SUN-044 Methylation Status and Gene Expression of Steroidogenic Enzymes in Benign Adrenocortical Tumors

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. 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 equation 2−ΔΔCt was used to calculate the fold changes in gene expression between the categories of samples. CYP11B2 was significantly hypomethylated in Conn’s adenoma, when compared to other adrenal tissues (P<0.001). 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). 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.