CTLA-4基因变异及表达异常在胸腺瘤患者发生重症肌无力中的作用及其机制研究

About 17%-50% of thymoma patients develop myasthenia gravis (MG), while 20% of MG patients have thymomas. The patient’s genetic profile and the thymic ability to export autoreactive T cells are equally important in developing MG.CTLA-4 plays important roles in immune tolerance, so produce autoreactive T cells, and CTLA-4 gene polymorphism is increased in thymoma associated MG. Our previous results showed that CTLA4 alleles rs733618*C and rs231775*A were associated with early onset of MG and thymoma status. We hypothesize that CTLA-4 gene polymorphism and aberrant expression play important roles in thymoma associated myasthenia gravis. To gain greater insight into the relationship between CTLA-4 status and thymoma associated MG, we design our research as follows: ⑴Identify every SNPs in the whole gene region of CTLA-4 through next generation sequencing technique, and verify the relationship between potential SNPs. ⑵Identification of CTLA-4 isoforms produced by alternative splicing and their association with CTLA-4 SNPs and thymoma associated myasthenia gravis. ⑶Detection of the CTLA-4 protein expression through immunohistochemical and immunofluorescence. ⑷Identification the percentage of CTLA-4+ T cells to suppressive T cells (Treg，CD4+CD25+Foxp3+）,evaluate the role of CTLA-4 in the immune tolerance,so the onset of MG.⑸Evaluation the role played by CTLA-4 gene polymorphism in the transcription，translation process and its potential role in regulation of immune tolerance and the onset/process of thymoma associated MG.This research will provide insights into the underlying mechanism of the disorder and may ultimately lead to the identification of novel targets for the therapeutic interventions.

为深入理解胸腺瘤相关重症肌无力（MG）的发病机制，我们检测发现细胞毒性T细胞相关抗原4（CTLA-4）基因单核苷酸多态（SNP）位点rs733618*C 和rs231775*A与胸腺瘤相关MG密切相关；结合国内外研究，我们认为CTLA-4基因变异和表达异常可能在胸腺瘤患者发生MG的机制中发挥重要作用。本项目中我们将⑴对CTLA-4全基因扫描测序，找到所有变异SNP并关联分析，分析其基因型与患者表型的关系；⑵检测CTLA-4 mRNA各剪切体的表达状态；⑶检测胸腺组织CTLA-4蛋白表达部位和水平；⑷检测CTLA-4+的T细胞占抑制性T细胞（Treg）的比例，评价CTLA-4对免疫耐受的调节作用。本项目将从基因、mRNA、蛋白及分子免疫等多水平解释CTLA-4在胸腺瘤患者发生MG中的作用，其顺利实施将使我们对胸腺瘤相关MG的发病机制有更深入的解释，并由此为新型靶向免疫治疗提供新的策略。

为深入理解胸腺瘤及相关重症肌无力（MG）的发病机制，我们检测发现细胞毒性T细胞相关抗原4 （CTLA-4）基因单核苷酸多态（SNP）位点rs733618*C 和rs231775*A与胸腺瘤相关MG密切相关 ；结合国内外研究，我们认为CTLA-4基因变异和表达异常可能在胸腺瘤患者发生MG的机制中发 挥重要作用。我们的研究发现，1） rs1863800*C，rs733618*C和rs231775*G与MG患者相关，并且与非胸腺瘤的MG患者密切相关；单倍型分析显示，rs1863800-rs733618-rs4553808-rs5742909-rs231775构成单倍型，其中CCACG是MG发病相关的危险单倍型；CTLA-4基因启动区-1722位点（rs733618）和启动区-318位点（ rs5742909）与伴胸腺瘤MG相关。2） rs733618*C是伴胸腺瘤MG发病的危险因素，而rs574290 9*T是伴胸腺瘤MG发病的保护因素。 而非胸腺瘤MG患者则与rs733618多态性相关，我们既往在168例MG患者的研究中发现 rs231775多态性与非胸腺瘤MG发病相关，在本研究中未能重复出阳性结果。 生物信息学分析和既往研究显示，rs733618*C影响基因的剪接，干扰蛋白的表达和 功能；而rs5742909为T等位基因时，（与C等位基因相比）具有更强的启动活性。GCs治疗不敏感组糖皮质激素受体（GR）基因rs9324921*A等位基因频率显著高于GC 治疗敏感组(24.13% vs 38.10%, p = 0.046, OR = 1.94, 95% CI = 1.00-3.74)；GCs治 疗不敏感组rs17209237*G等位基因显著低于GCs治疗敏感组(2% vs 17%, p = 0.013, OR = 0.119, 95% CI = 0.016-0.877)。3.TTF-1+/PAX8-免疫表型高度支持肺 脏来源的神经内分泌癌，而PAX8+则支持更可能是胸腺来源的神经内分泌癌。