Three Novel Heterozygous Point Mutations of NR3C1 Causing Glucocorticoid Resistance.
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.
INSERM UMR S 1185, Fac Med Paris Sud, Univ. Paris Sud, Université Paris-Saclay, Le Kremlin Bicêtre, F-94276, France.
Géraldine
G
Vitellius
INSERM UMR S 1185, Fac Med Paris Sud, Univ. Paris Sud, Université Paris-Saclay, Le Kremlin Bicêtre, F-94276, France.
Jérôme
J
Fagart
Service d'Endocrinologie, Hôpital Robert Debré, CHU Reims, Reims, F-51100, France.
Brigitte
B
Delemer
INSERM UMR S 1185, Fac Med Paris Sud, Univ. Paris Sud, Université Paris-Saclay, Le Kremlin Bicêtre, F-94276, France.|INSERM UMS-32, Institut Biomédical de Bicêtre, Univ. Paris Sud, Université Paris-Saclay, Le Kremlin Bicêtre, F-94275, France.
Larbi
L
Amazit
INSERM UMR S 1185, Fac Med Paris Sud, Univ. Paris Sud, Université Paris-Saclay, Le Kremlin Bicêtre, F-94276, France.
Nelly
N
Ramos
INSERM UMR S 1185, Fac Med Paris Sud, Univ. Paris Sud, Université Paris-Saclay, Le Kremlin Bicêtre, F-94276, France.|Service de Génétique Moléculaire, Pharmacogénétique et Hormonologie, Hôpitaux Universitaires Paris Sud, CHU Bicêtre, F-94275, France.
Jérôme
J
Bouligand
INSERM UMR S 1185, Fac Med Paris Sud, Univ. Paris Sud, Université Paris-Saclay, Le Kremlin Bicêtre, F-94276, France.
Florian
F
Le Billan
Service d'Endocrinologie, Hôpital de la Timone, CHU Marseille, Marseille, F-13385, France.
Frédéric
F
Castinetti
INSERM UMR S 1185, Fac Med Paris Sud, Univ. Paris Sud, Université Paris-Saclay, Le Kremlin Bicêtre, F-94276, France.|Service de Génétique Moléculaire, Pharmacogénétique et Hormonologie, Hôpitaux Universitaires Paris Sud, CHU Bicêtre, F-94275, France.
Anne
A
Guiochon-Mantel
INSERM UMR S 1185, Fac Med Paris Sud, Univ. Paris Sud, Université Paris-Saclay, Le Kremlin Bicêtre, F-94276, France.|Service de Génétique Moléculaire, Pharmacogénétique et Hormonologie, Hôpitaux Universitaires Paris Sud, CHU Bicêtre, F-94275, France.
Séverine
S
Trabado
INSERM UMR S 1185, Fac Med Paris Sud, Univ. Paris Sud, Université Paris-Saclay, Le Kremlin Bicêtre, F-94276, France.|Service d'Endocrinologie et des Maladies de la Reproduction, Hôpitaux Universitaires Paris Sud, CHU Bicêtre, Le Kremlin Bicêtre, F-94275, France.
Marc
M
Lombès
D000310:Adrenal Gland Neoplasms / Q000188:drug therapy / Q000235:genetics*; D000328:Adult; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D003480:Cushing Syndrome / Q000235:genetics; D003593:Cytoplasm / Q000378:metabolism; D004273:DNA, Neoplasm / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D005260:Female; D005938:Glucocorticoids / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008875:Middle Aged; D008958:Models, Molecular; D020125:Mutation, Missense; D017354:Point Mutation; D017433:Protein Structure, Secondary; D021381:Protein Transport; D011965:Receptors, Glucocorticoid / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D004273:DNA, Neoplasm; D005938:Glucocorticoids; C497713:NR3C1 protein, human; D011965:Receptors, Glucocorticoid; D003907:Dexamethasone
NR3C1; adrenal masses; glucocorticoid resistance; glucocorticoid signaling