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Glucocorticoids (GCs) exert potent antiproliferative and anti-inflammatory properties, explaining their therapeutic efficacy for skin diseases. GCs act by binding to the GC receptor (GR) and the mineralocorticoid receptor (MR), co-expressed in classical and non-classical targets including keratinocytes. Using knockout mice, we previously demonstrated that GR and MR exert essential nonoverlapping functions in skin homeostasis. These closely related receptors may homo- or heterodimerize to regulate transcription, and theoretically bind identical GC-response elements (GRE). We assessed the contribution of MR to GR genomic binding and the transcriptional response to the synthetic GC dexamethasone (Dex) using control (CO) and MR knockout (MREKO ) keratinocytes. GR chromatin immunoprecipitation (ChIP)-seq identified peaks common and unique to both genotypes upon Dex treatment (1 h). GREs, AP-1, TEAD, and p53 motifs were enriched in CO and MREKO peaks. However, GR genomic binding was 35% reduced in MREKO , with significantly decreased GRE enrichment, and reduced nuclear GR. Surface plasmon resonance determined steady state affinity constants, suggesting preferred dimer formation as MR-MR > GR-MR ~ GR-GR; however, kinetic studies demonstrated that GR-containing dimers had the longest lifetimes. Despite GR-binding differences, RNA-seq identified largely similar subsets of differentially expressed genes in both genotypes upon Dex treatment (3 h). However, time-course experiments showed gene-dependent differences in the magnitude of expression, which correlated with earlier and more pronounced GR binding to GRE sites unique to CO including near Nr3c1. Our data show that endogenous MR has an impact on the kinetics and differential genomic binding of GR, affecting the time-course, specificity, and magnitude of GC transcriptional responses in keratinocytes.
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Receptores de Glucocorticoides , Receptores de Mineralocorticoides , Animais , Camundongos , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Glucocorticoides/farmacologia , Glucocorticoides/metabolismo , Cinética , Queratinócitos/metabolismo , Camundongos Knockout , GenômicaRESUMO
Generalized glucocorticoid resistance is associated with glucocorticoid receptor (GR; NR3C1) mutations. Three novel heterozygous missense NR3C1 mutations (R477S, Y478C, and L672P) were identified in patients presenting with adrenal incidentalomas, glucocorticoid excess without Cushing syndrome. Dexamethasone (DXM) binding studies demonstrated that the affinity of GRR477S and GRY478C mutants, located in the DNA-binding domain (DBD) of GR, was similar to wild-type GR (Kd = 2-3 nM). In contrast, GRL672P mutant, located in the ligand-binding domain (LBD) of GR, was unable to bind glucocorticoids and was more sensitive to protein degradation. GR subcellular distribution revealed a marked decrease in DXM-induced nuclear translocation of GRR477S and GRY478C mutants, whereas GRL672P remained exclusively cytoplasmic. Chromatin immunoprecipitation demonstrated impaired recruitment of DBD mutants onto the regulatory sequence of FKBP5. Transactivation assays disclosed the lack of transcriptional activity of GRR477S and GRL672P , whereas GRY478C had a reduced transactivation capacity. Three-dimensional modeling indicated that R477S lost two essential hydrogen bonds with DNA, Y478C resulted in altered interaction with surrounding amino-acids, destabilizing DBD, whereas L672P altered the H8 helix folding, leading to unstructured LBD. This study identifies novel NR3C1 mutations with their molecular consequences on altered GR signaling and suggests that genetic screening of NR3C1 should be conducted in patients with subclinical hypercorticism.
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Neoplasias das Glândulas Suprarrenais/genética , Resistencia a Medicamentos Antineoplásicos , Glucocorticoides/farmacologia , Mutação Puntual , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Neoplasias das Glândulas Suprarrenais/tratamento farmacológico , Adulto , Animais , Sítios de Ligação , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Síndrome de Cushing/genética , Citoplasma/metabolismo , DNA de Neoplasias/metabolismo , Dexametasona/farmacologia , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Transporte Proteico , Receptores de Glucocorticoides/metabolismoRESUMO
The transition of young type 1 diabetic (T1D) patients from pediatric to adult healthcare is a high-risk period of loss to follow-up. Since 2015, we have implemented a transition program, involving both pediatric and adult clinicians. The main objective was to evaluate the number of patients who had succeeded this transition program at 1 year. We found that 86% of patients underwent the complete transition program. However, adverse outcomes occurred in 19.1% of patients at 1 year but decreased to 2.9% after 3 years. In 63% of patients their HbA1c level had deteriorated 1 year after the transition day and this level stabilized at around 8% in the following 2 and 3 years. In patients who had improved HbA1c levels the body mass index was lower (P = 0.03) and they lived alone (P = 0.04). Although our program seemed to allow a better follow-up than previously described, this study highlights the importance of further supporting this transition period.
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Context: Medullary thyroid cancer (MTC) is a rare disease. Objective: The main objective of our study was to analyze the incidence evolution of MTC with a follow-up of more than 40 years. Further, a descriptive and survival analysis was performed according to the Kaplan-Meier analysis. Design Setting and Patients: This is a retrospective epidemiological study using data from the Marne-Ardennes registry from 1975 to 2018. Two hundred sixty patients with MTC were included. Main Outcome Measures: The incidence was calculated in the territory of the register (Marne and Ardennes departments of France) and standardized on the demographic structure of France. Patient and tumor characteristics were described. An analysis in a subgroup comparing hereditary and sporadic forms was performed. An analysis of survival was performed. Results: The standardized incidence shows an increasing trend over time. The incidence increased significantly from 0.41 to 0.57/100 000 person-years between 1986 and 1996 and 2008 and 2018. The MTC was hereditary in 21.2% of cases. The sex ratio (males:females) was 0.73. The average age at diagnosis was 53 years. Ninety-seven patients (37.3%) were N1, 26 (10%) were M1, and 56 (21.5%) developed metastases during the follow-up. Complete remission was obtained in 58.5% of patients. The disease was refractory for 18.1% of patients. The 5-year survival rate was 88.4%. Sporadic cases had a poorer prognosis than hereditary MTC. Conclusion: Our study demonstrates a moderate increase in the incidence of MTC between 1975 and 2018. The prognosis remains worse for sporadic MTC than for hereditary MTC.
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CONTEXT: Prospective studies have demonstrated the efficacy of osilodrostat in Cushing disease. No study has evaluated osilodrostat in a series of patients with paraneoplastic Cushing syndrome/ectopic adrenocorticotropin syndrome (PNCS/EAS). OBJECTIVE: This work aimed to evaluate in France the real-world efficacy and safety of osilodrostat in patients with PNCS/EAS. METHODS: A total of 33 patients with PNCS/EAS with intense/severe hypercortisolism were involved in this retrospective, multicenter, real-world study. Patients received osilodrostat between May 2019 and March 2022 at a median initial dose (range) of 4 mg/day (1-60) and maximum dose, 20 mg/day (4-100), first under patient then cohort temporary authorizations and after marketing authorization. Regimens used titration (n = 6), block and replace (n = 16), or titration followed by block and replace (n = 11). RESULTS: In 11 patients receiving osilodrostat as first-line monotherapy, median 24-hour urinary free cortisol (24h-UFC) decreased dramatically (from 26 × upper limit of normal [ULN; 2.9-659] to 0.11 × ULN [0.08-14.9]; P < .001). In 9 of them, 24h-UFC normalization was achieved in 2 weeks (median). Thirteen additional patients were previously treated with classic steroidogenesis inhibitors but 10 of these 13 were not controlled. In these patients, osilodrostat monotherapy, used as second line, induced a significantly decreased of 24h-UFC (from 2.6 × ULN [1.1-144] to 0.22 × ULN [0.12-0.66]; P < .01). Nine additional patients received osilodrostat in combination with another anticortisolic drug, decreasing 24h-UFC from 11.8 × ULN (0.3-247) to 0.43 × ULN (0.33-2.4) (P < .01). In parallel, major clinical symptoms/comorbidities improved dramatically with improvement in blood pressure, hyperglycemia, and hypokalemia, allowing the discontinuation or dose reduction of patient treatments. Adrenal insufficiency (grade 3-4) was reported in 8 of 33 patients. CONCLUSION: Osilodrostat is a rapidly efficient therapy for PNCS/EAS with severe/intense hypercortisolism. Osilodrostat was generally well tolerated; adrenal insufficiency was the main side effect.
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Insuficiência Adrenal , Síndrome de Cushing , Humanos , Síndrome de Cushing/tratamento farmacológico , Estudos Prospectivos , Estudos Retrospectivos , Hormônio Adrenocorticotrópico , Hidrocortisona/uso terapêuticoRESUMO
BACKGROUND: Diabetes mellitus prevalence is increasing among women of child-bearing age. Diabetic pregnancy is associated with major maternal and fetal risks, and these can be reduced by preconception care. Pregnancy can be planned using appropriate effective contraception. The objective of this study was to assess diabetic patients' knowledge about pregnancy and to describe their contraceptive use. STUDY DESIGN: An observational study was conducted from February to July 2020 at Reims University Hospital, France. Inclusion criteria were: women aged 18 to 40years, with type 1 (T1D) or type 2 diabetes (T2D). Patients filled out a survey about contraceptive use and knowledge regarding diabetic pregnancy and data were completed from medical records. RESULTS: Eighty-nine T1D and 33 T2D patients were included, with mean ages of 27.9±6.3 and 32.6±4.6years, respectively. Seventy-five percent reported that they had been informed about pregnancy-related risks and 67% about the need to plan pregnancy. The preconception HbA1c target was known by 33% of patients. Appropriate knowledge about pregnancy was greater in T1D patients (65.9%, versus 36.4% in T2D patients; P=0.003). The rate of patients using an effective contraceptive method was 66.4%. Fifteen percent patients for whom contraception was recommended reported having no contraceptive method; 12.5% of contraception users were using a contraindicated method. CONCLUSION: A large majority of diabetic women were aware of pregnancy-related risks and the importance of pregnancy planning, but there are still gaps, especially in T2D patients. We need to improve our practices by providing more information and better access to appropriate effective contraception. GOV NUMBER: NCT04350879.
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Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Gravidez em Diabéticas , Adulto , Anticoncepção/métodos , Anticoncepcionais , Diabetes Mellitus Tipo 2/epidemiologia , Feminino , Humanos , Masculino , Gravidez , Gravidez em Diabéticas/epidemiologia , Adulto JovemRESUMO
French health insurance data showed that the incidence of type 1 diabetes mellitus (T1DM) in children increased over the years to 2015. The objective of our study was to assess the evolution of the number of incident cases of paediatric and adult type 1 diabetes in our institution, and to describe their clinical presentation and its evolution. All patients with T1DM managed at diagnosis at Reims University Hospital between 1997 and 2019 were included. The clinical and biological data were extracted from the Champagne-Ardenne Diabetes Network database. Included were 847 patients with a median age of 10.3 years. Diagnosis was established in 71% of cases before 15 years, 7.4% after 35 years. The number of newly diagnosed cases was 3.6-times higher in 2019 compared to 1997. Ketoacidosis, the frequency of which decreased with age (P < 0.0001), revealed diabetes in a total of 32% of cases and in 46% of children under 5 years. It was more severe in children than in adults (P = 0.03), and its frequency increased over the study period. Hypotrophy was found in 23% of children under 15 years of age, and was more pronounced before 5 years of age, with no improvement over time. We saw an increase in the frequency of obesity or overweight among adults. Our study showed an increase in incident cases of diabetes in our hospital that continued over time for both children and adults. Clinical features at diagnosis deteriorated during this period for those under 15 years of age with an increase in ketoacidosis frequency.
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Diabetes Mellitus Tipo 1 , Cetoacidose Diabética , Cetose , Adulto , Criança , Pré-Escolar , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/epidemiologia , Cetoacidose Diabética/diagnóstico , Cetoacidose Diabética/epidemiologia , Cetoacidose Diabética/etiologia , Hospitais , Humanos , Incidência , Estudos RetrospectivosRESUMO
Glucocorticoids (GC) such as cortisol regulate multiple physiological functions, notably those involved in development, metabolism, inflammatory processes and stress, and exert their effects upon binding to the glucocorticoid receptor (GR, encoded by NR3C1 gene in humans). GC signaling follows several consecutive steps leading to target gene transactivation, including ligand binding, nuclear translocation of ligand-activated GR complexes, DNA binding, and recruitment of functional transcriptional machinery. Generalized glucocorticoid resistance syndrome, due to GR loss-of-function mutations, may be related to the impairment of one of the GC signaling steps. To date, 31 NR3C1 loss-of-function mutations have been reported in patients presenting with various clinical signs such as hypertension, adrenal hyperplasia, hirsutism or metabolic disorders associated with biological hypercortisolism but without Cushing syndrome signs and no negative regulatory feedback loop on the hypothalamic-pituitary-adrenal axis. Functional characterization of GR loss-of-function mutations often demonstrates GR haploinsufficiency and a decrease of GR target gene induction in relevant cell types. The main signs at presentation are very variable from resistant hypertension, bilateral adrenal hyperplasia likely related to increased ACTH levels but not exclusively, hirsutism to isolated renin-angiotensin-aldosterone system abnormalities in a context of 11ßHSD2 deficiency. Some mutated GR patients are obese or overweight together with a healthier metabolic profile that remains to be further explored in future studies. Deciphering the molecular mechanisms altered by GR mutations should enhance our knowledge on GR signaling and ultimately facilitate management of GC-resistant patients. This review also focuses on the criteria facilitating identification of novel NR3C1 mutations in selected patients.
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Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/genética , Receptores de Glucocorticoides/deficiência , Hormônio Adrenocorticotrópico/sangue , Hormônio Adrenocorticotrópico/genética , Glucocorticoides/sangue , Glucocorticoides/genética , Humanos , Hidrocortisona/sangue , Hidrocortisona/genética , Erros Inatos do Metabolismo/sangue , Receptores de Glucocorticoides/sangue , Receptores de Glucocorticoides/genéticaRESUMO
Recent advances in genetic analysis technologies such as next-generation sequencing (NGS) have considerably increased the incidental discovery of genetic abnormalities. Six heterozygous missense mutations of the human glucocorticoid receptor (GR; encoded by the NR3C1 gene) have been identified in the context of genetic screening of endocrine pathologies. GR, a nuclear receptor, hormone-induced transcription factor, is involved in many physiological processes. Nevertheless, the pathogenic significance of incidentally discovered mutations remains obscure. The aim of this work was to characterize these variants by evaluating their functional impact on GR signaling. Six original GR variants, located in exon 2, led to amino acid substitutions of the N-terminal domain of GR (F65V, M86V, A229T, A304E, N374S, and R386Q), excluding mainly the activation function tau core 1 domain, the potential site of functional interaction with transcriptional coregulators. Transient cotransfection in HEK293T cells of mutated GR-expressing vectors and a luciferase reporter established dose-response curves for dexamethasone. This excluded any major transactivation abnormality of the mutated GRs (ligand concentration leading to 50% maximal transactivation capacity ≈ 0.2 nM), with maximal transactivation capacity identical to that of the wild-type (WT) GR and without modification of the potentiation of transcriptional coactivator steroid receptor coactivator 2 except in N374S. Moreover, protein expression of mutated GRs and their cytonuclear translocation studied by immunocytochemistry were almost unchanged compared with WT GR. These results underline the silent nature of these missense GR variants and call for cautious interpretation of the discovery of genetic incidentalomas by NGS in the absence of detailed characterization in order to appropriately assess their functional impact on a particular signaling pathway.
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CONTEXT: Six patients carrying heterozygous loss-of-function mutations of glucocorticoid (GC) receptor (GR) presented with hypercortisolism, associated with low kalemia, low plasma renin, and aldosterone levels, with or without hypertension, suggesting a pseudohypermineralocorticism whose mechanisms remain unclear. We hypothesize that an impaired activity of the 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2; encoded by the HSD11B2 gene), catalyzing cortisol (F) inactivation, may account for an inappropriate activation of a renal mineralocorticoid signaling pathway in these GC-resistant patients. OBJECTIVE: We aim at studying the GR-mediated regulation of HSD11B2. DESIGN: The HSD11B2 promoter was subcloned and luciferase reporter assays evaluated GR-dependent HSD11B2 regulation, and 11ß-HSD2 expression/activity was studied in human breast cancer MCF7 cells, endogenously expressing this enzyme. RESULTS: Transfection assays revealed that GR transactivated the long (2.1-kbp) HSD11B2 promoter construct, whereas a defective 501H GR mutant was unable to stimulate luciferase activity. GR-mediated transactivation of the HSD11B2 gene was inhibited by the GR antagonist RU486. A threefold increase in HSD11B2 mRNA levels was observed after dexamethasone (DXM) treatment of MCF7 cells, inhibited by RU486 or by actinomycin, supporting a GR-dependent transcription. Chromatin immunoprecipitation further demonstrated a DXM-dependent GR recruitment onto the HSD11B2 promoter. 11ß-HSD2 activity, evaluated by the cortisone/F ratio, quantified by liquid chromatography/tandem mass spectrometry, was 10-fold higher in the supernatant of DXM-treated cells than controls, consistent with a GR-dependent stimulation of 11ß-HSD2 catalytic activity. CONCLUSION: Collectively, we demonstrate that 11ß-HSD2 expression and activity are transcriptionally regulated by GR. In the context of GR haploinsufficiency, these findings provide evidence that defective GR signaling may account for apparent mineralocorticoid excess in GC-resistant patients.
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11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Regulação da Expressão Gênica , Receptores de Glucocorticoides/metabolismo , Adulto , Idoso , Linhagem Celular Tumoral , Dexametasona/administração & dosagem , Feminino , Células HEK293 , Humanos , Mutação com Perda de Função , Masculino , Pessoa de Meia-Idade , Síndrome de Excesso Aparente de Minerolocorticoides/genética , Síndrome de Excesso Aparente de Minerolocorticoides/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/genética , Transdução de Sinais , Síndrome de Excesso Aparente de MinerolocorticoidesRESUMO
Glucocorticoids (GC), such as cortisol or dexamethasone, control various physiological functions, notably those involved in development, metabolism, inflammatory processes and stress, and exert most of their effects upon binding to the glucocorticoid receptor (GR, encoded by NR3C1 gene). GC signaling follows several consecutive steps leading to target gene transactivation, including ligand binding, nuclear translocation of ligand-activated GR complexes, DNA binding, coactivator interaction and recruitment of functional transcriptional machinery. Any step may be impaired and may account for altered GC signaling. Partial or generalized glucocorticoid resistance syndrome may result in a reduced level of functional GR, a decreased hormone affinity and binding, a defect in nuclear GR translocation, a decrease or lack of DNA binding and/or post-transcriptional GR modifications. To date, 26 loss-of-function NR3C1 mutations have been reported in the context of hypertension, hirsutism, adrenal hyperplasia or metabolic disorders. These clinical signs are generally associated with biological features including hypercortisolism without negative regulatory feedback loop on the hypothalamic-pituitary-adrenal axis. Patients had often low plasma aldosterone and renin levels despite hypertension. Only one GR gain-of-function mutation has been described associating Cushing's syndrome phenotype with normal urinary-free cortisol. Some GR polymorphisms (ER22/23EK, GR-9ß) have been linked to glucocorticoid resistance and a healthier metabolic profile whereas some others seemed to be associated with GC hypersensitivity (N363S, BclI), increasing cardiovascular risk (diabetes type 2, visceral obesity). This review focuses on the earlier findings on the pathophysiology of GR signaling and presents criteria facilitating identification of novel NR3C1 mutations in selected patients.
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Glucocorticoides/metabolismo , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Receptores de Glucocorticoides/deficiência , Síndrome de Cushing/genética , Predisposição Genética para Doença , Humanos , Sistema Hipotálamo-Hipofisário/fisiologia , Mutação , Sistema Hipófise-Suprarrenal/fisiologia , Polimorfismo Genético , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Recently discovered mutations of NR3C1 gene, encoding for the GR, in patients with glucocorticoid resistance and bilateral adrenal incidentalomas prompted us to investigate whether GR mutations might be associated with adrenal hyperplasia. OBJECTIVE: The multicenter French Clinical Research Program (Muta-GR) was set up to determine the prevalence of GR mutations and polymorphisms in patients harboring bilateral adrenal incidentalomas associated with hypertension and/or biological hypercortisolism without clinical Cushing's signs. RESULTS: One hundred patients were included in whom NR3C1 sequencing revealed five original heterozygous GR mutations that impaired GR signaling in vitro. Mutated patients presented with mild glucocorticoid resistance defined as elevated urinary free cortisol (1.7 ± 0.7 vs 0.9 ± 0.8 upper limit of normal range, P = 0.006), incomplete 1 mg dexamethasone suppression test without suppressed 8-AM adrenocorticotrophin levels (30.9 ± 31.2 vs 16.2 ± 17.5 pg/mL) compared to the non-mutated patients. Potassium and aldosterone levels were lower in mutated patients (3.6 ± 0.2 vs 4.1 ± 0.5 mmol/L, P = 0.01, and 17.3 ± 9.9 vs 98.6 ± 115.4 pg/mL, P = 0.0011, respectively) without elevated renin levels, consistent with pseudohypermineralocorticism. Ex vivo characterization of mutated patients' fibroblasts demonstrated GR haploinsufficiency as revealed by below-normal glucocorticoid induction of FKBP5 gene expression. There was no association between GR polymorphisms and adrenal hyperplasia in this cohort, except an over-representation of BclI polymorphism. CONCLUSION: The 5% prevalence of heterozygous NR3C1 mutations discovered in our series is higher than initially thought and encourages GR mutation screening in patients with adrenal incidentalomas to unambiguously differentiate from Cushing's states and to optimize personalized follow-up.