Mutations in the melanocortin 2 receptor (MC2R) and its accessory protein (MRAP), in the ACTH signalling pathway, and the antioxidant genes nicotinamide nucleotide transhydrogenase (NNT) and thioredoxin reductase 2 (TXNRD2) have been associated with familial glucocorticoid deficiency (FGD). Using a tandem affinity purification and mass spectrometry approach to identify interacting partners of MC2R and MRAP failed to identify putative candidate genes for further FGD cases. However in a male patient a homozygous mutation in another antioxidant gene, glutathione peroxidase 1 (GPX1), was identified. In vitro studies showed H295R cells with knockdown of GPX1 had 50% less basal GPX activity and were less viable than wild-type when exposed to oxidative stress. Adrenals from Gpx1-/- mice showed no gross morphological changes and corticosterone levels were not significantly different from their wild-type counterparts (in contrast to the Nnt mutants). Sequencing of >100 FGD patients did not reveal any other GPX1 mutations. This equivocal data lead to the hypothesis that there could be a second gene defect present in this proband contributing to his disease. Whole exome sequencing revealed a homozygous loss-of-function mutation in peroxiredoxin 3 PRDX3 (p.Q67X) in this patient, that was also present in his unaffected brother. In vitro studies revealed both single and double knockdown of the two genes in H295R cells reduced cell viability, but redox homeostasis and cortisol production were unaffected. GPXs and PRDXs work simultaneously to reduce H2O2, preventing cellular damage. My data suggest that loss of PRDX3 alone is insufficient to cause adrenal failure and further that mutation in GPX1, either alone or in combination with PRDX3 mutation, may tip the redox balance to cause FGD.

Maintaining redox homeostasis is crucial for normal cellular functions. Electron leak by the cytochrome P450 enzymes renders steroidogenic tissues acutely vulnerable to redox imbalance and oxidative stress is implicated in several potentially lethal adrenal disorders. This thesis aims to further delineate the role of oxidative stress in triple A syndrome and familial glucocorticoid deficiency (FGD). Triple A syndrome incorporates adrenal failure and progressive neurodegenerative disease. The AAAS gene product is the nuclear pore complex protein ALADIN, of unknown function. Patient dermal fibroblasts are sensitive to oxidative stress, with failure of nuclear import of DNA repair proteins and ferritin heavy chain protein. To provide an adrenal and neuronal-specific disease model, I established AAAS-knockdown in H295R human adrenocortical tumour cells and SH-SY5Y human neuroblastoma cells. This had effects on cell viability, exacerbated by hydrogen peroxide treatment. Redox homeostasis was impaired in AAAS-knockdown H295R cells, with depletion of key components of the steroidogenic pathway and a significant reduction in cortisol production, with partial reversal following treatment with N-acetylcysteine. Mutations in the mitochondrial antioxidant, nicotinamide nucleotide transhydrogenase (NNT), causing FGD, have recently highlighted the importance of redox regulation in steroidogenesis. I investigated seven individuals from a consanguineous Kashmiri kindred, mutation negative for known causes of FGD. A stop gain mutation, p.Y447* in TXNRD2, encoding the mitochondrial selenoprotein thioredoxin reductase 2 segregated with the disease trait; with complete absence of the 56 kDa TXNRD2 protein in patients homozygous for the mutation. TXNRD2-knockdown led to impaired redox homeostasis in H295R cells. This is the first report of a homozygous mutation in any component of the thioredoxin antioxidant system leading to inherited disease in humans.

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. / 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.

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. / 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.

Objective: Description of the clinical, biochemical and genetic features of a Polish patient with familial glucocorticoid deficiency. Methods: Detailed clinical investigation, hormonal analysis and sequencing of the coding region of the melanocortin 2 receptor (MC2R) gene in this patient. Results: We report on a 3-month-old boy with familial glucocorticoid deficiency who presented at the age of 3 months with skin hyperpigmentation, muscle weakness, mild jaundice and constipation. Hormonal analyses revealed high ACTH and TSH serum concentrations, low serum cortisol concentration along with normal blood electrolytes. On hydrocortisone supplementation, the disease symptoms disappeared and the child recovered completely. His physical and mental development progresses normally. Genetic analysis disclosed a novel compound heterozygous MC2R mutation p.Leu46fs and p.Val49Met. Conclusion: The heterozygous p.Leu46fs mutation adds to the small number of MC2R nonsense mutations and is the first frameshift mutation within the first transmembrane domain of the receptor. According to molecular modeling the Val49Met mutation results in a structural change of the first transmembrane domain and in a potential novel interaction of the transmembrane domains I and VII. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Objective: Description of the clinical, biochemical and genetic features of a Polish patient with familial glucocorticoid deficiency. Methods: Detailed clinical investigation, hormonal analysis and sequencing of the coding region of the melanocortin 2 receptor (MC2R) gene in this patient. Results: We report on a 3-month-old boy with familial glucocorticoid deficiency who presented at the age of 3 months with skin hyperpigmentation, muscle weakness, mild jaundice and constipation. Hormonal analyses revealed high ACTH and TSH serum concentrations, low serum cortisol concentration along with normal blood electrolytes. On hydrocortisone supplementation, the disease symptoms disappeared and the child recovered completely. His physical and mental development progresses normally. Genetic analysis disclosed a novel compound heterozygous MC2R mutation p.Leu46fs and p.Val49Met. Conclusion: The heterozygous p.Leu46fs mutation adds to the small number of MC2R nonsense mutations and is the first frameshift mutation within the first transmembrane domain of the receptor. According to molecular modeling the Val49Met mutation results in a structural change of the first transmembrane domain and in a potential novel interaction of the transmembrane domains I and VII. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.