Hypomethylation of CYP11B2 in Aldosterone-Producing Adenoma

Abstract

The purpose of this study was to evaluate the DNA methylation levels of steroidogenic enzyme genes in aldosterone-producing adenoma (APA), and the effects of gene mutations in APA on the DNA methylation levels. DNA methylation array analysis was conducted using non-functioning adrenocortical adenoma (NF, n=12), and APA (n=35) samples including some with a KCNJ5 mutation (n=21), an ATP1A1 mutation (n=5), and without known mutations (n=9). The quantitative polymerase chain reaction assay was performed for the detection of CYP11B2 and CYP11B1 expression levels in NF and APA. We introduced the KCNJ5 T158A mutation using lentivirus delivery in the human adrenocortical cell line (HAC15), and analyzed the effects of the mutation on DNA methylation levels. We analyzed the 83 presumed DNA methylation sites of steroidogenic enzymes. In APA, we found seven hypo-methylated sites in CYP11B2 and one hypo-methylated six hyper-methylated sites in CYP11B1. There were no differences in the steroidogenic enzymes gene DNA methylation of peripheral leucocytes between NF and APA. No CYP11B2 methylation level was associated with CYP11B2 transcription levels in APA. All methylation sites, except for a CYP11B2 region, showed no difference among APAs with or without gene mutations. HAC15 cells with the KCNJ5 mutation showed no changes in CYP11B2 or CYP11B1 methylation levels compared to control cells. We demonstrated that CYP11B2 in APA was extensively hypo-methylated, and CYP11B2 methylation in the region with hypo-methylation was not induced by KCNJ5 or ATP1A1 mutations that cause aldosterone over-production in APA and a KCNJ5 mutation HAC15 cells.

Hypothesis on a relationship between hyperaldosteronism, inflammation, somatic mutations, and autoimmunity

“…This hypothesis is supported by the observation of a possible reversal of PA in some patients after long‐term treatment with spironolactone or its derivatives . A reversal of the DNA methylation or of somatic mutations can be proposed to explain the recovery and normal aldosterone secretion in PA …”

“…This hypothesis is supported by the observation of a possible reversal of PA in some patients after long-term treatment with spironolactone or its derivatives. 21,22 A reversal of the DNA methylation or of somatic mutations can be proposed to explain the recovery and normal aldosterone secretion in PA. 23 The recent finding of increased titers of AT1R-AAs in PA suggests two other possible pathological hypotheses: (1) hyperaldosteronism induces AT1R-AAs, or (2) AT1R-AAs are responsible for PA. Considering the first hypothesis, hyperaldosteronism or its increased activity (as occurs in preeclampsia) could cause a proinflammatory systemic state that induces the formation of AT1R-AAs.…”

Human Adrenal Cortex: Epigenetics and Postnatal Functional Zonation

“…The human CYP11B2 and CYP11B1 gene promoters possess a number of target sites for DNA methylation that are closely associated with gene silencing [37-40]. The CYP11B1 gene promoter was found to be predominantly unmethylated in ZF while completely methylated in both ZG and ZR.…”

“…Pathways responsible for tissue and cell development and the pathology of cancer often coincide. In this regard, CYP11B2-regulating miRNAs and CYP11B2 promoter methylation are deregulated in aldosterone producing adenoma tissues [38, 40, 51, 52]. However, no information regarding epigenetic changes in childhood ACT and their association with the steroid profile is available.…”

The E3 ubiquitin ligase Siah1 regulates adrenal gland organization and aldosterone secretion

“…Genetic mutations in a number of genes, including those encoding ion channels (KCNJ5, ATP1A1, ATP2B3, CACNA1D, and CACNA1H) and proteins involved in cell-cell and protein-protein (ARMC5, CTNNB1) interactions, have been implicated in PA. Finally, perturbations in epigenetic regulatory mechanisms have also been associated with overproduction of aldosterone (4,5).…”