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1.
Hum Mutat ; 40(7): 975-982, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30908763

RESUMO

D-2-hydroxyglutaric aciduria Type I (D-2-HGA Type I), a neurometabolic disorder with a broad clinical spectrum, is caused by recessive variants in the D2HGDH gene encoding D-2-hydroxyglutarate dehydrogenase (D-2-HGDH). We and others detected 42 potentially pathogenic variants in D2HGDH of which 31 were missense. We developed functional studies to investigate the effect of missense variants on D-2-HGDH catalytic activity. Site-directed mutagenesis was used to introduce 31 missense variants in the pCMV5-D2HGDH expression vector. The wild type and missense variants were overexpressed in HEK293 cells. D-2-HGDH enzyme activity was evaluated based on the conversion of [2 H4 ]D-2-HG to [2 H4 ]2-ketoglutarate, which was subsequently converted into [2 H4 ]L-glutamate and the latter quantified by LC-MS/MS. Eighteen variants resulted in almost complete ablation of D-2-HGDH activity and thus, should be considered pathogenic. The remaining 13 variants manifested residual activities ranging between 17% and 94% of control enzymatic activity. Our functional assay evaluating the effect of novel D2HGDH variants will be beneficial for the classification of missense variants and determination of pathogenicity.


Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Encefalopatias Metabólicas Congênitas/genética , Mutação de Sentido Incorreto , Encefalopatias Metabólicas Congênitas/metabolismo , Cromatografia Líquida , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Espectrometria de Massas em Tandem , Anormalidades Urogenitais
2.
Am J Hum Genet ; 92(4): 627-31, 2013 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-23561848

RESUMO

The Krebs cycle is of fundamental importance for the generation of the energetic and molecular needs of both prokaryotic and eukaryotic cells. Both enantiomers of metabolite 2-hydroxyglutarate are directly linked to this pivotal biochemical pathway and are found elevated not only in several cancers, but also in different variants of the neurometabolic disease 2-hydroxyglutaric aciduria. Recently we showed that cancer-associated IDH2 germline mutations cause one variant of 2-hydroxyglutaric aciduria. Complementary to these findings, we now report recessive mutations in SLC25A1, the mitochondrial citrate carrier, in 12 out of 12 individuals with combined D-2- and L-2-hydroxyglutaric aciduria. Impaired mitochondrial citrate efflux, demonstrated by stable isotope labeling experiments and the absence of SLC25A1 in fibroblasts harboring certain mutations, suggest that SLC25A1 deficiency is pathogenic. Our results identify defects in SLC25A1 as a cause of combined D-2- and L-2-hydroxyglutaric aciduria.


Assuntos
Proteínas de Transporte de Ânions/genética , Encefalopatias Metabólicas Congênitas/etiologia , Ácido Cítrico/metabolismo , Genes Recessivos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Mutação/genética , Sequência de Aminoácidos , Biomarcadores/análise , Encefalopatias Metabólicas Congênitas/metabolismo , Encefalopatias Metabólicas Congênitas/patologia , Estudos de Casos e Controles , Células Cultivadas , Cromatografia Líquida , Exoma/genética , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Glutaratos/urina , Humanos , Masculino , Dados de Sequência Molecular , Transportadores de Ânions Orgânicos , Fenótipo , Estrutura Terciária de Proteína , Estudos Retrospectivos , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Espectrometria de Massas em Tandem
3.
J Inherit Metab Dis ; 35(4): 571-87, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22391998

RESUMO

The organic acidurias D: -2-hydroxyglutaric aciduria (D-2-HGA), L-2-hydroxyglutaric aciduria (L-2-HGA), and combined D,L-2-hydroxyglutaric aciduria (D,L-2-HGA) cause neurological impairment at young age. Accumulation of D-2-hydroxyglutarate (D-2-HG) and/or L-2-hydroxyglutarate (L-2-HG) in body fluids are the biochemical hallmarks of these disorders. The current review describes the knowledge gathered on 2-hydroxyglutaric acidurias (2-HGA), since the description of the first patients in 1980. We report on the clinical, genetic, enzymatic and metabolic characterization of D-2-HGA type I, D-2-HGA type II, L-2-HGA and D,L-2-HGA, whereas for D-2-HGA type I and type II novel clinical information is presented which was derived from questionnaires.


Assuntos
Encefalopatias Metabólicas Congênitas/genética , Encefalopatias Metabólicas Congênitas/metabolismo , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Encefalopatias Metabólicas Congênitas/patologia , Humanos , Doenças do Sistema Nervoso/patologia
4.
Science ; 330(6002): 336, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20847235

RESUMO

Heterozygous somatic mutations in the genes encoding isocitrate dehydrogenase-1 and -2 (IDH1 and IDH2) were recently discovered in human neoplastic disorders. These mutations disable the enzymes' normal ability to convert isocitrate to 2-ketoglutarate (2-KG) and confer on the enzymes a new function: the ability to convert 2-KG to d-2-hydroxyglutarate (D-2-HG). We have detected heterozygous germline mutations in IDH2 that alter enzyme residue Arg(140) in 15 unrelated patients with d-2-hydroxyglutaric aciduria (D-2-HGA), a rare neurometabolic disorder characterized by supraphysiological levels of D-2-HG. These findings provide additional impetus for investigating the role of D-2-HG in the pathophysiology of metabolic disease and cancer.


Assuntos
Encefalopatias Metabólicas Congênitas/genética , Mutação em Linhagem Germinativa , Glutaratos/metabolismo , Isocitrato Desidrogenase/genética , Adolescente , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Criança , Pré-Escolar , Feminino , Glutaratos/urina , Heterozigoto , Humanos , Lactente , Isocitrato Desidrogenase/química , Isocitrato Desidrogenase/metabolismo , Masculino , Neoplasias/genética , Neoplasias/metabolismo , Adulto Jovem
5.
Hum Mutat ; 31(3): 279-83, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20020533

RESUMO

We performed molecular, enzyme, and metabolic studies in 50 patients with D-2-hydroxyglutaric aciduria (D-2-HGA) who accumulated D-2-hydroxyglutarate (D-2-HG) in physiological fluids. Presumed pathogenic mutations were detected in 24 of 50 patients in the D-2-hydroxyglutarate dehydrogenase (D2HGDH) gene, which encodes D-2-hydroxyglutarate dehydrogenase (D-2-HGDH). Enzyme assay of D-2-HGDH confirmed that all patients with mutations had impaired enzyme activity, whereas patients with D-2-HGA whose enzyme activity was normal did not have mutations. Significantly lower D-2-HG concentrations in body fluids were observed in mutation-positive D-2-HGA patients than in mutation-negative patients. These results imply that multiple genetic loci may be associated with hyperexcretion of D-2-HG. Accordingly, we suggest a new classification: D-2-HGA Type I associates with D-2-HGDH deficiency, whereas idiopathic D-2-HGA manifests with normal D-2-HGDH activity and higher D-2-HG levels in body fluids compared with Type I patients. It remains possible that several classifications for idiopathic D-2-HGA patients with diverse genetic loci will be revealed in future studies.


Assuntos
Oxirredutases do Álcool/genética , Glutaratos/sangue , Glutaratos/urina , Deficiência Múltipla de Acil Coenzima A Desidrogenase/genética , Algoritmos , Líquidos Corporais , Análise Mutacional de DNA , Genótipo , Glutaratos/líquido cefalorraquidiano , Homozigoto , Humanos , Modelos Genéticos , Mutação , Reprodutibilidade dos Testes , Ácido gama-Aminobutírico/análogos & derivados , Ácido gama-Aminobutírico/genética
6.
J Sep Sci ; 28(14): 1796-802, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16224975

RESUMO

LC-ESI-MS is applied frequently in pharmaceutical analysis. The sample amount is generally not restricted, however with LC-ESI-MS, a lack of sensitivity may still be observed with standard-bore LC columns in isocratic mode. Therefore, it was investigated whether increased sensitivity could be achieved by using miniaturized LC-ESI-MS. Seven columns ranging from 0.1 to 4.6 mm ID were tested using several instrument setups. For proper comparison, a sensitivity gain factor (SGF) was introduced. The SGF expresses the extra sensitivity that may be obtained on top of the normal increase of peak concentration, which can be expected when the column ID is reduced. Desogestrel, mirtazapine, and sugammadex sodium were used as test compounds. For desogestrel and sugammadex sodium, the SGF increased up to a factor of 5-13 when the column ID was reduced, indicating enhanced ionization efficiencies at lower flow rates. Optimum sensitivity was found for the 0.3 mm column coupled in combination with a microinjection valve and a dedicated low flow rate interface. For mirtazapine, no increase of SGF was observed when the column ID was decreased. Apparently, the ionization efficiency of this compound is not affected by the flow rate and the spray quality.


Assuntos
Preparações Farmacêuticas/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Cromatografia Líquida de Alta Pressão/métodos , Desogestrel/química , Desogestrel/isolamento & purificação , Mianserina/análogos & derivados , Mianserina/química , Mianserina/isolamento & purificação , Miniaturização , Mirtazapina , Preparações Farmacêuticas/química , Preparações Farmacêuticas/isolamento & purificação , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Propriedades de Superfície
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