大动脉转位候选基因DYNC2LI1的发现与功能分析

Discovery and Functional Analysis of The Candidate Gene DYNC2LI1 for Transposition of Great Arteries

目的通过遗传学分析及功能学实验发现新的大动脉转位候选基因。方法对381例散发大动脉转位患者进行全外显子组测序,利用基因负荷分析筛选候选基因。针对患者中发现的不同突变,进行突变功能分析:构建体外表达质粒,并采用实时荧光定量PCR及Western blot检测mRNA及蛋白质的表达情况。在NIH3T3成纤维细胞中敲低目的基因,并对表达谱进行通路富集分析。结果与人群突变相比,大动脉转位组中DYNC2LI1基因突变负荷显著增加,提示DYNC2LI1基因突变与大动脉转位的发生存在必然联系。相比于野生型质粒,DYNC2LI1基因突变c.A239C,p.D80A的mRNA及蛋白的表达水平均显著降低,差异有统计学意义(P<0.0001,P<0.01);c.T240A,p.D80E和c.T389C,p.L130R的mRNA的表达量同样明显降低(P<0.001,P<0.05),但蛋白表达没有明显差异(P>0.05)。对DYNC2LI1敲低后的细胞转录组分析发现,伪结构组装相关通路的基因显著上调;而与心脏结构发育及体轴形态等通路相关的基因发生下调。结论与纤毛结构和功能相关的DYNC2LI1基因是大动脉转位的潜在致病基因。

Objective To explore novel candidate genes for transposition of the great arteries by genetic analysis and functional experiments.Methods Whole exome sequencing and gene burden analysis to screen the candidate genes were performed in 381 patients with sporatic transposition of the great arteries.We carried out functional analysis for different mutations found in patients:constructed in vitro expression plasmids,and used real-time fluorescent quantitative PCR and Western blot to detect the expression of mRNA and protein.The target gene was knocked down in NIH3 T3 fibroblasts,and the expression profile was analyzed by pathway enrichment.Results Compared with the population,the mutation burden of DYNC2 LI1 gene in the transposition of the great artery group was significantly increased,suggesting that the mutation of DYNC2 LI1 gene was necessarily related to the occurrence of the transposition of the great artery.Compared with NIH3 T3 cells transfected with wild-type plasmids,the c.A239 C,p.D80 A mutations of Dync2 li1 gene significantly reduced the expression levels of mRNA and protein,and the difference was statistically significant(P<0.0001,P<0.01).The mRNA expression levels of c.T240 A,p.D80 E and c.T389 C,p.L130 R were also significantly decreased(P<0.01,P<0.05),but there were no significant difference in protein expression(P>0.05).Analysis of the cell transcriptome after Dync2 li1 knockdown revealed that genes for pseudopodium assembly and cell shape were significantly upregulated;genes related to pathways such as outflow tract and ventricular septum morphogenesis,and axis specification were down-regulated.Conclusion The DYNC2 LI1 gene,which is related to ciliary structure and function,is a potential pathogenic gene for transposition of the great arteries.