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Three Novel Heterozygous Point Mutations of NR3C1 Causing Glucocorticoid Resistance
Author(s) -
Vitellius Géraldine,
Fagart Jérôme,
Delemer Brigitte,
Amazit Larbi,
Ramos Nelly,
Bouligand Jérôme,
Billan Florian,
Castinetti Frédéric,
GuiochonMantel Anne,
Trabado Séverine,
Lombès Marc
Publication year - 2016
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.23008
Subject(s) - transactivation , glucocorticoid receptor , biology , mutant , point mutation , glucocorticoid , chromatin immunoprecipitation , mutation , wild type , microbiology and biotechnology , genetics , gene , transcription factor , endocrinology , gene expression , promoter
ABSTRACT 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 GR R477S and GR Y478C mutants, located in the DNA‐binding domain (DBD) of GR, was similar to wild‐type GR ( K d = 2–3 nM). In contrast, GR L672P 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 GR R477S and GR Y478C mutants, whereas GR L672P 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 GR R477S and GR L672P , whereas GR Y478C 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.