甲状腺相关眼病易感基因与病情严重程度的相关性研究

Thyroid associated ophthalmopathy (TAO) is an autoimmune inflammatory disorder with clinical manifestation in the orbital region, characterized by eyelid retraction, edema, erythema, conjunctivitis, proptosis, and in its most severe form, orbital neuropathy. At least 85% of TAO is associated with Graves' disease (GD), while it can occur with Hashimoto thyroditis and hypothyroidism. Because the understanding of the mechanistic regulator of TAO remains incomplete, prediction of disease course awaits improvement and clinical management focuses on symptom relief. As a result, TAO claims the NO.1 cause of orbital inflammation and treatments are often less than satisfactory with high relapsing rate and in some cases leads to vision loss. Our group selected 7 genes (HLA, CTLA-4, IL-12, IL-13, IL23R, TNF-α and IFN-γ) most representative of the three signals of antigen-specific T cell activation as well as the three directions of T cell polarization in autoimmune inflammatory response. A total of 18 gene polymorphisms on these 7 genes in an esitmated 400 participants will be evaluated. Three categories of subjects, namely, GD+/TAO-, GD+/TAO+ and healthy controls will be recruited and the GD+/TAO+ groups will be further divided into light, mild/severe and sight-threatening, 3 categories based on the Clinical Activity Score (CAS) of TAO. We, for the first time, apply nested-PCR combined with high resolution melting analysis and gene sequencing to test the polymorphism of interest for association with susceptibility to GD, TAO and mild/severe type TAO. The applied technique is highly sensitive and accurate. Thus the aim of this study is to explore the theoretical basis and provide the technical possibility of predicting TAO onset in GD patients and likelihood of progression into sight-threatening phase in TAO patients, in order to engage in early clinical intervention.

甲状腺相关眼病（TAO）居眼眶病首位，近年发病率上升，其中85%来自Graves病（GD）。病情轻者外观改变，重者可致盲。部分GD发生TAO，部分TAO病情呈重度表现，机制尚不清楚。TAO发生发展过程中，免疫炎症反应起重要作用。本研究选取免疫炎症反应时抗原递呈细胞与T细胞相互作用中识别，粘附及活化（Th1，Th2，Th17三个方向）中的7个较具代表性基因（HLA、CTLA-4、IL-12、IL-13、IL-23R、TNF-α、IFN-γ）及其上的18个位点，率先在国际上选取多基因、多位点，对正常组、GD无眼症、TAO三组人群进行相关性研究，首次将巢式PCR扩增结合高分辨熔解曲线基因分型技术应用于TAO。目的是明确TAO易感基因，阐明GD与TAO易感基因相关性，TAO易感基因与TAO临床严重程度相关性，为TAO的早期预测、干预及靶向治疗奠定基础，构建TAO相关基因标准品库，搭建临床检测平台。

背景：甲状腺相关眼病（TAO）是一种常见的眼眶病，约90%TAO来自格雷夫斯病（GD）患者。25%~50%GD患者发生眼部病变，轻者外观改变，重者危及视力。目前TAO发病机制尚不明确，研究普遍认为TAO是基因和环境共同作用的结果。本项目在国内外原有基因研究的基础上，选取了6个基因片段上14个单核苷酸多态性（SNP）位点，旨在发现TAO易感基因及其与TAO严重程度的相关性。.方法：纳入研究对象495人：健康对照组152人，GD无眼征组189人，TAO组154人。TAO组：轻度52人，中重度55人，危及视力47人。用全血基因组DNA提取试剂盒（磁珠法）提取外周血中DNA。 Taqman 实时荧光定量PCR分析技术检测14个SNP位点的基因型。.结果：采用Fisher精确检验对健康对照组、GD组、TAO组14个SNP的基因型进行单因素分析，差异无统计学意义。将研究对象分为健康对照组（152人，组1）、GD+轻度TAO组（241人，组2）、中重度危及视力TAO组（102人，组3）进行分析：TNF alpha-863C/A位点CC基因型频率分别为：70.3%,68.1%,54.9%，组3 CC基因型频率低于组2（P＜0.05，OR=0.57）和组1（P＜0.05，OR=0.51）。TNF alpha -1031T/C位点TT基因型频率分别为：66.4%,62.8%,48.0%，组3 TT基因型频率低于组2（P＜0.05，OR=0.55）和组1（P＜0.05，OR=0.47）。CTLA-4 CT60A/G位点AA基因型频率分别为：3.9%,0.8%,4.9%，组3 AA基因型频率高于组2（P＜0.05，OR=6.1）。采用logistic回归对组3和组2进行分析，筛选出显著因素CTLA-4 CT60A/G、性别、年龄，并构建预测模型logit(P)=ln〖P/(1-P)〗=-2.040-0.560×Gender+0.049×Age+0.629×M。.结论：TNF alpha-863C/A、TNF alpha -1031T/C、CTLA-4 CT60A/G是TAO易感基因，与TAO严重程度有关。年龄是TAO严重程度的危险因素，女性是保护性因素。.该项研究结果对于患者的诊疗和随访有一定的临床指导意义。