Glucocorticoid Resistance Syndrome as a Hidden Cause of Nonneoplastic Hypercortisolism

Abstract

Cases of hypercortisolemia without physical signs of Cushing's syndrome (CS), suggestive of nonneoplastic hypercortisolism (NNH), often remain partially unexplained. We present a unique case that was initially misdiagnosed as ACTH-dependent CS due to abnormal laboratory findings, despite the absence of Cushingoid features. Molecular and functional analyses ultimately led to a diagnosis of glucocorticoid resistance syndrome (GRS). A 54-year-old female patient underwent endocrinological evaluation for an adrenal incidentaloma associated with hypokalemia, which revealed hypercortisolemia. Subsequent endocrinological testing was consistent with ACTH-dependent CS; however, no Cushingoid features were observed on physical examination, suggesting NNH. As no apparent cause of NNH was identified, we hypothesized a functional disorder of the glucocorticoid receptor (GR) and performed a genetic analysis of NR3C1, which encodes GR. This revealed a novel germline heterozygous variant, p.L670P, located in the ligand-binding domain of the GR. Structural analyses revealed that Leu670 forms a hydrophobic core near the ligand-binding pocket. The p.L670P variant disrupted the secondary structure, suggesting a potential compromise in the structural stability of the ligand-binding site. In vitro experiments showed that this GR variant failed to suppress the transcriptional activity of the proopiomelanocortin promoter following dexamethasone administration. These findings confirmed that the patient had a loss-of-function variant in GR, leading to a diagnosis of GRS and ruling out ACTH-dependent CS. This case highlights that GRS may underline cases of NNH without a clear etiology, and genetic testing for GR can aid in its diagnosis.

Keywords: Cushing's syndrome; NR3C1; glucocorticoid receptor; glucocorticoid resistance syndrome; nonneoplastic hypercortisolism.

Figure 1.

Clinical findings on patient imaging and physical examination. (A) Findings of abdominal CT scan. The white arrow indicates the left adrenal tumor, which is 20 mm in size and has a CT value of 10 HU. (B) Photo of the patient. No signs suggestive of Cushingoid symptoms, including moon face, central obesity, purple striae, skin atrophy, proximal myopathy, peripheral edema, or hirsutism, are observed. (C) MRI of the pituitary gland with a contrast reagent. Abbreviations: CT, computed tomography; HU, Hounsfield units; MRI, magnetic resonance imaging.

Figure 2.

Results of NR3C1 gene analysis by Sanger sequencing. The c.2009 T > C (p.L670P) variant is detected in the patient's whole blood. (A) Patient and (B) wild-type (healthy subjects).

Figure 3.

Structural presentation of Leu670 in the LBD and the impact of the p.L670P variant. (A) Overall structure of the LBD bound to Dex, based on the crystal structure of the LBD–Dex complex (PDB: 1M2Z). Leu670 and Dex are highlighted and indicated by labels. (B) Close-up view of the Dex-binding pocket, showing that Leu670 forms a hydrophobic core with Leu603, Phe606, Leu722, and Met725, all of which are labeled accordingly. (C) Predicted structures of the wild-type and p.L670P variant. Both structures were first generated by AlphaFold 3, followed by 5 ns of molecular dynamics simulations using GROMACS. The α-helical region around residues 659 to 673 is shown in the lower panels. Abbreviations: Dex, dexamethasone; LBD, ligand binding domain.

Figure 4.

Functional analysis of the hGRα p.L670P variant. (A) Expression of the hGRα protein from each plasmid is confirmed. HEK293T cells are transfected with 0.8 μg plasmids encoding for hGRα WT, hGRα L670P, or hGRα L672P. The empty vector, pcDNA3.1, is used as a negative control. Transfected cells are lysed, and protein extracts are analyzed by SDS-PAGE. hGRα proteins are detected by Western blotting using a mouse monoclonal antibody. (B) Functional analysis of the hGRα variant. HEK293T cells are transfected with expression plasmids encoding for hGRα WT, hGRα L670P, or hGRα L672P, along with Pomc-luc. The empty vector, pGL4.10 [luc2], serves as a negative control. Transfected cells are subsequently treated with vehicle, Dex 10 nM, or Dex 100 nM for 24 hours, and Pomc-promoter activity is analyzed using a luciferase reporter assay. Normalized Pomc- promoter activity (luciferase signal corrected by NanoLuc internal control) in cells expressing hGRα wild-type and treated with vehicle was defined as 100%, corresponding to a mean relative luciferase value of 17.22 ± 1.96. Bars indicate the mean values of relative luciferase activity, and error bars indicate SDs. Statistical significance is calculated using one-way ANOVA with post hoc Tukey–Kramer test. *P < .05. Abbreviations: Dex, dexamethasone; hGRα, human glucocorticoid receptor α; NS, not significant.