DNA Methylation of Steroidogenic Enzymes in Benign Adrenocortical Tumors: New Insights in Aldosterone-Producing Adenomas

Context: DNA methylation has been identified among putative regulatory mechanisms for CYP1182 expression in primary aldosteronism. Objective: The objective of this work is to investigate DNA methylation and expression of genes encoding steroidogenic enzymes in benign adrenocortical tumors. Design and Setting: This cross-sectional study took place at university hospitals. Patients: We collected fresh-frozen tissues from patients with benign adrenocortical adenomas (n = 48) (nonfunctioning n = 9, autonomous cortisol secretion n = 9, Cushing syndrome n = 17, aldosterone-producing [APA] n = 13) and adrenal cortex adjacent to APA (n = 12).We collected formalin-fixed, paraffin-embedded (FFPE) specimens of paired APA and concurrent aldosterone-producing cell clusters (APCCs) (n = 6). Intervention: DNA methylation levels were evaluated by quantitative bisulfite nextgeneration sequencing in fresh-frozen tissues (CYP11A1, CYP11131, CYP1162, CYP17A1, CYP21A2, HSD3B1, HSD362, NR5A1, STAR, and TSPO) and FFPE APA/APCC paired samples (CYP11B2). CYP11B1, CYP11B2, CYP17, CYP21, and STAR gene expressions were examined by quantitative real-time polymerase chain reaction. Main Outcome Measure: The main outcome measure was DNA methylation. Results: CYP11B2 methylation levels were significantly lower in APA than in other adrenal tissues (P < .001). Methylation levels of the remaining genes were comparable among groups. Overall, CYP1182 expression and DNA methylation were negatively correlated (p = -0.379; P= .003). In FFPE-paired APA/APCC samples, CYP1182 methylation level was significantly lower in APA than in concurrent APCCs (P= .028). Conclusions: DNA methylation plays a regulatory role for CYP11B2 expression and may contribute to aldosterone hypersecretion in APA. Lower CYP11B2 methylation levels in APA than in APCCs may suggest an APCC-to-APA switch via progressive CYP1182 demethylation. Conversely, DNA methylation seems not to be relevant in regulating the expression of genes encoding steroidogenic enzymes other than CYP1182.