AID与Sε区相互作用增强IgE类别转换重组在哮喘中的作用机制

Our previous studies have found that Sε mutation frequency of patients with asthma was significantly higher than that of the control group, and serum IgE, AID mRNA, εGLT, ε mRNA levels were significantly increased compared with the control group, suggesting that Sε mutations may lead to the hyperfunction of IgE class switch recombination, however, the effects need to be further verified and the molecular mechanism remains to be explored. Sε region represents the important regulatory sequences determining immunoglobulin class switching to IgE isotype, its unique sequence characteristics are essential for recruiting AID. So we surmised that mutations in Sε region may cause the changes of sequence, especially the RGYW/WRCY motif and / or GC repetitive sequences, leading to the enhancement of interaction between AID and Sε region, and the hyperfunction of IgE class switch. To verify this, we will catch sight of the important mutations by analyzing and comparing Sε sequences of asthma patients and controls; confirm the mutation forms which causing AID hyperfunction by EMSA and validate in vitro; explore the interactions between Sε mutants and AID and its mechanisms by the use of Sε mutant knock-in mice, so as to lay solid foundation for the intervention of IgE production and the development of new asthma drugs.

课题组前期研究发现，哮喘患者Sε区突变频率显著高于对照组，且血清IgE水平及AID mRNA、εGLT、ε mRNA水平与对照组相比均明显升高，提示Sε区突变可能致IgE类别转换重组亢进，但该作用尚需进一步验证，分子机制尚待探讨。Sε区是决定免疫球蛋白类别转换向IgE方向进行的重要调控序列，其特有的序列特征是募集类别转换启动酶即AID的关键所在。由此推测：Sε区突变可能致其序列特征改变，尤其是与AID作用的RGYW/WRCY基序和/或GC重复序列，促使AID与Sε区作用增强，致IgE类别转换特异性亢进。为证实以上假设，本项目通过分析患者Sε区序列，发现重要突变型；应用凝胶迁移等实验，明确致AID作用亢进的突变型，并通过构建AID和 Sε双表达质粒，在体外进行验证；利用Sε突变型knock-in小鼠，探讨Sε突变型与AID相互作用及其机制，为干预IgE产生及研发新型哮喘治疗药物奠定基础。

哮喘是以血清IgE水平升高为特征的免疫功能紊乱性疾病，IL-4/IL-13信号通路在IgE的产生即IgE类别转换重组的过程中发挥着重要作用。本项目主要进行了以下研究：1)IL-4/IL-13信号通路基因多态性参与IgE类别转换重组的机制；2)IL-4/IL-13信号通路基因间相互作用在IgE类别转换重组中发挥的作用；3)Sε区突变在高血清IgE哮喘患儿中的作用机制。通过分析500例哮喘患儿和523例健康对照儿童的7个SNP位点(IL-4 rs2243250, IL-13 rs1800925, IL-4RA rs1805010, STAT6 rs324011, FCERIA rs2494262, rs2427837 和 rs2251746)，发现IL-13 rs1800925位点与儿童哮喘高度相关（OR=0.578，95%CI 0.442-0.756）。同时，对FCERIA rs2494262, rs2427837 和 rs2251746三个位点的单倍型分析表明，rs2427837 和 rs2251746存在连锁不平衡（D’=0.94，logarithm of odds 105.15, r2=0.83）。采用SIEMENS immunoglobulin E kit检测哮喘患儿血清IgE水平，分析位点突变对血清IgE水平的影响。结果表明7个SNP位点中，除了IL-13 rs1800925外，其余全部与IgE水平升高相关，其中IL-4 rs2243250和IL-4RA rs1805010差异最显著，P值分别为0.0012和0.0013。多位点分析表明，哮喘组中同时携带多个风险等位基因的患儿比例显著高于健康对照组，且同时携带多个风险等位基因的患儿其血清IgE水平显著升高，IgE均值从最低的 71.07 KU/L（不携带任何等位基因） 升高到 901.7 KU/L（同时携带7个风险等位基因）。同时携带4 个SNPs位点 (IL-4 rs2243250, IL-13 rs1800925, IL-4RA rs1805010, STAT6 rs3224011) 风险等位基因的患儿IL-4, IL-13 和 STAT6 基因表达水平显著升高。结果表明，IL-4/IL-13通路多基因位点突变可放大对疾病风险的作用。