甲型H1N1流感病毒感染致病候选基因的鉴定

Identification of candidate genes for pathogenicity of influenza A H1N1 virus infection

目的通过C57BL/6J(B6)和DBA/2J(D2)亲本品系小鼠流感感染后相关表型及杂交子代BXD小鼠肺脏转录本数据的系统性分析进行基因筛选,以找到在流感病毒感染中发挥作用的候选基因.方法共选取41个BXD小鼠及亲本品系B6和D2的雌性小鼠,在8~12周龄之间给予流感病毒甲型鼠肺适应株(H1N1,PR8M)得到相关表型及基因表达量数据,对基因表达量进行聚类分析及加权基因共表达网络分析,构建模块特征值与小鼠病毒载量的相关性,经表达数量性状座位分析,皮尔逊相关性分析,结合全基因组关联研究(GWAS)、小鼠基因组信息学(MGI)、国际小鼠表型联合会(IMPC)三个基因数据库中的信息分析筛选流感病毒感染相关的候选功能基因及确定该基因的遗传调控方式;最后将挑选出的基因进行通路富集分析.结果通过聚类分析和加权基因共表达网络分析,将24219个基因分为23个模块;通过模块与表型的相关性分析,发现MEred、MEgreen、MEma-genta模块与感染密切相关,将其作为核心模块;对核心模块基因通过KEGG和MPO富集分析,结果显示流感病毒相关基因显著参与MAPK信号通路、TNF信号通路等多个细胞免疫相关通路;对核心模块内基因与BXD小鼠流感病毒感染的3种表型(体质量减轻,平均死亡时间和感染后体质量减轻中位数)进行相关性分析,共发现21个基因的肺mRNA表达水平与3种表型均相关(P<0.05);进一步通过GeneNetwork中GEMMA算法对这21个基因行表达数量性状座位分析,结果表明Acpl2受顺式遗传调控,Dusp12、Ovol2、Catsper4、Tmc5、Acsm 1、Fam81a、Rtn4、Rhpn1受反式遗传调控;通过结合MGI,IMPC,GWAS三个基因数据库中的信息分析发现部分基因在免疫通路方面发挥作用.结论通过系统遗传学分析,在小鼠基因组上鉴定了影响流感病毒感染相关的21个候选基因,后续可做进一步的功能验证来阐明其参与流感病毒感染的遗传调控机制.

OBJECTIVE To conduct a genetic screen by systematic analysis of the phenotypes associated with influ-enza infection in the C57BL/6J(B6)and DBA/2J(D2)parental strains of mice and the lung transcript data of in-bred offspring BXD mice,and in order to find candidate genes that play a role in influenza virus infection.METHODS A total of 41 female BXD mice and parental strains B6 and D2 were selected and given influenza virus type A murine lung-adapted strains(H1N1,PR8M)between 8 and 12 weeks of age to obtain relevant phenotypic and gene expression quantity data,which were subjected to clustering analysis and weighted gene co-expression network analysis to conduct the correlations between modular trait values and viral load in mice.After expression quantitative trait seating analysis,Pearson correlation analysis,combined with information from three gene data-bases,namely,Genome-Wide Association Study(GWAS),Mouse Genome Informatics(MGI),and International Mouse Phenotyping Consortium(IMPC),the candidate functional genes related to influenza virus infection were analyzed and screened,and the genetic regulation of the genes was determined.Finally,the selected genes were subjected to pathway enrichment analysis.RESULTS The 24219 genes were classified into 23 modules by cluste-ring analysis and weighted gene co-expression network analysis.Through the correlation analysis between modules and phenotypes,the MEred,MEgreen,and MEmagenta modules were found to be closely related to infection,and were considered as the core modules.The results KEGG and MPO enrichment analysis of the genes in the core module showed that influenza virus-related genes were significantly involved in several cellular immune-related pathways such as MAPK signaling pathway and TNF signaling pathway.Correlation analysis of genes in the core module with three phenotypes of influenza virus infection in BXD mice(body mass reduction,mean time to death and median body mass reduction after infection)revealed that the lung mRNA expression levels of 21 genes were correlated with all three phenotypes(P<0.05).Further,the quantitative trait seating analysis of these 21 genes by GEMMA algorithm in GeneNetwork showed that Acpl2 was regulated by cis-genetic inheritance,and Dusp12,Ovol2,Catsper4,Tmc5,Acsm1,Fam81a,Rtn4,Rhpn1 were regulated by trans-genetic inheritance.Some of the genes were found to play a role in the immune pathway by combining the information from three gene databas-es,MGI,IMPC,and GWAS.CONCLUSION Through phylogenetic analysis,21 candidate genes related to influ-enza virus infection were identified in the mouse genome,and further functional validation should be performed to elucidate the genetic regulatory mechanisms involved in influenza virus infection.