title: 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 nonfunctioning adrenocortical adenoma (n=12) and APA (n=35) samples, including some with a KCNJ5 mutation (n=21), an ATP1A1 mutation (n=5), and without the known mutations (n=9). The quantitative polymerase chain reaction assay was performed for the detection of CYP11B2 and CYP11B1 expression levels in nonfunctioning adrenocortical adenoma and APA. We introduced the KCNJ5 T158A mutation using lentivirus delivery in the human adrenocortical 15 cell line, 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 7 hypomethylated sites in CYP11B2 and 1 hypomethylated and 6 hypermethylated sites in CYP11B1 There were no differences in the steroidogenic enzymes gene DNA methylation of peripheral leukocytes between nonfunctioning adrenocortical adenoma 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. Human adrenocortical 15 cells with the KCNJ5 mutation showed no changes in CYP11B2 or CYP11B1 methylation levels compared with control cells. We demonstrated that CYP11B2 in APA was extensively hypomethylated, and CYP11B2 methylation in the region with hypomethylation was not induced by KCNJ5 or ATP1A1 mutations that cause aldosterone overproduction in APA and a KCNJ5 mutation human adrenocortical 15 cells.
authors.affiliation: From the Department of Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan (Y.Y., K.O., H.O., K.I., K.K., M.Y.); and Division of Endocrinology, G.V. (Sonny) Montgomery VA Medical Center, University of Mississippi Medical Center, Jackson (C.E.G.-S.).
authors.forename: Yoko
authors.initials: Y
authors.lastname: Yoshii
authors.affiliation: From the Department of Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan (Y.Y., K.O., H.O., K.I., K.K., M.Y.); and Division of Endocrinology, G.V. (Sonny) Montgomery VA Medical Center, University of Mississippi Medical Center, Jackson (C.E.G.-S.). kenjioki@hiroshima-u.ac.jp.
authors.forename: Kenji
authors.initials: K
authors.lastname: Oki
authors.affiliation: From the Department of Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan (Y.Y., K.O., H.O., K.I., K.K., M.Y.); and Division of Endocrinology, G.V. (Sonny) Montgomery VA Medical Center, University of Mississippi Medical Center, Jackson (C.E.G.-S.).
authors.forename: Celso E
authors.initials: CE
authors.lastname: Gomez-Sanchez
authors.affiliation: From the Department of Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan (Y.Y., K.O., H.O., K.I., K.K., M.Y.); and Division of Endocrinology, G.V. (Sonny) Montgomery VA Medical Center, University of Mississippi Medical Center, Jackson (C.E.G.-S.).
authors.forename: Haruya
authors.initials: H
authors.lastname: Ohno
authors.affiliation: From the Department of Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan (Y.Y., K.O., H.O., K.I., K.K., M.Y.); and Division of Endocrinology, G.V. (Sonny) Montgomery VA Medical Center, University of Mississippi Medical Center, Jackson (C.E.G.-S.).
authors.forename: Kiyotaka
authors.initials: K
authors.lastname: Itcho
authors.affiliation: From the Department of Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan (Y.Y., K.O., H.O., K.I., K.K., M.Y.); and Division of Endocrinology, G.V. (Sonny) Montgomery VA Medical Center, University of Mississippi Medical Center, Jackson (C.E.G.-S.).
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Kobuke
authors.affiliation: From the Department of Molecular and Internal Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan (Y.Y., K.O., H.O., K.I., K.K., M.Y.); and Division of Endocrinology, G.V. (Sonny) Montgomery VA Medical Center, University of Mississippi Medical Center, Jackson (C.E.G.-S.).
authors.forename: Masayasu
authors.initials: M
authors.lastname: Yoneda
mesh_terms: D018246:Adrenocortical Adenoma / Q000235:genetics*; D000450:Aldosterone / Q000096:biosynthesis; D019175:DNA Methylation / Q000235:genetics*; D005260:Female; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics*; D005838:Genotype; D006801:Humans; D006929:Hyperaldosteronism / Q000235:genetics*; D008297:Male; D009154:Mutation; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013252:Steroid 11-beta-Hydroxylase / Q000235:genetics*
chemical_list: D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; C484274:KCNJ5 protein, human; D012333:RNA, Messenger; D000450:Aldosterone; D013252:Steroid 11-beta-Hydroxylase; C499076:ATP1A1 protein, human; D000254:Sodium-Potassium-Exchanging ATPase
keywords: DNA methylation; aldosterone; hyperaldosteronism; mutation