ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Endocrine Abstracts (2019) 63 GP98 | DOI: 10.1530/endoabs.63.GP98

Methylation status and gene expression of steroidogenic enzymes in benign adrenocortical tumors

Guido Di Dalmazi1, Luca Morandi2, Beatrice Rubin3, Catia Pilon3, Sofia Asioli2, Valeria Maffeis3, Valentina Vicennati1, Uberto Pagotto1 & Francesco Fallo3

1Endocrinology Unit - Department of Medical and Surgical Sciences, Bologna, Italy; 2Research Laboratory, Department of Biomedical and Neuromotor Sciences, Bologna, Italy; 3Department of Medicine, University of Padova, Padova, Italy.

Background: DNA methylation has been recognized as a putative regulatory mechanism for CYP11B2 in primary hyperaldosteronism. We aimed to investigate the DNA methylation and the expression of a panel of genes encoding several enzymes involved in steroidogenesis in adrenocortical benign tumors.

Methods: We collected a total of 60 adrenocortical tissues, including 9 non-functioning adrenal adenomas, 9 adenomas associated with autonomous cortisol secretion, 17 adenomas associated with Cushing’s syndrome, 13 Conn’s adenomas and 12 tissues derived from adrenal gland adjacent to the Conn’s adenomas. Non-functioning tumors and autonomous cortisol secretion were defined according to cortisol levels after 1 mg dexamethasone suppression test ≤ or > 50 nmol/L, respectively. The DNA methylation level of CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP21A2, DHCR24, HSD3B1, HSD3B2, NR5A1, STAR, and TSPO was evaluated by quantitative Bisulfite Next Generation Sequencing (bisulfite-NGS). Bioinformatic analysis was performed in a GalaxyProject environment and processed by BSPAT (Bisulfite Sequencing Pattern Analysis Tool). Spearman correlation coefficients were calculated using IBM SPSS 21 (IBM). CYP11B1, CYP11B2, CYP17, CYP21, STAR and β-actin gene expressions were examined by quantitative Real-Time PCR using a Sybr Green Assay kit (Thermo Fisher Scientific). The Default 2−ΔΔCt was used to calculate the fold changes in gene expression between the categories of samples.

Results: When compared to other adrenal tissues (P<0.001), CYP11B2 was significantly hypomethylated in Conn’s adenoma. No difference in methylation status was found among groups for the remaining genes. CYP11B2 mRNA levels were significantly higher in Conn’s adenoma than in the remaining adrenal tissues (P=0.001). CYP21 mRNA was significantly higher in all but Conn’s adenomas, when compared to normal adrenal tissues (P<0.001). Overall, we found a negative correlation between CYP11B2 expression and DNA methylation (rho=−0.379; P=0.003).

Conclusion: DNA methylation seems to be a pivotal regulatory mechanism for CYP11B2 expression. It is feasible that epigenetic mechanisms may be responsible for aldosterone hypersecretion in Conn’s adenoma.

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