Abstract
DNA methylation has been identified among putative regulatory mechanisms for CYP11B2 expression in primary aldosteronism. The objective of this work is to investigate DNA methylation and expression of genes encoding steroidogenic enzymes in benign adrenocortical tumors. This cross-sectional study took place at university hospitals. 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). DNA methylation levels were evaluated by quantitative bisulfite next-generation sequencing in fresh-frozen tissues (CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP21A2, HSD3B1, HSD3B2, 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. The main outcome measure was DNA methylation. 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, CYP11B2 expression and DNA methylation were negatively correlated (ρ = -0.379; P = .003). In FFPE-paired APA/APCC samples, CYP11B2 methylation level was significantly lower in APA than in concurrent APCCs (P = .028). 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 CYP11B2 demethylation. Conversely, DNA methylation seems not to be relevant in regulating the expression of genes encoding steroidogenic enzymes other than CYP11B2.
Highlights
Epigenetic changes in DNA are well-known heritable mechanisms of regulation of gene expression, not caused by modification of DNA
The mean CYP11B2 methylation level was significantly lower in aldosterone-producing adenoma (APA), when compared to other adrenal tumoral tissues (P
We found a negative correlation between CYP11B2 expression and respective DNA methylation, whereas no correlation was found between mRNA and DNA methylation levels for CYP11B1, CYP17, CYP21A2, and STAR
Summary
Epigenetic changes in DNA are well-known heritable mechanisms of regulation of gene expression, not caused by modification of DNA. Genome-wide [5, 6] and targeted methylation analysis [7, 8] have investigated the methylation signature of benign adrenocortical tumors, showing that DNA methylation may be a putative regulatory mechanism of CYP11B1 and CYP11B2 expression in cortisol-producing and aldosterone-producing adenoma (APA), respectively. Previous studies in H295R human adrenocortical cell lines showed that angiotensin II or potassium may change the DNA methylation status around transcription factor binding sites and transcription start site, activating CYP11B2 expression and promoting aldosterone secretion [9]. No studies have investigated the methylation levels of CYP11B2 in APCCs. The first aim of our study was to investigate the DNA methylation of a selected panel of genes encoding key enzymes involved in steroidogenesis in benign adrenocortical tumors, including adenomas associated with autonomous cortisol secretion (ACS). The second aim was to assess the DNA methylation of CYP11B2 in APA and concomitant APCCs
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