TCN2基因及其致病变异在麻风发病中的分子机制研究

TCN2 has been identified as one of susceptibility genes of leprosy in our group, and previous studies confirmed that the gene was involved in the NF-κB immune pathways through increasing TCN2 expression. Our previous RNA sequencing and Elisa results showed that TCN2 was over expressed in leprosy; Next generation sequencing confirmed that the rs75680863 located in the coding region of TCN2 correlated with leprosy most; The protein structural analysis suggests that the rs75680863 might play a role in the binding of TCII to the receptor and the stability of TCII. These results indicated that TCN2 may be involved in pathogenesis of leprosy, however, the mechanism of TCN2 gene and its causative variants in the pathogenesis of leprosy remains unclear. To figure out that question, the rare variant rs75680863 will be validated in 1,000 leprosy cases and 1,000 health controls by fine maping; Elisa will be conducted to investigate the TCII expression affected by leprosy infection; to investigate the interaction of TCII with the receptor, molecular experiments such as immunoprecipitation and indirect immunofluorescence assay will be performed; further, luciferase reporter assay and zebrafish model will be performed to elucidate the functional role of both TCN2 and its regulatory variant in the regulation of NF-κB and in the pathogenesis of leprosy.

TCN2为课题组发现的麻风易感基因，既往研究证实该基因通过上调钴胺素转运蛋白表达进而影响NF-κB发挥免疫保护作用。前期转录组测序和血清ELISA结果显示TCN2在麻风病例中表达升高，对该基因进行深度测序发现其编码区域的变异rs75680863与疾病的相关性最强，蛋白结构分析显示该变异可以影响钴胺素转运蛋白的稳定性以及其与受体的结合。以上均提示TCN2及其致病变异在麻风发生、发展中发挥重要作用，具体机制尚待证实。因此课题组后续拟采用体外、体内功能实验对其机理进行阐释，采用ELISA方法研究病例、对照间、不同基因型样本间TCN2蛋白表达差异；免疫共沉淀和共聚焦显微镜技术探讨变异对钴胺素转运蛋白与受体结合的影响；荧光素酶报告实验探讨TCN2基因及其变异位点对NF-κB转录活性的影响；斑马鱼感染模型探讨TCN2变异位点在海鱼分枝杆菌感染致肉芽肿形成中的作用，以及对NF-κB免疫通路的调节作用。

TCN2为课题组发现的麻风易感基因，既往研究证实该基因通过上调钴胺素转运蛋白表达进而影响NF-κB发挥免疫保护作用。前期转录组测序和血清ELISA结果显示TCN2在麻风病例中表达升高，对该基因进行深度测序发现其编码区域的变异rs75680863与疾病的相关性最强，蛋白结构分析显示该变异可以影响钴胺素转运蛋白的稳定性以及其与受体的结合。接下来课题组利用体外细胞感染模型以及qPCR检测发现TCN2差异表达来源于皮损，而非血细胞；体外细胞感染模型以及蛋白印迹检测显示TCN2对p65蛋白入核无影响，课题组进而利用荧光素酶报告实验发现rs75680863变异位点一定程度上影响p65转录活性，推测该变异位点通过其他中间途径影响NF-κB信号通路；为证实上述现象，课题组利用MSD以及体外细胞感染模型发现，麻风患者以及对照PBMC体外麻风菌刺激后，TNF-α（NF-κB下游细胞因子）表达均明显上升，但在加入B12后，对照组TNF-α表达出现一定程度下降，提示rs75680863变异影响B12的转运，进而影响NF-κB下游因子TNF-α的表达，从而调控疾病的发生。