自身免疫性甲状腺疾病易感基因DNA甲基化与疾病发生机制的关系

Autoimmune thyroid diseases (AITD) are the commonest organ-specific autoimmune endocrine diseases, including two clinically opposing syndromes: Hashimoto's thyroiditis (HT) manifesting by hypothyroidism and Graves' disease (GD) manifesting by hyperthyroidism. Significant progress has been made in our understanding of the mechanisms leading to AITD. The susceptibility genes interact with environmental factors, such as iodine intake, infection, likely through epigenetic mechanisms to trigger AITD. Excess iodine intake increass the incidence of thyroid diseases by strengthening organism immune respones. S0 our hypothesis is that epigenetic changes of AITD susceptibility genes in response to environmental stimulus would contribute to the etiology of AITD by immune function and genes expression abnormality of Th1、Th2、Th17、Treg resulting in autoimmune respones unbalance. The project will analyze DNA methylation level of AITD susceptibility genes of peripheral blood leukocytes in AITD cases and controls, and analyze the association among iodine intake level, polymorphisms of AITD susceptibility genes and DNA methylation level in vivo and in vitro, and compare the difference of DNA methylation patterns among AITD clinical cases，and analyze the effect of DNA methylation level on the immune functiong and genes expression of Th1,Th2，Th17,Treg in vitro. By the study, the effect of epigenetic changes on the etiology of AITD and the association between DNA methylation difference and AITD clinical characteristics will be investigated initially to look for epigenetic markers of the etiology of AITD and to search for clues of predicting clinical turnover of AITD.

自身免疫性甲状腺疾病(AITD)是一组最常见的器官特异性的自身免疫性疾病，主要包括Graves病（GD）和桥本甲状腺炎（HT）。近年AITD病因学研究的最新重大进展是，发现环境与遗传因素相互作用的表观遗传学改变可能是AITD的病因，但尚需进一步验证。过量碘使机体免疫反应增强而增加甲状腺疾病的发病率。因此，本项目拟分析AITD病例及对照者外周血白细胞的易感基因DNA甲基化水平，通过体内、外实验分析碘摄入量、AITD易感基因多态与DNA甲基化间的关系，验证易感基因DNA甲基化水平对机体Th1、Th2、Th17、Treg的免疫学功能及基因表达的影响，并比较疾病样本间DNA甲基化修饰模式差异，探讨表观遗传学对AITD发病和临床特征的影响，为寻找AITD的表观遗传标志及临床转归的预测标志提供新的线索。

近年自身免疫性甲状腺疾病(AITD)病因学研究的最新重大进展是，发现环境与遗传因素相互作用的表观遗传学改变可能是其病因，但尚需进一步验证。过量碘使机体免疫反应增强而增加甲状腺疾病的发病率。Graves病（GD）和桥本甲状腺炎（HT）是AITD不同表现形式，均有一个共同的自身免疫起源，发病机制相似可同时或先后存在于同一患者，并可互相转变，故本项目以AITD为研究对象，应用焦磷酸盐测序法检测易感基因HLA-DR、CTLA-4、CD40、PTPN22、FOXP3、CD25共24个位点的甲基化状态，发现病例对照组间易感基因CTLA-4、CD40、CD25上游启动子区的甲基化修饰差异有统计学意义，进一步选择代表性位点扩大样本量验证，证实在AITD病例组CD40启动子区存在低甲基化率，同时行qRT-PCR证实AITD病例组mRNA表达明显上调，首次证实CD40基因启动子区DNA甲基化修饰可能参与了AITD的发生。对2000余例流行病学调查的成年人行尿碘测定分析，该人群的尿碘中位数（MUI）133.4 μg/L，但未发现甲状腺功能及抗体水平与尿碘明显相关，但碘过量组的甲状腺结节检出率（35.3%）明显高，尤其在女性组（38.5%）更显著；未发现尿碘水平与候选易感基因启动子区的DNA甲基化明显相关。同时，应用流式细胞技术分析了596例流行病学调查获取的成人AITD病例对照者的外周血白细胞CD4+ T细胞亚群（Th1、Th2、Treg、Th17）比例变化，发现HT患者Th1/Th2比值、Th17/Treg比值明显低于对照组，Treg细胞比例HT组明显高于对照组，提示除了已知的Th1/Th2外，Treg与Th17间的相互作用和平衡可能与AITD的患病相关。上述结果提示CD40基因启动子区的低甲基化率可能与AITD相关或者是易感甲基化区，为AITD表观遗传标志物筛查及初步机制探讨提供了线索。