Hypomethylation of CYP11B2 in Aldosterone-Producing Adenoma.
Hypomethylation of CYP11B2 in Aldosterone-Producing Adenoma.
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DOI：
10.1161/hypertensionaha.116.08313
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发表时间：
2016-12
期刊：
影响因子：
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通讯作者：
Yoneda M
中科院分区：
文献类型：
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作者：
Yoshii Y;Oki K;Gomez-Sanchez CE;Ohno H;Itcho K;Kobuke K;Yoneda M
本研究旨在探讨醛固酮腺瘤（aldosterone-producing adenoma，阿帕）中类固醇生成酶基因的DNA甲基化水平，以及阿帕基因突变对DNA甲基化水平的影响。使用无功能肾上腺皮质腺瘤（NF，n=12）和阿帕（n=35）样本进行DNA甲基化阵列分析，其中包括一些具有KCNJ 5突变（n=21），ATP 1A 1突变（n=5）和没有已知突变（n=9）的样本。采用定量聚合酶链反应（PCR）检测NF和阿帕中CYP 11B 2和CYP 11B 1的表达水平。我们使用慢病毒递送在人肾上腺皮质细胞系（HAC 15）中引入KCNJ 5 T158 A突变，并分析突变对DNA甲基化水平的影响。我们分析了类固醇生成酶的83个假定的DNA甲基化位点。在阿帕中，我们发现CYP 11B 2有7个低甲基化位点，CYP 11B 1有1个低甲基化位点和6个高甲基化位点。NF和阿帕患者外周血白细胞类固醇生成酶基因甲基化水平无差异。阿帕中CYP 11B 2甲基化水平与CYP 11B 2转录水平无关。除了CYP 11B 2区域，所有甲基化位点在有或没有基因突变的APA之间没有差异。与对照细胞相比，具有KCNJ 5突变的HAC 15细胞显示CYP 11B 2或CYP 11B 1甲基化水平没有变化。我们证明阿帕中的CYP 11B 2是广泛的低甲基化，并且在低甲基化区域的CYP 11B 2甲基化不是由导致阿帕和KCNJ 5突变的HAC 15细胞中醛固酮过量产生的KCNJ 5或ATP 1A 1突变诱导的。
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.