隐球菌脑膜炎患者染色体9q33.1区域易感位点的定位与验证

Cryptococcal infection has been an ever escalating threaten to the health of the general public.One of the most concerned issues has been the mechanism by which immunocompetent hosts may become susceptible to cryptococcosis, and the potential underlying genetic deficiency. In our previous study, we have collected clinical and DNA database from case-control series of cryptococcal meningitis and identified that several genetic immune deficiencies in apparently healthy patients with cryptococcal meningitis. We have also found for the first time that polymorphisms of rs10759932 and rs1927907, two loci located in the non-coding area of TLR4 gene at chromosome 9q33.1, were significantly associated with susceptibility to cryptococcal meningitis. The absence of functional role identified for these 2 SNPs suggested that there exist other functional variants that are within the linkage disequilibrium region marked by the loci. Therefore, we plan to perform target region sequencing of the 198kb region of interest through next generation sequencing technique, to fine map variations that alters susceptibility, and to validate their roles by both a prospective replicate population, transcriptome sequencing, and functional study on transcription activity and cell phenotypes to elucidate the effect of variants upon genes, and that of genes to host defense against Cryptococcus. This work will bring new insight into the molecular genetic mechanism of the pathogenesis of cryptococcosis, and provide information regarding risk prediction and prevention of cryptococcal meningitis.

隐球菌感染已成为威胁普通人群生命健康的全球性公共卫生问题。其中，健康宿主为何对隐球菌易感、是否具有潜在的免疫缺陷备受关注。本课题组前期已建立健康成人隐球菌脑膜炎病例-对照临床资料数据库和DNA标本库，并经研究证实健康患者存在免疫遗传缺陷，且首次发现位于染色体9q33.1区域TLR4基因非编码区的rs10759932和rs1927907位点与隐球菌脑膜炎易感性显著相关，然而该位点并无功能报道，提示该区域存在连锁不平衡的其他相关功能位点。为此，本课题拟采用二代测序技术对该区域的198kb基因片段进行目标序列捕获测序，实现对其易感位点的精细定位，并通过前瞻性病例对照研究、转录组测序进行验证，以及从转录活性及细胞表型等角度明确变异位点对基因功能的影响，及相应基因对宿主免疫防御功能的影响，以期为隐球菌脑膜炎分子遗传学发病机制提供新的理论依据，从而为隐球菌感染风险的预测及高危人群的预防提供参考。

本课题以健康体检者和隐球菌脑膜炎患者为对象，通过大样本量的病例对照遗传关联研究，成功筛选出多个隐球菌脑膜炎患者染色体9q33.1区域易感性关联的SNPs位点（位于TLR1、TLR2、TLR4、TLR6、TLR9），再通过外显子及其侧翼深度测序技术，精确定位染色体9q33.1区域的高频和低频易感SNP位点，发现其分布于TLR2（外显子区）、TLR4（UTR3区和基因间区）、TLR6（UTR区和外显子区）和TLR9（侧翼）。进一步通过某特定群体（福建省）进行易感位点的病例对照遗传易感性验证，病例组来自福建的AIDS相关隐脑患者，对照组为福建非隐脑的HIV感染者，结果显示TLR6的SNP与隐球菌脑膜炎患者的易感性显著相关，且为外显子2区非同义编码位点，提示TLR基因与隐球菌脑膜炎的易感性密切相关；为进一步明确这些基因位点的功能性，我们比较这些易感位点的基因多态性与隐球菌脑膜炎患者脑脊液细胞因子表达量高低的关联性，结果显示该位点与脑脊液细胞因子表达量密切相关，提示TLR6易感基因位点不但与隐球菌脑膜炎的易感性相关，也与隐球菌脑膜炎的致病性也密切相关。也就意味着宿主TLR在基因水平的改变，与脑脊液炎症反应蛋白水平表达量的高低密切关联，表明TLR参与隐球菌脑膜炎患者脑脊液的炎症反应。因此，本课题研究结果首次揭示TLR基因与隐球菌脑膜炎的发生发展密切相关。