联合GWAS与RNA-Seq技术深入挖掘麻风相关信号通路与关键基因

In the past seven years, we have conducted genome-wide association study (GWAS) and identified 20 susceptibility genes associated with leprosy. However, these genes can only explain 13.53% of the leprosy risk, which showed a limited effect for risk prediction. To avoid false positive correlation, GWAS adopts very strict P values and some signals related to the disease may be missed. This is one of the key factors that too few susceptibility genes were found. At the same time, we found 1,791 differentially expressed genes based on RNA-Seq data of 27 leprosy lesions and 18 healthy controls’ normal skin. But the large number of differential expressed genes made it difficult for the choice of disease susceptibility genes. In order to overcome the disadvantages of these research methods, this study intends to integrate GWAS and RNA-Seq data of leprosy using Fisher's combined method, Permutation test etc. After the calculating of associated P values in the GO and KEGG pathways database, genes/signaling pathways showed suggestive association will be validated in leprosy and normal skin tissues/peripheral blood. This study may be helpful to pathogenesis elucidation, risk prediction and intervention of this disease.

课题组前期采用全基因组关联分析方法(GWAS)先后定位了20个麻风易感基因，但这仅能够解释13.53%的患病风险，发病风险预测效力有限。GWAS为避免假阳性关联，采用非常严格的P值界定，因此会漏掉部分确实与疾病相关的单核苷酸多态性(SNP)位点，这是造成发现易感基因过少的关键原因之一。同时，课题组利用27例麻风皮损组织、18例正常对照皮肤组织的转录组测序（RNA-Seq）发现了1,791个差异表达基因，数量过多的此类基因也给疾病易感基因的选择造成了困难。为克服以上研究方法的缺陷，本研究拟利用Fisher联合法、置换检验等方法对课题组前期麻风GWAS数据及RNA-Seq数据进行整合，通过计算GO和KEGG数据库中通路的关联P值，筛选麻风候选基因/信号通路并进一步在麻风和正常人皮肤组织、外周血中进行验证，以阐明麻风发病机理，为疾病风险预测、疾病干预等累积基础。

课题组前期采用全基因组关联分析方法(GWAS)先后定位了20个麻风易感基因，但这仅能够解释13.53%的患病风险，发病风险预测效力有限。GWAS为避免假阳性关联，采用非常严格的P值界定，因此会漏掉部分确实与疾病相关的单核苷酸多态性(SNP)位点，这是造成发现易感基因过少的关键原因之一。同时，课题组利用27例麻风皮损组织、18例正常对照皮肤组织的转录组测序（RNA-Seq）发现了1,791个差异表达基因，数量过多的此类基因也给疾病易感基因的选择造成了困难。为克服以上研究方法的缺陷，本研究利用Fisher联合法对课题组前期麻风GWAS数据及RNA-Seq数据进行整合，得到在GWAS和RNA结果中P值均小于0.0001，并且联合关联分析P值小于5E-8的47个基因作为候选基因。通过KEGG数据库富集通路的结果筛选得出HLA-E为目标基因。麻风和正常对照皮肤单细胞转录组测序的免疫细胞结果显示，HLA-E在朗格汉斯细胞中患者表达显著高于正常人。进一步的多重免疫组化结果显示，在患者中该基因的表达增加，而且与朗格汉斯细胞共定位。据报道，HLA-E可能向CD8 + T细胞呈递数十种结核分枝杆菌（Mtb）衍生的非异构肽，比对结果发现Mlep衍生的5种非肽段与这些Mtb同源物100％相同，因此，朗格汉斯细胞还可以通过HLA-E向CD8 + T细胞呈递Mlep肽，表明MHC一类分子可以通过朗格汉斯细胞呈递Mlep抗原。该发现为进一步理解麻风发病的分子遗传学机制、风险预测及干预措施的制定累积基础。