Micro- and macroadenomas causing primary aldosteronism: molecular and biochemical studies

Yang, Yuhong (2021): Micro- and macroadenomas causing primary aldosteronism: molecular and biochemical studies. Dissertation, LMU München: Medizinische Fakultät

Abstract

Aldosterone-producing adenomas are the surgically correctable form of primary aldosteronism. Next generation sequencing targeted to aldosterone-producing adenomas with positive expression of CYP11B2 has unveiled the existence of somatic mutations and explained aldosterone overproduction. However, pathogenic mechanisms contributing to the diverse adenoma sizes and the associated biochemical features remain largely unclear. In this thesis, we utilized steroid and transcriptome profiling of aldosterone-producing adenomas of distinctly different diameter (micro and macro aldosterone-producing adenomas) to identify circulating biomarkers and key modulators that regulate adrenal cell growth of aldosterone-producing adenomas, and performed functional characterization. These findings have been published in three publications. In paper I, we compared peripheral venous plasma steroid profiles of patients with micro, macro aldosterone-producing adenomas and bilateral adrenal hyperplasia measured by LC-MS/MS. Steroid profiling highlighted the steroids, including aldosterone, 18-oxocortisol, 18-hydroxycortisol, and dehydroepiandrosterone sulfate, with distinct concentrations among the 3 groups. Steroid profiling achieved a high predictive accuracy of the 3 groups while a low specificity for micro aldosterone-producing adenomas. The hypothetical diagnostic algorithm that integrated steroid profiling, CT and adrenal venous sampling limited to patients with conflicting steroid pro- filing and CT results would have improved the detection rate of micro aldosterone-producing adenomas. It would have also elevated the overall predictive accuracy of aldosterone-producing adenoma and bilateral adrenal hyperplasia to a comparable level achieved by adrenal venous sampling-based management. Thus, peripheral venous plasma steroid profiles are highly different between patients with micro and macro aldosterone-producing adenomas, and are potentially useful for the identification of patients with micro aldosterone-producing adenomas in whom adrenal venous sampling should be considered mandatory. In paper II, the comparison of transcriptomes of aldosterone-producing adenomas of distinctly different sizes (diameter ≤10 mm versus ≥30 mm) by mRNA sequencing analysis highlighted a number of differentially expressed genes between these 2 groups of aldosterone-producing adenomas with different genotypes (no mutation detected versus KCNJ5 mutations). Gene ontology enrichment analysis highlighted different patterns of differentially expressed gene sets in aldosterone-producing adenomas with different genotypes, indicating the distinct biology that underlies their development. Cell death was the most significantly enriched biological process in aldosterone-producing adenomas with no mutation detected. We validated the differential expression of 10 genes with a known function in cell death and proliferation in an expanded sample set. The gene BEX1, with an unknown function in the adrenal gland, was selected for functional characterization due to its differential expression in the aldosterone-producing adenomas with different diameter and genotypes. Stable BEX1 expression in human adrenocortical cells did not alter cell cycle progression or sensitivity to apoptosis, but conferred protection from ferroptosis. The higher expression profile of BEX1 in micro aldosterone-producing adenomas may indicate a high level of anti-ferroptotic mechanism in these adrenal tissues that produce notably high pathological levels of aldosterone. This postulation was supported by the higher BEX1 expression in micro aldosterone-producing adenomas and aldosterone-producing micronodules compared with the paired adjacent cortex. These findings demonstrate that micro and macro aldosterone-producing adenomas display distinct transcriptome profiles that is helpful for the identification of potential modulators of the deregulated cell growth in aldosterone-producing adenomas. BEX1 promotes cell survival of adrenal cells by mediating the inhibition of ferroptosis and indicate a pathogenic role for BEX1 in aldosterone-producing adenomas. In paper III, we aimed to investigate the functional role of KCNJ5 mutations in aldosterone-producing adenomas. Therefore, we performed in vitro functional and immunohistochemical characterization of KCNJ5. The expression of KCNJ5-G151R, L168R and G151E in human adrenocortical cells caused apoptosis, whereas T158A had no such effect. Instead, compared with control cells, a low-level induction of T158A induced a significant increase in cell proliferation, but an increased level of induction ablated this difference. The use of our newly developed KCNJ5 antibody demonstrated a lower expression of KCNJ5 in aldosterone-producing adenomas carrying KCNJ5 mutations compared with aldosterone-producing adenomas with other genotypes or aldosterone-producing micronodules. We also identified a decreased KCNJ5 expression in cells expressing CYP11B2 compared with CYP11B2-negative cells in aldosterone-producing adenomas with KCNJ5 mutations. Collectively, these findings indicate that KCNJ5 mutations induce cell toxicity and their cell growth effects are likely to be determined by the expression level of the mutated KCNJ5 channel. In summary, this thesis has identified circulating steroid biomarkers for aldosterone-producing adenomas with different adenoma sizes and potential modulators (BEX1 and KCNJ5 mutations) that may contribute to such size differences of aldosterone-producing adenomas. It provides an alternative method for subtype differentiation of primary aldosteronism that could be especially helpful for clinical centers with limited access to the technically demanding procedure of adrenal venous sampling. It also deepens our understanding of the pathogenesis of aldosterone-producing adenomas that could be useful for future perspectives in the diagnosis and clinical management of primary aldosteronism.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: primary aldosteronism, aldosterone-producing adenoma, steroid profiling, transcriptome profiling, ferroptosis, apoptosis, KCNJ5 mutations
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 25. November 2021
• 1. Bericht­erstatter:in: Reincke, Martin