Confirmation Of Methylation Microarray Results Of Aldosterone-producing Adenoma(APA),Analysing The Different Expression And Methylation Of Dopamine Receptor2between APA And NA

ObjectivesPrimary aldosteronism is one of the most reason of symptomatic hypertension? The clinical manifestation of Primary aldosteronism is high blood pressure, hyperaldosteronemia, and hypokalemic. The most common clinical subtypes of primary aldosteronism are aldosterone-producing adrenocortical adenoma(APA) and adrenal cortical hyperplasia(or idiopathic hyperaldosteronism, IHA). The prevalence of APA may be as high as60%-90%, but the pathogenesis of APA is still not clear. Dedicated to the study of Epigenetics, mainly about DNA methylation of producing-adrenocortical adenoma, We previously used the methylation microarray to analyse hypermethylated genes.The expression of these genes in APA was much less than that in the normal adrenal cortex.Now we start to verify the results we got from the microarray. According to the literatures, Dopamine receptor2(DRD2) plays a key role in the aldosterone secretion of APA. The expression of DRD2was different between APA and NA.So we explore the mechanism associated with aldosterone-producing adenomas by using technology of DNA methylation.MethodsDNA were extracted from5aldosterone-producing adenomas and5normal adrenal tissues.Then all the DNA samples were bisulfite converted and used to re-check the rusults of DNA methylation microarry by BSP.There were3locuses (BECN1, ACAT1, MCCC1), ranking as the top methylated genes, selected and found to be differentially expressed in APA compared with NA in the previous study. Secondly, Real-time PCR and MSP were used to test the expression of Dopamine receptor2and the level of DNA methylation by screening of15APA which were established by the clinical manifestations and confirmed histologically, compared with3NA(Adrenal medulla were dissected out).RusultsWe found the results we tested were so inconsistent with what came from results of microarray that the locuses(BECN1、ACAT1、MCCC1)were demethylated. The levels of DRD2expressed in APA and normal adrenal cortex from18patients were examined. The mRNA levels of the DRD2in APA were significantly lower than in the normal adrenal cortex (△C(t)12.59±2.45vs.9.44±1.96, P=0.002<0.01).And then the methylation of Dopamine receptor2were tested.9cases out of15APA were rmethylated, and the left were demethylated in comparison with the normal adrenal cortex, which were all demethylated. Interestingly, The mRNA levels of the DRD2in methylated cases of APA were lower than in demethylated cases(△C(t)12.27±2.65vs11.24±2.44. P=0.553>0.01). The mRNA levels of the DRD2in methylated cases were significantly lower than NA (P=0.002<0.01), and the same with demethylated APA and NA((P=0.005<0.01)Conclusions1. The previous study showed that BECN1,ACAT1and MCCC1were hypermethylated.However, the results were inconsistent with what we get from BSP.2. The expression of DRD2was significantly lower in APA,compared with NA.3. The methylated DRD2plays a partial role in the expression of APA.