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Master's Dissertation
DOI
10.11606/D.17.2018.tde-23032018-131353
Document
Author
Full name
Aline Faccioli Bodoni
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2017
Supervisor
Committee
Antonini, Sonir Roberto Rauber (President)
Júnior, Gil Guerra
Leopoldino, Andréia Machado
Scrideli, Carlos Alberto
Title in Portuguese
Estresse oxidativo mitocondrial e deficiência familiar de glicocorticoide: caracterização fenotípica e funcional da nova variante p.G866D no gene Nicotinamide Nucleotide Transidrogenase (NNT)
Keywords in Portuguese
Deficiência Familiar de glicocorticoide
Estresse oxidativo
Gene NNT
Insuficiência Adrenal
Mutação
Abstract in Portuguese
Introdução: a proteína mitocondrial codificada pelo gene Nicotinamide Nucleotide Transidrogenase (NNT) regula o processo intramitocondrial de desintoxicação de espécies reativas de oxigênio (EROs). Defeitos na homeostase desse sistema (sistema Redox) podem comprometer a esteroidogênese adrenal. Objetivo: caracterizar o impacto funcional da nova variante missense NNT p.G866D (c.2597G>A) e sua associação com o fenótipo de deficiência familiar de glicocorticoide (DFG). Indivíduos e Métodos: Caso índice: lactente com DFG diagnosticada aos 18 meses, seus pais e irmão assintomáticos e controles saudáveis. O ácido desoxirribonucleico (DNA) genômico do caso índice foi submetido a sequenciamento exômico que revelou, entre algumas variantes gênicas-candidatas, a variante (c.2597G> A; p.G866D) em homozigose no exon 17 do gene NNT. Avaliou-se a segregação familiar dessa variante com o fenótipo de DFG. A análise funcional in vitro dessa variante foi realizada em cultura transitória de células mononucleares sanguíneas do paciente, heterozigotos e controles em condição basal e após a indução do estresse oxidativo com 100?m de H2O2 por cinco horas. A expressão do RNAm do gene NNT nessas células foi realizada por meio de qPCR. Os parâmetros mitocondriais avaliados foram: a produção intracelular de espécies reativas de oxigênio (EROS) detectadas por CMDCFDA, a concentração de glutationa reduzida (GSH) e ATP celular medidos ensaios luminescentes, , além da massa e potencial de membrana mitocondrial avaliados com o probe MitoTracker. Resultados: A análise de segregação familiar confirmou a segregação desta variante em homozigose com o fenótipo de DFG. A expressão do NNT RNAm foi semelhante entre paciente, heterozigotos e controles. Em homozigose a variante p.G866D no gene NNT causa aumento na produção de EROs, diminuição de GSH e diminuição da massa e potencial de membrana mitocondrial, tanto em condições basais, quanto após a indução de estresse oxidativo. Após a indução de estresse oxidativo as concentrações de ATP foram menores no homozigoto NNT p.G866D do que nos controles. Conclusão: este estudo confirma a associação da variante NNT em homozigose com o fenótipo de DFG Tipo quatro. In vitro o mutante homozigoto NNT p.G866D reduz significativamente os mecanismos de defesas antioxidantes, comprometendo o sistema da glutationa redutase, levando ao acúmulo de EROs.
Title in English
Mitochondrial oxidative stress and glucocorticoid family deficiency: phenotypic and functional characterization of the new variant p.G866D in the gene Nicotinamide Nucleotide Transidrogenase (NNT)
Keywords in English
Familial Glucocorticoid Deficiency
Mutation
NNT gene
Oxidative stress
Abstract in English
Background: mitochondrial Nicotinamide Nucleotide Transidrogenase (NNT) is essential in the intracellular reactive oxygen species (ROS) detox process. Defects in this system may impair adrenal steroidogenesis by yet not fully understood mechanism. Aim: we sought to characterize the functional impact of the new NNT p.G866D (c.2597G>A) variant and its association with familial glucocorticoid deficiency (FGD). Subjects and Methods: evaluated subjects included the index case diagnosed with FGD at the age of 18 months, his asymptomatic parents and young brother and healthy controls. The Index case genomic DNA was evaluated by whole exome sequencing, which revealed several potential candidate variants, including the homozygous NNT p.G866D. We then analyzed the family pedigree to confirm the segregation of this variant with FDG phenotype. To prove the genotype-phenotype association, this new variant was comprehensive evaluated in vitro. Transient mononuclear cell cultures from the patient, family members and controls were performed both in basal conditions and after oxidative stress induced by 5-hours treatment with 100?m de H2O2. We analyzed NNT RNAm expression by qPCR and mitochondrial parameters including ROS intracellular production by 20, 70-diacetate dichlorodihydrofluorescein, reduced glutathione (GSH) and cellular ATP levels by luminescence, and mitochondrial mass and mitochondrial membrane potential by Mitotraker Probe system. Results: family pedigree analysis confirmed the segregation of the homozygous NNT p.G866D variant with FGD. In vitro, no differences in RNAm expression was found among patients, heterozygous carriers and healthy controls. Both in basal and under stress conditions, the homozygous p.G866D NNT variant exhibited significantly increased ROS production, decreased GSH levels and decreased mitochondrial parameters. In addition, under H2O2 oxidative stress, the homozygous p.G866D NNT also leaded in decreased ATP intracellular . Conclusion: this study clearly confirms the association of the homozygous NNT p.G866D variant with the phenotype of FGD. In vitro, this variant significantly impairs anti-oxidants mechanisms and affects the glutathione reductase systems resulting in increased ROS accumulation.
 
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Publishing Date
2018-07-12
 
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