Endocrine Abstracts

Background: Previous studies have shown an association between low CYP11B2 DNA methylation levels and high CYP11B2 mRNA levels in aldosterone-producing adenoma (APA), suggesting that epigenetic mechanisms play a pivotal role for regulating CYP11B2 function in primary aldosteronism. It has been proposed that aldosterone-producing cell clusters (APCCs) may become a source of autonomous aldosterone production when evolving into APA.

Aim: Our aim was to determine whether the CYP11B2 DNA is differentially methylated in APAs and in their concurrent APCCs, and to assess the relationship with CYP11B2 tissue expression.

Methods: A series of 12 formalin-fixed paraffin-embedded (FFPE) adrenal tissues from patients with APA were studied. Immunohistochemical staining was performed using anti-CYP11B1 and anti-CYP11B2 monoclonal antibodies, and anti-CYP17A1 polyclonal antibody. Staining was quantified by the McCarty’s H-scoring system. APA, APA-adjacent adrenal cortex, and satellite APCCs identified by immunohistochemistry were microdissected using manual core drilling, and genomic DNA was extracted and assessed for CYP11B2 methylation analysis. CYP11B2 DNA methylation level was measured by quantitative Bisulfite Next Generation Sequencing. Bioinformatic analysis was performed in a GalaxyProject environment and processed by BSPAT (Bisulfite Sequencing Pattern Analysis Tool). The equation 2^-ddCt was used to calculate the fold changes in gene expression between the different intra-adrenal cell structures. Analysis of DNA mutations in aldosterone-driver genes KCNJ5, ATP1A1, ATP2B3 and CACNA1D was performed by Sanger sequencing.

Results: 9/12 APA specimens showed at least one concurrent APCC. A wide range of CYP11B2, CYP11B1 and CYP17A1 immunohistochemical expression was detected in tumor cells of APA, while positive CYP11B2 and negative CYP11B1/CYP17A1 staining was uniformly found in APCCs. H-score of CYP11B2 expression in 3/9 APAs was lower than in their satellite APCCs and was generally negative or very low in APA-adjacent adrenal cortex. CYP11B2 DNA methylation levels were significantly lower in APA than in concurrent APCCs and APA-adjacent adrenal cortex [mean ± standard deviation] 0.51 ± 0.25 vs 0.87 ± 0.11 vs 0.70 ± 0.27; P < 0.05 in all but one CpG island considered). Five KCNJ5 and one ATP2B3 mutations were found overall in 12 APAs, including four KCNJ5 and one ATP2B3 mutations among the 9 APAs with concurrent APCCs. No somatic mutations were found in APCCs.

Conclusion: Lower CYP11B2 methylation levels in APA than in concurrent APCCs may sustain the hypothesis of a progressive demethylation process of APCCs, causing their switch to an autonomous aldosterone production. Somatic aldosterone-driver gene mutations do not affect the CYP11B2 DNA methylation rate in APA.