Pregnancy, Primary Aldosteronism, and Adrenal CTNNB1 Mutations

Abstract

Recent discoveries of somatic mutations permit the recognition of subtypes of aldosterone-producing adenomas with distinct clinical presentations and pathological features. Here we describe three women with hyperaldosteronism, two who presented in pregnancy and one who presented after menopause. Their aldosterone-producing adenomas harbored activating mutations of CTNNB1, encoding β-catenin in the Wnt cell-differentiation pathway, and expressed LHCGR and GNRHR, encoding gonadal receptors, at levels that were more than 100 times as high as the levels in other aldosterone-producing adenomas. The mutations stimulate Wnt activation and cause adrenocortical cells to de-differentiate toward their common adrenal-gonadal precursor cell type. (Funded by grants from the National Institute for Health Research Cambridge Biomedical Research Centre and others.).

Figure 1. Adrenal Imaging and Histologic Features in Three Patients with Aldosterone-Producing Adenomas

The images in Panel A show the adenomas in the three patients (arrows). Histologic examination of the adenomas after adrenalectomy (Panel B, hematoxylin and eosin) shows spironolactone bodies (red arrows), which are characteristic of zona glomerulosa–like aldosterone-producing adenomas. No spironolactone bodies were observed in the adenoma in Patient 2, but it nevertheless resembled a zona glomerulosa–like aldosterone-producing adenoma. Scale bars denote 50 μm.

Figure 2. Relative TCF/LEF Activity and Immunoblots after Transfection with Control and CTNNB1 Constructs

Panel A shows relative TCF/LEF activity, measured as the ratio of firefly luciferase to renilla luciferase, with the use of a luciferase-tagged TCF/LEF-responsive promoter in HEK293T cells. Results are from five experiments performed 48 hours after transfection with empty vector and with wild-type and mutated CTNNB1 constructs. I bars indicate standard errors, and P<0.001 for all comparisons of the mutated constructs with wild-type CTNNB1. Panel B shows immunoblots for active endogenous β-catenin in HEK293T cells 72 hours after transfection with empty vector and with wild-type and mutated CTNNB1 constructs. Values are percentages of total endogenous β-catenin that was active (i.e., nonphosphorylated at residues Ser33, Ser37, and Thr41). Immunoblots obtained 24 hours after transfection showed similar results (Fig. S3 in the Supplementary Appendix). GAPDH denotes glyceraldehyde-3-phosphate dehydrogenase.

Figure 3. Ex Vivo Studies of Aldosterone-Producing Adenomas

Panel A shows the results of a microarray analysis of LHCGR and GNRHR expression in the aldosterone-producing adenoma (APA) in Patient 1, as compared with six zona glomerulosa (ZG)–like control adenomas and seven zona fasciculata (ZF)–like control adenomas. T bars indicate standard errors. Panel B shows the results of a quantitative polymerase-chain-reaction (qPCR) assay, which confirms that expression of both genes is much higher in the APAs of the three patients than in the six ZG-like and seven ZF-like control APAs. The log₂ factor change refers to the difference between the tumor and the adjacent nontumorous adrenal tissue in the expression of LHCGR and GNRHR. Panel C shows immunohistochemical staining for β-catenin and luteinizing hormone–chorionic gonadotropin receptor (LHCGR) on adenoma cells after adrenalectomy in the three patients, as compared with adjacent adrenal tissue. (For each patient, adenoma cells are shown on the left, and adjacent adrenal tissue is shown on the right.) Scale bars denote 50 μm. Panel D shows immunofluorescence staining for LHCGR proteins in ZG-like adenoma cells transfected with mutant (Ser33Cys) CTNNB1. Red indicates LHCGR, green GFP (green fluorescent protein)–tagged mutant β-catenin, and blue nuclear DNA stained with 4′,6-diamidino-2-phenylindole (DAPI).