RESUMEN
Using 3D organotypic rat brain cell cultures in aggregates we recently identified 2-methylcitrate (2-MCA) as the main toxic metabolite for developing brain cells in methylmalonic aciduria. Exposure to 2-MCA triggered morphological changes and apoptosis of brain cells. This was accompanied by increased ammonium and decreased glutamine levels. However, the sequence and causal relationship between these phenomena remained unclear. To understand the sequence and time course of pathogenic events, we exposed 3D rat brain cell aggregates to different concentrations of 2-MCA (0.1, 0.33 and 1.0mM) from day in vitro (DIV) 11 to 14. Aggregates were harvested at different time points from DIV 12 to 19. We compared the effects of a single dose of 1mM 2-MCA administered on DIV 11 to the effects of repeated doses of 1mM 2-MCA. Pan-caspase inhibitors Z-VAD FMK or Q-VD-OPh were used to block apoptosis. Ammonium accumulation in the culture medium started within few hours after the first 2-MCA exposure. Morphological changes of the developing brain cells were already visible after 17h. The highest rate of cleaved caspase-3 was observed after 72h. A dose-response relationship was observed for all effects. Surprisingly, a single dose of 1mM 2-MCA was sufficient to induce all of the biochemical and morphological changes in this model. 2-MCA-induced ammonium accumulation and morphological changes were not prevented by concomitant treatment of the cultures with pan-caspase inhibitors Z-VAD FMK or Q-VD-OPh: ammonium increased rapidly after a single 1mM 2-MCA administration even after apoptosis blockade. We conclude that following exposure to 2-MCA, ammonium production in brain cell cultures is an early phenomenon, preceding cell degeneration and apoptosis, and may actually be the cause of the other changes observed. The fact that a single dose of 1mM 2-MCA is sufficient to induce deleterious effects over several days highlights the potential damaging effects of even short-lasting metabolic decompensations in children affected by methylmalonic aciduria.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Compuestos de Amonio/metabolismo , Lesiones Encefálicas/metabolismo , Citratos/toxicidad , Clorometilcetonas de Aminoácidos/farmacología , Errores Innatos del Metabolismo de los Aminoácidos/inducido químicamente , Errores Innatos del Metabolismo de los Aminoácidos/fisiopatología , Compuestos de Amonio/toxicidad , Animales , Apoptosis/efectos de los fármacos , Lesiones Encefálicas/inducido químicamente , Lesiones Encefálicas/patología , Caspasa 3/metabolismo , Técnicas de Cultivo de Célula , Medios de Cultivo/química , Glutamina/metabolismo , Humanos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Quinolinas/farmacología , RatasRESUMEN
Inherited peripheral neuropathies are a genetically heterogeneous group of disorders characterized by distal muscle weakness and sensory loss. Mutations in genes encoding aminoacyl-tRNA synthetases have been implicated in peripheral neuropathies, suggesting that these tRNA charging enzymes are uniquely important for the peripheral nerve. Recently, a mutation in histidyl-tRNA synthetase (HARS) was identified in a single patient with a late-onset, sensory-predominant peripheral neuropathy; however, the genetic evidence was lacking, making the significance of the finding unclear. Here, we present clinical, genetic, and functional data that implicate HARS mutations in inherited peripheral neuropathies. The associated phenotypic spectrum is broad and encompasses axonal and demyelinating motor and sensory neuropathies, including four young patients presenting with pure motor axonal neuropathy. Genome-wide linkage studies in combination with whole-exome and conventional sequencing revealed four distinct and previously unreported heterozygous HARS mutations segregating with autosomal dominant peripheral neuropathy in four unrelated families (p.Thr132Ile, p.Pro134His, p.Asp175Glu and p.Asp364Tyr). All mutations cause a loss of function in yeast complementation assays, and p.Asp364Tyr is dominantly neurotoxic in a Caenorhabditis elegans model. This study demonstrates the role of HARS mutations in peripheral neuropathy and expands the genetic and clinical spectrum of aminoacyl-tRNA synthetase-related human disease.
Asunto(s)
Ligamiento Genético/genética , Neuropatías Hereditarias Sensoriales y Autónomas/genética , Histidina-ARNt Ligasa/genética , Mutación/genética , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedad de Charcot-Marie-Tooth/genética , Femenino , Humanos , Masculino , LinajeRESUMEN
Charcot-Marie-Tooth disease (CMT) comprises a clinically and genetically heterogeneous group of peripheral neuropathies characterized by progressive distal muscle weakness and atrophy, foot deformities and distal sensory loss. Following the analysis of two consanguineous families affected by a medium to late-onset recessive form of intermediate CMT, we identified overlapping regions of homozygosity on chromosome 1p36 with a combined maximum LOD score of 5.4. Molecular investigation of the genes from this region allowed identification of two homozygous mutations in PLEKHG5 that produce premature stop codons and are predicted to result in functional null alleles. Analysis of Plekhg5 in the mouse revealed that this gene is expressed in neurons and glial cells of the peripheral nervous system, and that knockout mice display reduced nerve conduction velocities that are comparable with those of affected individuals from both families. Interestingly, a homozygous PLEKHG5 missense mutation was previously reported in a recessive form of severe childhood onset lower motor neuron disease (LMND) leading to loss of the ability to walk and need for respiratory assistance. Together, these observations indicate that different mutations in PLEKHG5 lead to clinically diverse outcomes (intermediate CMT or LMND) affecting the function of neurons and glial cells.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/genética , Genes Recesivos , Factores de Intercambio de Guanina Nucleótido/deficiencia , Factores de Intercambio de Guanina Nucleótido/genética , Adulto , Edad de Inicio , Animales , Niño , Cromosomas Humanos Par 1/genética , Codón sin Sentido , Femenino , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Enfermedad de la Neurona Motora/genética , Mutación Missense , Neuroglía/metabolismo , Neuroglía/fisiología , Neuronas/metabolismo , Adulto JovenRESUMEN
Deep intronic mutations are often ignored as possible causes of human diseases. A deep intronic mutation in the MTRR gene, c.903+469T>C, is the most frequent mutation causing the cblE type of homocystinuria. It is well known to be associated with pre-mRNA mis-splicing, resulting in pseudoexon inclusion; however, the pathological mechanism remains unknown. We used minigenes to demonstrate that this mutation is the direct cause of MTRR pseudoexon inclusion, and that the pseudoexon is normally not recognized due to a suboptimal 5' splice site. Within the pseudoexon we identified an exonic splicing enhancer (ESE), which is activated by the mutation. Cotransfection and siRNA experiments showed that pseudoexon inclusion depends on the cellular amounts of SF2/ASF and in vitro RNA-binding assays showed dramatically increased SF2/ASF binding to the mutant MTRR ESE. The mutant MTRR ESE sequence is identical to an ESE of the alternatively spliced MST1R proto-oncogene, which suggests that this ESE could be frequently involved in splicing regulation. Our study conclusively demonstrates that an intronic single nucleotide change is sufficient to cause pseudoexon activation via creation of a functional ESE, which binds a specific splicing factor. We suggest that this mechanism may cause genetic disease much more frequently than previously reported.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Exones/genética , Ferredoxina-NADP Reductasa/genética , Homocistinuria/genética , Intrones/genética , Mutación/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Secuencia de Bases , Células COS , Chlorocebus aethiops , Biología Computacional , Homocistinuria/clasificación , Homocistinuria/enzimología , Datos de Secuencia Molecular , Proteínas Mutantes/genética , Unión Proteica , Proto-Oncogenes Mas , Sitios de Empalme de ARN/genética , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Empalme Serina-Arginina , Vitamina B 12/metabolismoRESUMEN
The cblE type of homocystinuria is a rare autosomal recessive disorder caused by impaired reductive activation of methionine synthase. Although earlier biochemical studies proposed that the methionine synthase enzyme might be activated by two different reducing systems, mutations were reported in only the methionine synthase reductase gene (MTRR) in cblE patients. The pathogenicity of MTRR mutations, however, has not yet been tested functionally. We report on nine patients of European origin affected by the cblE type of homocystinuria. They presented between 2 weeks and 3 years of age (median age 4 weeks) with anemia, which was macrocytic in only three patients, and with neurological involvement in all but two cases. Bone marrow examination performed in seven patients showed megaloblastic changes in all but one of them. All patients exhibited moderate to severe hyperhomocysteinemia (median plasma total homocysteine [Hcy] 92 mumol/L, range 44-169), while clearly reduced methionine was observed only in four cases. Pathogenic mutations were identified in both parental alleles of the MTRR gene in all patients. Five known (c.903+469T>C, c.1361C>T, c.1459G>A, c.1557-4_1557+3del7, and c.1622_1623dupTA) and three novel mutations (c.7A>T, c.1573C>T, and c.1953-6_1953-2del5) were detected. Importantly, transfection of fibroblasts of cblE patients with a wild-type MTRR minigene expression construct resulted in a significant approximately four-fold increase of methionine synthesis, indicating correction of the enzyme defect. Our study shows a link between a milder predominantly hematological presentation and homozygosity for the c.1361C>T mutation, but no other obvious genotype-phenotype correlation. The identification of mutations in the MTRR gene, together with restoration of methionine synthesis following MTRR minigene expression in cblE cells confirms that this disease is caused by defects in the MTRR gene.
Asunto(s)
5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/deficiencia , Ferredoxina-NADP Reductasa/deficiencia , Terapia Genética , Homocistinuria/genética , Sustitución de Aminoácidos , Betaína/uso terapéutico , Encéfalo/patología , Línea Celular Transformada/enzimología , Línea Celular Transformada/patología , Codón sin Sentido , Análisis Mutacional de ADN , Ferredoxina-NADP Reductasa/genética , Fibroblastos/enzimología , Fibroblastos/patología , Ácido Fólico/uso terapéutico , Genes Sintéticos , Prueba de Complementación Genética , Haplotipos/genética , Homocisteína/sangre , Homocistinuria/sangre , Homocistinuria/clasificación , Homocistinuria/tratamiento farmacológico , Homocistinuria/enzimología , Homocistinuria/patología , Homocistinuria/terapia , Humanos , Hidroxocobalamina/uso terapéutico , Mutación Missense , Mutación Puntual , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de Restricción , Proteínas Recombinantes de Fusión/fisiología , Eliminación de Secuencia , Transfección , Población Blanca/genéticaRESUMEN
To facilitate the association studies in complex diseases characterized by hyperhomocysteinemia, we collected structural and frequency data on single-nucleotide polymorphism (SNPs) in 24 genes relating to homocysteine metabolism. Firstly, we scanned approximately 1.2 Mbp of sequence in the NCBI SNP database (dbSNP) build 110 and we detected 1353 putative SNPs with an average in silico genic density of 1:683. Out of 112 putative SNPs in coding regions (cSNPs), we selected a subset of 42 cSNPs and we assessed the applicability of the NCBI dbSNP to the Czech population - a typical representative of European Caucasians - by determining the frequency of the putative cSNPs experimentally by PCR-RFLP or ARMS-PCR in at least 110 control Czech chromosomes. As only 25 of the 42 analyzed cSNPs met the criterion of >/=1% frequency, the positive predictive value of the NCBI data set for our population reached 60%, which is similar to other studies. The correlation of SNP frequency between Czechs and other Caucasians - obtained from NCBI and/or literature - was stronger (r(2)=0.90 for 20 cSNPs) than between Czechs and general NCBI database entries (r(2)=0.73 for 27 cSNPs). Moreover, frequencies of all 20 putative cSNPs, for which data in Caucasians were available, were congruently below or above the 1% frequency criterion both in Czechs and in other Caucasians. In summary, our study shows that the NCBI dbSNP is a useful tool for selecting cSNPs for genetic studies of hyperhomocysteinemia in European populations, although experimental validation of SNPs should be performed, especially if the cSNP entry lacks any frequency data in Caucasians.
Asunto(s)
ADN Complementario/química , Marcadores Genéticos/genética , Genoma Humano , Homocisteína/metabolismo , Polimorfismo de Nucleótido Simple/genética , Población Blanca/genética , Checoslovaquia , Bases de Datos Genéticas , Etiquetas de Secuencia Expresada , Frecuencia de los Genes/genética , Humanos , Sector PúblicoRESUMEN
BACKGROUND: Methylmalonic aciduria is an inborn error of metabolism characterized by accumulation of methylmalonate (MMA), propionate and 2-methylcitrate (2-MCA) in body fluids. Early diagnosis and current treatment strategies aimed at limiting the production of these metabolites are only partially effective in preventing neurological damage. METHODS: To explore the metabolic consequences of methylmalonic aciduria on the brain, we used 3D organotypic brain cell cultures from rat embryos. We challenged the cultures at two different developmental stages with 1 mM MMA, propionate or 2-MCA applied 6 times every 12 h. In a dose-response experiment cultures were challenged with 0.01, 0.1, 0.33 and 1 mM 2-MCA. Immunohistochemical staining for different brain cell markers were used to assess cell viability, morphology and differentiation. Significant changes were validated by western blot analysis. Biochemical markers were analyzed in culture media. Apoptosis was studied by immunofluorescence staining and western blots for activated caspase-3. RESULTS: Among the three metabolites tested, 2-MCA consistently produced the most pronounced effects. Exposure to 2-MCA caused morphological changes in neuronal and glial cells already at 0.01 mM. At the biochemical level the most striking result was a significant ammonium increase in culture media with a concomitant glutamine decrease. Dose-response studies showed significant and parallel changes of ammonium and glutamine starting from 0.1 mM 2-MCA. An increased apoptosis rate was observed by activation of caspase-3 after exposure to at least 0.1 mM 2-MCA. CONCLUSION: Surprisingly, 2-MCA, and not MMA, seems to be the most toxic metabolite in our in vitro model leading to delayed axonal growth, apoptosis of glial cells and to unexpected ammonium increase. Morphological changes were already observed at 2-MCA concentrations as low as 0.01 mM. Increased apoptosis and ammonium accumulation started at 0.1 mM thus suggesting that ammonium accumulation is secondary to cell suffering and/or cell death. Local accumulation of ammonium in CNS, that may remain undetected in plasma and urine, may therefore play a key role in the neuropathogenesis of methylmalonic aciduria both during acute decompensations and in chronic phases. If confirmed in vivo, this finding might shift the current paradigm and result in novel therapeutic strategies.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/patología , Apoptosis/efectos de los fármacos , Encéfalo/patología , Citratos/farmacología , Ácido Metilmalónico/orina , Compuestos de Amonio Cuaternario/metabolismo , Animales , Western Blotting , Encéfalo/enzimología , Encéfalo/metabolismo , Caspasa 3/metabolismo , Células Cultivadas , Medios de Cultivo , Femenino , Inmunohistoquímica , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
Glutaric aciduria type I (glutaryl-CoA dehydrogenase deficiency) is an inborn error of metabolism that usually manifests in infancy by an acute encephalopathic crisis and often results in permanent motor handicap. Biochemical hallmarks of this disease are elevated levels of glutarate and 3-hydroxyglutarate in blood and urine. The neuropathology of this disease is still poorly understood, as low lysine diet and carnitine supplementation do not always prevent brain damage, even in early-treated patients. We used a 3D in vitro model of rat organotypic brain cell cultures in aggregates to mimic glutaric aciduria type I by repeated administration of 1 mM glutarate or 3-hydroxyglutarate at two time points representing different developmental stages. Both metabolites were deleterious for the developing brain cells, with 3-hydroxyglutarate being the most toxic metabolite in our model. Astrocytes were the cells most strongly affected by metabolite exposure. In culture medium, we observed an up to 11-fold increase of ammonium in the culture medium with a concomitant decrease of glutamine. We further observed an increase in lactate and a concomitant decrease in glucose. Exposure to 3-hydroxyglutarate led to a significantly increased cell death rate. Thus, we propose a three step model for brain damage in glutaric aciduria type I: (i) 3-OHGA causes the death of astrocytes, (ii) deficiency of the astrocytic enzyme glutamine synthetase leads to intracerebral ammonium accumulation, and (iii) high ammonium triggers secondary death of other brain cells. These unexpected findings need to be further investigated and verified in vivo. They suggest that intracerebral ammonium accumulation might be an important target for the development of more effective treatment strategies to prevent brain damage in patients with glutaric aciduria type I.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Errores Innatos del Metabolismo de los Aminoácidos/patología , Encefalopatías Metabólicas/metabolismo , Encefalopatías Metabólicas/patología , Encéfalo/metabolismo , Encéfalo/patología , Compuestos de Amonio Cuaternario/metabolismo , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Muerte Celular/efectos de los fármacos , Medios de Cultivo/química , Modelos Animales de Enfermedad , Glutaratos/farmacología , Glutaril-CoA Deshidrogenasa/deficiencia , Glutaril-CoA Deshidrogenasa/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Oligodendroglía/patología , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND: The human condition known as Premature Ovarian Failure (POF) is characterized by loss of ovarian function before the age of 40. A majority of POF cases are sporadic, but 10-15% are familial, suggesting a genetic origin of the disease. Although several causal mutations have been identified, the etiology of POF is still unknown for about 90% of the patients. METHODOLOGY/PRINCIPAL FINDINGS: We report a genome-wide linkage and homozygosity analysis in one large consanguineous Middle-Eastern POF-affected family presenting an autosomal recessive pattern of inheritance. We identified two regions with a LOD(max) of 3.26 on chromosome 7p21.1-15.3 and 7q21.3-22.2, which are supported as candidate regions by homozygosity mapping. Sequencing of the coding exons and known regulatory sequences of three candidate genes (DLX5, DLX6 and DSS1) included within the largest region did not reveal any causal mutations. CONCLUSIONS/SIGNIFICANCE: We detect two novel POF-associated loci on human chromosome 7, opening the way to the identification of new genes involved in the control of ovarian development and function.
Asunto(s)
Cromosomas Humanos Par 7/genética , Consanguinidad , Ligamiento Genético/genética , Sitios Genéticos/genética , Insuficiencia Ovárica Primaria/genética , Árabes , Femenino , Humanos , Escala de Lod , Repeticiones de Microsatélite/genética , Linaje , Análisis de Secuencia de ADNRESUMEN
Intracellular cobalamin is converted to adenosylcobalamin, coenzyme for methylmalonyl-CoA mutase and to methylcobalamin, coenzyme for methionine synthase, in an incompletely understood sequence of reactions. Genetic defects of these steps are defined as cbl complementation groups of which cblC, cblD (described in only two siblings), and cblF are associated with combined homocystinuria and methylmalonic aciduria. Here we describe three unrelated patients belonging to the cblD complementation group but with distinct biochemical phenotypes different from that described in the original cblD siblings. Two patients presented with isolated homocystinuria and reduced formation of methionine and methylcobalamin in cultured fibroblasts, defined as cblD-variant 1, and one patient with isolated methylmalonic aciduria and deficient adenosylcobalamin synthesis in fibroblasts, defined as cblD-variant 2. Cell lines from the cblD-variant 1 patients clearly complemented reference lines with the same biochemical phenotype, i.e. cblE and cblG, and the cblD-variant 2 cell line complemented cells from the mutant classes with isolated deficiency of adenosylcobalamin synthesis, i.e. cblA and cblB. Also, no pathogenic sequence changes in the coding regions of genes associated with the respective biochemical phenotypes were found. These findings indicate heterogeneity within the previously defined cblD mutant class and point to further complexity of intracellular cobalamin metabolism.