Cortisol homeostasis and glucocorticoid signaling are altered in FMR1 full mutation and premutation carriers.
Fragile X syndrome (FXS) and fragile X-associated tremor/ataxia syndrome (FXTAS) are caused by CGG-repeat expansions in the FMR1 gene. Although the genetic mechanisms underlying these disorders are well established, the contribution of glucocorticoid signaling pathways and neuroendocrine regulation remains unclear. We investigated whether FMR1 CGG-repeat expansions are associated with alterations in cortisol homeostasis and glucocorticoid signaling pathways. A clinically referred cohort of 891 individuals was screened for FMR1 CGG-repeat expansions using conventional and methylation-specific polymerase chain reaction (PCR). Molecular screening identified full mutation (FM) alleles in five males and five females and premutation (PM) alleles in seven males and three females. Endocrine, transcriptional, and epigenetic analyses were subsequently performed in 20 molecularly confirmed carriers (10 PM and 10 FM). Hypothalamic-pituitary-adrenal (HPA) axis activity was assessed by measuring plasma cortisol concentrations using enzyme-linked immunosorbent assay (ELISA), and the expression of stress-responsive genes (NR3C1 and FKBP5) was quantified by real-time PCR. Epigenetic regulation was further evaluated by assessing NR3C1 promoter methylation using methylation-specific PCR. To investigate the direct effects of expanded CGG repeats on glucocorticoid signaling independent of systemic influences, complementary in vitro studies were performed in HEK293 cells transiently expressing the expanded CGG repeats. Cells were subsequently subjected to a 12-hr dexamethasone challenge, followed by analysis of NR3C1 and FKBP5 expression. In this preliminary cohort of 20 molecularly confirmed carriers, PM and FM carriers exhibited elevated cortisol concentrations compared with controls. Carriers showed reduced NR3C1 expression, increased FKBP5 expression, and increased methylation of the NR3C1 promoter. Correlation analysis revealed significant relationships between cortisol, NR3C1, and FKBP5 expression. Expression of expanded CGG repeats in HEK293 cells reproduced the glucocorticoid signaling signature observed in patient samples, characterized by reduced NR3C1 and increased FKBP5 expression. Following dexamethasone challenge, FKBP5 expression increased in all experimental groups, with the greatest relative induction observed in CGG90-expressing cells, while NR3C1 expression remained substantially lower in CGG90-expressing cells than in wild-type cells. These findings indicate altered glucocorticoid-responsive transcription in the CGG-repeat-expressing cellular model. Although these findings require validation in larger independent cohorts, they identify the NR3C1-FKBP5 axis as a potential molecular component of stress-axis dysfunction in fragile X-associated disorders and provide a foundation for future mechanistic studies.