Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
Int J Mol Sci ; 25(5)2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38474060

RESUMO

The pathophysiology of nonketotic hyperglycinemia (NKH), a rare neuro-metabolic disorder associated with severe brain malformations and life-threatening neurological manifestations, remains incompletely understood. Therefore, a valid human neural model is essential. We aimed to investigate the impact of GLDC gene variants, which cause NKH, on cellular fitness during the differentiation process of human induced pluripotent stem cells (iPSCs) into iPSC-derived astrocytes and to identify sustainable mechanisms capable of overcoming GLDC deficiency. We developed the GLDC27-FiPS4F-1 line and performed metabolomic, mRNA abundance, and protein analyses. This study showed that although GLDC27-FiPS4F-1 maintained the parental genetic profile, it underwent a metabolic switch to an altered serine-glycine-one-carbon metabolism with a coordinated cell growth and cell cycle proliferation response. We then differentiated the iPSCs into neural progenitor cells (NPCs) and astrocyte-lineage cells. Our analysis showed that GLDC-deficient NPCs had shifted towards a more heterogeneous astrocyte lineage with increased expression of the radial glial markers GFAP and GLAST and the neuronal markers MAP2 and NeuN. In addition, we detected changes in other genes related to serine and glycine metabolism and transport, all consistent with the need to maintain glycine at physiological levels. These findings improve our understanding of the pathology of nonketotic hyperglycinemia and offer new perspectives for therapeutic options.


Assuntos
Hiperglicinemia não Cetótica , Células-Tronco Pluripotentes Induzidas , Humanos , Hiperglicinemia não Cetótica/genética , Hiperglicinemia não Cetótica/patologia , Glicina Desidrogenase (Descarboxilante)/genética , Astrócitos/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Glicina , Serina
2.
J Inherit Metab Dis ; 46(2): 261-272, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36564894

RESUMO

Coenzyme A (CoA) is an essential cofactor involved in a range of metabolic pathways including the activation of long-chain fatty acids for catabolism. Cells synthesize CoA de novo from vitamin B5 (pantothenate) via a pathway strongly conserved across prokaryotes and eukaryotes. In humans, it involves five enzymatic steps catalyzed by four enzymes: pantothenate kinase (PANK [isoforms 1-4]), 4'-phosphopantothenoylcysteine synthetase (PPCS), phosphopantothenoylcysteine decarboxylase (PPCDC), and CoA synthase (COASY). To date, inborn errors of metabolism associated with all of these genes, except PPCDC, have been described, two related to neurodegeneration with brain iron accumulation (NBIA), and one associated with a cardiac phenotype. This paper reports another defect in this pathway (detected in two sisters), associated with a fatal cardiac phenotype, caused by biallelic variants (p.Thr53Pro and p.Ala95Val) of PPCDC. PPCDC enzyme (EC 4.1.1.36) catalyzes the decarboxylation of 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine in CoA biosynthesis. The variants p.Thr53Pro and p.Ala95Val affect residues highly conserved across different species; p.Thr53Pro is involved in the binding of flavin mononucleotide, and p.Ala95Val is likely a destabilizing mutation. Patient-derived fibroblasts showed an absence of PPCDC protein, and nearly 50% reductions in CoA levels. The cells showed clear energy deficiency problems, with defects in mitochondrial respiration, and mostly glycolytic ATP synthesis. Functional studies performed in yeast suggest these mutations to be functionally relevant. In summary, this work describes a new, ultra-rare, severe inborn error of metabolism due to pathogenic variants of PPCDC.


Assuntos
Carboxiliases , Cardiomiopatia Dilatada , Humanos , Carboxiliases/genética , Coenzima A/genética , Coração , Saccharomyces cerevisiae/genética
4.
Hum Mutat ; 38(6): 678-691, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28244183

RESUMO

The rapid analysis of genomic data is providing effective mutational confirmation in patients with clinical and biochemical hallmarks of a specific disease. This is the case for nonketotic hyperglycinemia (NKH), a Mendelian disorder causing seizures in neonates and early-infants, primarily due to mutations in the GLDC gene. However, understanding the impact of missense variants identified in this gene is a major challenge for the application of genomics into clinical practice. Herein, a comprehensive functional and structural analysis of 19 GLDC missense variants identified in a cohort of 26 NKH patients was performed. Mutant cDNA constructs were expressed in COS7 cells followed by enzymatic assays and Western blot analysis of the GCS P-protein to assess the residual activity and mutant protein stability. Structural analysis, based on molecular modeling of the 3D structure of GCS P-protein, was also performed. We identify hypomorphic variants that produce attenuated phenotypes with improved prognosis of the disease. Structural analysis allows us to interpret the effects of mutations on protein stability and catalytic activity, providing molecular evidence for clinical outcome and disease severity. Moreover, we identify an important number of mutants whose loss-of-functionality is associated with instability and, thus, are potential targets for rescue using folding therapeutic approaches.


Assuntos
Glicina Desidrogenase (Descarboxilante)/genética , Hiperglicinemia não Cetótica/genética , Mutação de Sentido Incorreto/genética , Relação Estrutura-Atividade , Éxons/genética , Regulação Enzimológica da Expressão Gênica , Glicina/metabolismo , Glicina Desidrogenase (Descarboxilante)/química , Humanos , Hiperglicinemia não Cetótica/patologia , Recém-Nascido , Conformação Molecular , Fenótipo , Estabilidade Proteica
5.
Genet Med ; 19(1): 104-111, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27362913

RESUMO

PURPOSE: The study's purpose was to delineate the genetic mutations that cause classic nonketotic hyperglycinemia (NKH). METHODS: Genetic results, parental phase, ethnic origin, and gender data were collected from subjects suspected to have classic NKH. Mutations were compared with those in the existing literature and to the population frequency from the Exome Aggregation Consortium (ExAC) database. RESULTS: In 578 families, genetic analyses identified 410 unique mutations, including 246 novel mutations. 80% of subjects had mutations in GLDC. Missense mutations were noted in 52% of all GLDC alleles, most private. Missense mutations were 1.5 times as likely to be pathogenic in the carboxy terminal of GLDC than in the amino-terminal part. Intragenic copy-number variations (CNVs) in GLDC were noted in 140 subjects, with biallelic CNVs present in 39 subjects. The position and frequency of the breakpoint for CNVs correlated with intron size and presence of Alu elements. Missense mutations, most often recurring, were the most common type of disease-causing mutation in AMT. Sequencing and CNV analysis identified biallelic pathogenic mutations in 98% of subjects. Based on genotype, 15% of subjects had an attenuated phenotype. The frequency of NKH is estimated at 1:76,000. CONCLUSION: The 484 unique mutations now known in classic NKH provide a valuable overview for the development of genotype-based therapies.Genet Med 19 1, 104-111.


Assuntos
Aminometiltransferase/genética , Complexo Glicina Descarboxilase/genética , Glicina Desidrogenase (Descarboxilante)/genética , Hiperglicinemia não Cetótica/genética , Alelos , Di-Hidrolipoamida Desidrogenase/genética , Éxons/genética , Feminino , Testes Genéticos , Genótipo , Glicina/genética , Glicina/metabolismo , Humanos , Hiperglicinemia não Cetótica/diagnóstico , Hiperglicinemia não Cetótica/patologia , Íntrons , Masculino , Mutação de Sentido Incorreto
6.
Orphanet J Rare Dis ; 17(1): 243, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35729663

RESUMO

BACKGROUND: Monocarboxylate transporter 1 (MCT1) deficiency has recently been described as a rare cause of recurrent ketosis, the result of impaired ketone utilization in extrahepatic tissues. To date, only six patients with this condition have been identified, and clinical and biochemical details remain incomplete. RESULTS: The present work reports a patient suffering from severe, recurrent episodes of metabolic acidosis and psychomotor delay, showing a pathogenic loss-of-function variation c.747_750del in homozygosity in SLC16A1 (which codes for MCT1). Persistent ketotic and lactic acidosis was accompanied by an abnormal excretion of organic acids related to redox balance disturbances. Together with an altered bioenergetic profile detected in patient-derived fibroblasts, this suggests possible mitochondrial dysfunction. Brain MRI revealed extensive, diffuse bilateral, symmetric signal alterations for the subcortical white matter and basal ganglia, together with corpus callosum agenesia. CONCLUSIONS: These findings suggest that the clinical spectrum of MCT1 deficiency not only involves recurrent atacks of ketoacidosis, but may also cause lactic acidosis and neuromotor delay with a distinctive neuroimaging pattern including agenesis of corpus callosum and other brain signal alterations.


Assuntos
Acidose Láctica , Acidose Láctica/genética , Agenesia do Corpo Caloso/patologia , Corpo Caloso/patologia , Metabolismo Energético/genética , Humanos , Mitocôndrias
7.
Stem Cell Res ; 39: 101503, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31349202

RESUMO

A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with nonketotic hyperglycinemia bearing the biallelic changes c.1742C > G (p.Pro581Arg) and c.2368C > T (p.Arg790Trp) in the GLDC gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. This cellular model provides a good resource for disease modeling and drug discovery.


Assuntos
Hiperglicinemia não Cetótica/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Humanos , Recém-Nascido , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
8.
Stem Cell Res ; 38: 101469, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31132581

RESUMO

A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with propionic acidemia that has a homozygous mutation (c.1218_1231del14ins12 (p.G407 fs)) in the PCCB gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. The generated iPSC line represents a useful tool to study the pathomechanisms underlying the deficiency.


Assuntos
Homozigoto , Células-Tronco Pluripotentes Induzidas , Metilmalonil-CoA Descarboxilase , Mutação , Acidemia Propiônica , Linhagem Celular , Humanos , Células-Tronco Pluripotentes Induzidas/enzimologia , Células-Tronco Pluripotentes Induzidas/patologia , Fator 4 Semelhante a Kruppel , Metilmalonil-CoA Descarboxilase/genética , Metilmalonil-CoA Descarboxilase/metabolismo , Acidemia Propiônica/enzimologia , Acidemia Propiônica/genética , Acidemia Propiônica/patologia
9.
J Clin Med ; 8(11)2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31683770

RESUMO

Congenital lactic acidosis (CLA) is a rare condition in most instances due to a range of inborn errors of metabolism that result in defective mitochondrial function. Even though the implementation of next generation sequencing has been rapid, the diagnosis rate for this highly heterogeneous allelic condition remains low. The present work reports our group's experience of using a clinical/biochemical analysis system in conjunction with genetic findings that facilitates the taking of timely clinical decisions with minimum need for invasive procedures. The system's workflow combines different metabolomics datasets and phenotypic information with the results of clinical exome sequencing and/or RNA analysis. The system's use detected genetic variants in 64% of a cohort of 39 CLA-patients; these variants, 14 of which were novel, were found in 19 different nuclear and two mitochondrial genes. For patients with variants of unknown significance, the genetic analysis was combined with functional genetic and/or bioenergetics analyses in an attempt to detect pathogenicity. Our results warranted subsequent testing of antisense therapy to rescue the abnormal splicing in cultures of fibroblasts from a patient with a defective GFM1 gene. The discussed system facilitates the diagnosis of CLA by avoiding the need to use invasive techniques and increase our knowledge of the causes of this condition.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA