基于同胞兄妹肺隐球菌病易感基因的精细定位与功能分析

Pulmonary cryptococcosis (PC) has been the third most common pulmonary fungal disease in China. During the past few years, we have established a detailed clinical database for pulmonary cryptococcosis corhort study. And in our previous study, we have found that genetic polymorphism of C-lectin receptor Dectin-2 was significantly associated with susceptibility to cryptococcal pneumonia. Though it is significantly difficult to screen and identify the exact genetic deficiency locus for this complicated fungal disease, the obtainment of an index family will make it much more easier. To obtain the candidate susceptibility genes, we plan to conduct among an index family including two brothers and two sisters, exon sequencing, RNA sequencing and miRNA microarray screening. Then, we will validate them in a larger population that includes PC patients and healthy volunteers. By prospective case-control association study and comparison for miRNA level using qRT-PCR, we can finally get several genes that are most associated with PC. Functional study will be conducted through flowcytometry, Luminex, gene overexpression and knock out to validate the influence of these genes on host immune defense. With the founding of associated genes and validation of their function, 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 pneumonia.

肺隐球菌病已上升为我国第3大最常见肺真菌病，本课题组前期已建立肺隐球菌病临床病例研究队列，并经研究发现C型凝集素受体Dectin-2与肺隐球菌病的遗传易感性密切相关。但肺隐球菌病众多易感基因位点的筛选和鉴定极为困难，本课题组新近获得的肺隐球菌病同胞兄妹家系样本，为我们精细定位肺隐球菌病的候选易感基因提供了可能。本课题拟采用全基因外显子组、转录组测序技术、miRNA芯片检测技术对同胞兄妹家系易感基因位点的筛选，实现肺隐球菌病易感基因的精细定位，并通过病例对照遗传关联研究和miRNA qRT-PCR差异性表达分析验证候选基因，进一步用流式细胞术、Luminex细胞因子检测术、基因敲除或转染过表达等技术分析易感基因位点突变对免疫细胞生物学功能的影响，以期发现肺隐球菌病相关的易感基因，为肺隐球菌病的分子免疫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。

隐球菌病是一种威胁生命的真菌感染。肺部通常是隐球菌进入机体的门户，故对肺隐球菌病的发病机制研究尤为重要。本课题组通过对同胞兄妹的全基因组进行外显子组和转录组测序，并采用miRNA芯片检测技术对同胞兄妹家系易感基因位点进行筛选，成功锚定了数个易感基因位点和相关的代谢通路，实现了隐球菌病易感位点的精细定位。同时本课题组还通过前瞻性的病例对照研究对候选基因进行了大规模的人群验证，并通过细胞因子的检测、流式细胞术、巨噬细胞功能的检测等方面明确了变异位点对基因功能的影响，以及候选基因对宿主免疫防御功能的影响，为隐球菌病的遗传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高危人群的预防提供参考。