基因SLC29A3的致病变异通过影响巨噬细胞功能导致麻风的分子病理机制研究

Gene SLC29A3, essential for the proper function of macrophage, is a susceptibility gene of leprosy. Resequencing of the coding sequence of SLC29A3 determined rs780668 as the most significant variant associated with leprosy. Structure ananlysis showed that rs780668 impair the stability of protein encoded by SLC29A3. These results imply that rs780668 may lead to the onset of leprosy by disordering the function of macrophage and influencing the immune response of host. However, no expermental evidences are available to support these implications. In this study, RNA-seq, qPCR and southern blot will be used to determine the differential expressions of SLC29A3 between leprosy cases and health controls. And the effects of casual variant on proliferation, phagocytosis and bacteria killing will be evaluated using THP-1 and RAW264.7 cells in vitro. Furthermore, to study the effects of casual variant on macrophage function and granuloma formation in vivo, knock down of zebrafish SLC29A3 by morpholino antisense oligonucleotides and rescue of human SLC29A3 carrying variant will be performed in mycobacterium marinum infected zebrafish. This study will reveal the molecular pathogenesis of SLC29A3 casual variant in leprosy and will also provide suggestions for the risk prediction and intervention strategy of leprosy.

SLC29A3是课题组新近发现的麻风易感基因，其对于维持巨噬细胞的正常功能具有重要作用。该基因的重测序显示其编码区的rs780668与麻风的关联性最强，分析发现该变异影响SLC29A3编码蛋白的结构稳定性，提示rs780668可能通过引起巨噬细胞的功能紊乱、影响宿主的免疫应答导致麻风的发生，但具体机制有待实验证实。因此后续课题组拟利用转录组测序、qPCR、免疫印迹技术探讨SLC29A3基因在病例和正常对照中的差异表达情况；并通过细胞学实验研究该致病变异对巨噬细胞的增殖、吞噬和杀菌能力的影响；同时利用吗啉代反义寡核苷酸对海鱼分枝杆菌感染的斑马鱼进行敲降及拯救，在体内条件下探讨SLC29A3及其致病变异对巨噬细胞功能及肉芽肿形成的影响，以最终阐释麻风的发病机理，为疾病风险预测、疾病干预等累积基础。

SLC29A3是课题组新近发现的麻风易感基因，其突变可造成以皮肤色素过度沉着、多毛症等为特征的H Syndrome，但是否影响麻风发病有待实验证实。我们对该基因的编码区进行了深度测序，发现5个与麻风具有遗传关联性的SNP，其中变异rs780668与麻风的关联性最强，分析发现该变异影响SLC29A3编码蛋白的结构稳定性，细胞学实验研究发现位点rs780668并不是通过影响细胞增殖对细胞功能产生影响。SLC29A3基因在病例和正常对照中的差异表达分析发现该基因在麻风病例中表达显著提高。以麻风分枝杆菌刺激巨噬细胞发现此基因被显著抑制，且敲降该基因显著降低巨噬细胞的吞噬病原体的功能。在斑马鱼中对SLC29A3的同源基因zslc29a3进行基因敲除后的转录组测序发现多个自噬相关关键基因下调表达，说明巨噬细胞中该基因的缺陷可能导致其自噬通路对麻风菌的抑制作用。且基因敲除导致斑马鱼胚胎色素表达消失，这与该基因突变可造成H Syndrome中的色素沉着过度可能相关。该研究证实基因SLC29A3的缺陷可导致巨噬细胞吞噬病原体功能缺陷、自噬通路受抑制进而增加麻风发病风险，且该基因的缺失可能导致色素沉着异常为特征的H Syndrome。本研究是遗传学基因关联性研究的继续和深入，为此类研究打通了研究思路和技术路线，为后续更多疾病的易感基因的深入研究奠定了基础。