结核感染免疫应答差异基因的eQTL定位及功能研究

Host immune response is the key factor to determine whether tuberculosis infection would eventually develop into active disease. In previous studies, we have compared the differences of cytokines and transcriptomics in peripheral blood mononuclear cells (PBMCs) between latent tuberculosis infection and active tuberculosis. We have found and verified significant differences in both expressional and transcriptional levels of a number of genes and cytokines, including C-X-C motif chemokine 10 (CXCL10), suggesting these genes might be associated with tuberculosis infection and pathogenesis. The result was confirmed by several overseas research teams. With these key genes, we intend to use the expression quantitative trait loci (eQTL) analysis strategy to do tubercular genetic association analysis and positioning in Chinese population, taking the expression of these genes as quantitative traits. After locating SNPs associated with expression, we plan to verify them in tuberculosis patients in cellular level, which are expected to clarify the susceptibility genes of tuberculosis in Chinese population and to explain the mechanisms of susceptibility as well.

结核感染后最终是否发展为活动性结核病与宿主免疫系统对结核的免疫应答强度有关。申请人前期发现，潜伏性结核感染和活动性结核患者外周血单个核细胞在特异性结核多肽抗原刺激后，存在细胞因子表达及转录组水平上的差异，筛选并验证了包括CXCL10 在内的多个免疫应答相关基因在转录水平及表达水平上存在显著差异，提示这些特定免疫应答相关基因与结核感染后发病密切相关。我们拟进一步运用表达数量性状座位（eQTL）分析策略，将前期发现与结核发病显著相关的关键基因的表达量作为数量性状，在活动性结核及潜伏性结核感染人群中进行遗传基因关联分析及定位；将定位的SNPs在人群队列中进行遗传学验证，最终选择与结核活动显著相关的SNP作进一步的功能研究。本课题在前期发现的结核感染免疫应答差异基因基础上，有望定位到中国人群的结核遗传易感基因，对其功能的鉴定将进一步揭示导致结核易感的分子机制。

申请人通过双重荧光反转录多重连接依赖式探针扩增技术检测了潜伏性结核感染和活动性结核患者外周血21个靶基因的表达量，验证了包括IL8，TNFRSF1B, BLR1, TIMP2, IL7R和TNFRSF10C等在内的多个免疫应答相关基因在转录水平及表达水平上存在显著差异。提示这些特定免疫应答相关基因与结核感染后发病密切相关。我们进一步运用表达数量性状座位（expression Quantitative Trait Loci, eQTLs）分析策略，通过PLINK 的混合线性模型进行数量性状关联分析。基于关联分析结果，发现与关联基因的表达水平显著关联的染色体SNP 区段(P<1e-5)。通过STRING 蛋白相互作用和蛋白功能相关性数据检索分析，发现差异表达的基因及其eQTLs关联位点TACR3，ADRA2C，FPR1，IL8，CCL19，PTPRU，CALM2处于一个互作调控网络中。研究人员进一步募集具有活动性结核患者103 例及结核潜伏感染者208 例，订制SNP 芯片，将eQTL 中筛选到的表达差异相关的SNPs 在两类人群中进行检测，通过验证，rs4654400 及rs62292160位点在两组人群中差异表达，提示其可能为中国人群的结核遗传易感基因，进一步揭示导致结核易感的分子机制。