorders covers a wide spectrum of phenotypes and are characterized by a wide array of clinical presentations. We discuss the major mitochondrial FAO disorders and the impact they have on maternal health and neonatal outcomes; diagnostic tools and the value of genetic screening are reviewed; and current therapeutic approaches and management strategies are discussed.