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OR11-03 A Novel Mutation In The Ligand-binding Domain Of The NR3C1 Gene Associated With Fluctuating Resistance To Glucocorticoids

Disclosure: M. Laulhé: None. E. Kuhn: None. J. Bouligand: None. L. Amazit: None. J. Perrot: None. P. Kamenicky: None. M. Lombes: None. J. Fagart: None. S. Viengchareun: None. L. Martinerie: None. Introduction: Constitutive glucocorticoid resistance is associated with mutations in the NR3C1 gene, enc...

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Autores principales: Laulhé, Margaux, Kuhn, Emmanuelle, Bouligand, Jerome, Amazit, Larbi, Perrot, Julie, Kamenicky, Peter, Lombes, Marc, Fagart, Jerome, Viengchareun, Say, Martinerie, Laetitia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10553904/
http://dx.doi.org/10.1210/jendso/bvad114.1736
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author Laulhé, Margaux
Kuhn, Emmanuelle
Bouligand, Jerome
Amazit, Larbi
Perrot, Julie
Kamenicky, Peter
Lombes, Marc
Fagart, Jerome
Viengchareun, Say
Martinerie, Laetitia
author_facet Laulhé, Margaux
Kuhn, Emmanuelle
Bouligand, Jerome
Amazit, Larbi
Perrot, Julie
Kamenicky, Peter
Lombes, Marc
Fagart, Jerome
Viengchareun, Say
Martinerie, Laetitia
author_sort Laulhé, Margaux
collection PubMed
description Disclosure: M. Laulhé: None. E. Kuhn: None. J. Bouligand: None. L. Amazit: None. J. Perrot: None. P. Kamenicky: None. M. Lombes: None. J. Fagart: None. S. Viengchareun: None. L. Martinerie: None. Introduction: Constitutive glucocorticoid resistance is associated with mutations in the NR3C1 gene, encoding the Glucocorticoid Receptor (GR). We identified a novel heterozygous point mutation causing fluctuating resistance to glucocorticoid depending on ligand concentrations. Initially, the propositus, a 45-year-old man, was diagnosed with a right adrenal incidentaloma and an ACTH-independent biological hypercortisolism. Shortly after adrenal surgery, plasma cortisol increased again (612 nmol/L) associated with inadequate ACTH levels (122 pg/mL) without any evidence of pituitary adenoma or ectopic ACTH-secreting tumor, suggesting a glucocorticoid resistance syndrome. Methods: We sequenced NR3C1 gene and performed in vitro analysis to assess contribution of this variant to the patient’s phenotype and how it affects biological activity of GR. Results: Exon sequencing of the NR3C1 gene identified a novel missense variant located on exon 5 (NM_000176.3, c.1706 G>A) resulting in substitution of arginine to glutamine at amino acid 569 (GR R569Q) in the Ligand-Binding Domain (LBD) of GR. We characterized transcriptional activity of GR R569Q in HEK 293T cells transiently co-transfected with either GR WT or GR R569Q, together with a luciferase reporter gene construct placed under control of a promoter containing two glucocorticoid responsive elements. We observed a decreased transcriptional activity of GR R569Q (EC(50) 4.55 nM [3.8;5.3]) compared to GR WT (EC(50) 0.19 nM [0.15; 0.24]), in response to dexamethasone. Interestingly, GR R569Q exerted a dominant negative effect on GR WT, with a decrease in transcriptional activity of 80% when stimulated with 1 nM dexamethasone (2-way ANOVA test, P<0.0001), which was blunted when stimulated with a 100-fold higher concentration of dexamethasone. As the variant is located within the second nuclear localization signal, we studied nuclear translocation in response to 1 nM cortisol for 1 h. As expected, nuclear-cytoplasmic ratio was lower in patients’ fibroblasts (2.3 ± 0.14 vs 3.0 ± 0.98, ANOVA 2-Way, P < 0.0001) compared with control fibroblasts, as quantified by automated high-throughput microscopy. Three-dimensional modeling revealed that arginine substitution leads to loss of a hydrogen bond stabilizing the LBD structure. This may result in a lower ligand affinity that can be bypassed by high concentrations of agonist. We are currently testing receptor affinity and reversibility of phenotype after hydrogen bond restoration by directed mutagenesis (c.1705 C>A, p.Q569K). Conclusion: Overall, we identified a novel variant of the NR3C1 gene associated with fluctuating GR sensitivity depending on ligand concentration. This finding may provide new insights into mechanisms involved in GR sensitivity. Presentation: Friday, June 16, 2023
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spelling pubmed-105539042023-10-06 OR11-03 A Novel Mutation In The Ligand-binding Domain Of The NR3C1 Gene Associated With Fluctuating Resistance To Glucocorticoids Laulhé, Margaux Kuhn, Emmanuelle Bouligand, Jerome Amazit, Larbi Perrot, Julie Kamenicky, Peter Lombes, Marc Fagart, Jerome Viengchareun, Say Martinerie, Laetitia J Endocr Soc Steroid Hormones, Nuclear Receptors And Coregulators Disclosure: M. Laulhé: None. E. Kuhn: None. J. Bouligand: None. L. Amazit: None. J. Perrot: None. P. Kamenicky: None. M. Lombes: None. J. Fagart: None. S. Viengchareun: None. L. Martinerie: None. Introduction: Constitutive glucocorticoid resistance is associated with mutations in the NR3C1 gene, encoding the Glucocorticoid Receptor (GR). We identified a novel heterozygous point mutation causing fluctuating resistance to glucocorticoid depending on ligand concentrations. Initially, the propositus, a 45-year-old man, was diagnosed with a right adrenal incidentaloma and an ACTH-independent biological hypercortisolism. Shortly after adrenal surgery, plasma cortisol increased again (612 nmol/L) associated with inadequate ACTH levels (122 pg/mL) without any evidence of pituitary adenoma or ectopic ACTH-secreting tumor, suggesting a glucocorticoid resistance syndrome. Methods: We sequenced NR3C1 gene and performed in vitro analysis to assess contribution of this variant to the patient’s phenotype and how it affects biological activity of GR. Results: Exon sequencing of the NR3C1 gene identified a novel missense variant located on exon 5 (NM_000176.3, c.1706 G>A) resulting in substitution of arginine to glutamine at amino acid 569 (GR R569Q) in the Ligand-Binding Domain (LBD) of GR. We characterized transcriptional activity of GR R569Q in HEK 293T cells transiently co-transfected with either GR WT or GR R569Q, together with a luciferase reporter gene construct placed under control of a promoter containing two glucocorticoid responsive elements. We observed a decreased transcriptional activity of GR R569Q (EC(50) 4.55 nM [3.8;5.3]) compared to GR WT (EC(50) 0.19 nM [0.15; 0.24]), in response to dexamethasone. Interestingly, GR R569Q exerted a dominant negative effect on GR WT, with a decrease in transcriptional activity of 80% when stimulated with 1 nM dexamethasone (2-way ANOVA test, P<0.0001), which was blunted when stimulated with a 100-fold higher concentration of dexamethasone. As the variant is located within the second nuclear localization signal, we studied nuclear translocation in response to 1 nM cortisol for 1 h. As expected, nuclear-cytoplasmic ratio was lower in patients’ fibroblasts (2.3 ± 0.14 vs 3.0 ± 0.98, ANOVA 2-Way, P < 0.0001) compared with control fibroblasts, as quantified by automated high-throughput microscopy. Three-dimensional modeling revealed that arginine substitution leads to loss of a hydrogen bond stabilizing the LBD structure. This may result in a lower ligand affinity that can be bypassed by high concentrations of agonist. We are currently testing receptor affinity and reversibility of phenotype after hydrogen bond restoration by directed mutagenesis (c.1705 C>A, p.Q569K). Conclusion: Overall, we identified a novel variant of the NR3C1 gene associated with fluctuating GR sensitivity depending on ligand concentration. This finding may provide new insights into mechanisms involved in GR sensitivity. Presentation: Friday, June 16, 2023 Oxford University Press 2023-10-05 /pmc/articles/PMC10553904/ http://dx.doi.org/10.1210/jendso/bvad114.1736 Text en © The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. https://creativecommons.org/licenses/by-nc-nd/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Steroid Hormones, Nuclear Receptors And Coregulators
Laulhé, Margaux
Kuhn, Emmanuelle
Bouligand, Jerome
Amazit, Larbi
Perrot, Julie
Kamenicky, Peter
Lombes, Marc
Fagart, Jerome
Viengchareun, Say
Martinerie, Laetitia
OR11-03 A Novel Mutation In The Ligand-binding Domain Of The NR3C1 Gene Associated With Fluctuating Resistance To Glucocorticoids
title OR11-03 A Novel Mutation In The Ligand-binding Domain Of The NR3C1 Gene Associated With Fluctuating Resistance To Glucocorticoids
title_full OR11-03 A Novel Mutation In The Ligand-binding Domain Of The NR3C1 Gene Associated With Fluctuating Resistance To Glucocorticoids
title_fullStr OR11-03 A Novel Mutation In The Ligand-binding Domain Of The NR3C1 Gene Associated With Fluctuating Resistance To Glucocorticoids
title_full_unstemmed OR11-03 A Novel Mutation In The Ligand-binding Domain Of The NR3C1 Gene Associated With Fluctuating Resistance To Glucocorticoids
title_short OR11-03 A Novel Mutation In The Ligand-binding Domain Of The NR3C1 Gene Associated With Fluctuating Resistance To Glucocorticoids
title_sort or11-03 a novel mutation in the ligand-binding domain of the nr3c1 gene associated with fluctuating resistance to glucocorticoids
topic Steroid Hormones, Nuclear Receptors And Coregulators
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10553904/
http://dx.doi.org/10.1210/jendso/bvad114.1736
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