Characterisation of a GNAS variant linked to cortisol-producing adrenocortical adenoma

Adrenocortical adenomas are frequent in the general population and can be associated with autonomous cortisol excess, increasing morbidity and mortality. Altered cAMP/PKA signalling is common in sporadic cortisol-producing adenomas, typically due to somatic activating mutations in the catalytic subunit α of PKA (PRKACA) or the G-protein α subunit, Gα_s (GNAS), which activate cAMP signalling. We previously identified a novel p.Lys58Gln GNAS somatic variant in a patient with a 5.3 cm adenoma and overt Cushing's syndrome. This novel mutation was not charactersised before but provided enough evidence to warrant further investigation.

Using HEK293 cells depleted of GNAS, we established wild-type (WT) Gα_s and Gα_s-Lys58Gln stable cell lines and evaluated adrenocorticotropic hormone (ACTH) receptor signalling using a cAMP GloSensor assay, measured CREB transcription factor phosphorylation (pCREB) by AlphaLISA and assessed CRE luciferase reporter activity. Cell viability and apoptosis were also assessed over 5 days.

The Gα_s-Lys58Gln variant showed a significantly higher basal cAMP, pCREB and CRE luciferase reporter concentration and a greater response to ACTH (0-10 nM, P < 0.001) compared to WT Gα_s. The variant had no effect on ligand potency. There was also significantly enhanced cell viability and apoptosis in cells with the Gα_s-Lys58Gln variant.

In conclusion, our study demonstrated that the Gα_s-Lys58Gln variant is associated with constitutive activation of GNAS signalling, similar to Arg201 mutations previously reported in adrenocortical adenomas, potentially representing a new pathogenic mechanism in a subset of patients with adrenal Cushing syndrome. This variant may also affect cell proliferation and requires further study.