摘要/Abstract
摘要： 目的·探究完全性肺静脉异位引流（total anomalous pulmonary venous connection，TAPVC）的发病机制，通过全外显子测序筛查出致病基因ARHGEF16，并对其进行功能验证。方法·收集78例TAPVC患儿及100例正常对照儿童的血液、临床资料及辅助检查结果，抽提全血DNA进行ARHGEF16突变筛查。构建ARHGEF16野生型及突变型表达质粒，转染293T细胞，通过实时荧光定量PCR (quantitative real-time PCR，RT-qPCR)和蛋白质印迹法（Western blotting）分别检测mRNA和蛋白的表达水平；通过软件Cytoscape进行蛋白与蛋白之间的相互作用分析。结果·在TAPVC患儿中发现ARHGEF16基因2个未报道的突变位点c.C236>T（A79V）和c.G619>C（G207R），在对照组中未发现。与野生型相比，突变型ARHGEF16 mRNA和蛋白表达量均上调。蛋白相互作用分析显示，ARHGEF16与RAC1可以直接相关联；通过RT-qPCR检测，发现在过表达ARHGEF16时，RAC1表达上调。结论·ARHGEF16的错义突变影响了ARHGEF16 mRNA及蛋白表达水平，过表达ARHGEF16可上调RAC1的表达，提示其可能通过对RAC1的调控参与TAPVC的形成及发展。
关键词: 完全性肺静脉异位引流, ARHGEF16, 错义突变, RAC1, 发育
Abstract:
Objective · To investigate the pathogenesis of total anomalous pulmonary venous connection (TAPVC), and to identify ARHGEF16 gene through full exon sequencing screening and analyze its mutation function. Methods · The blood, clinical data and auxiliary examination results of 78 children with TAPVC and 100 healthy controls were collected, and the genomic DNA was extracted for ARHGEF16 mutation screening. ARHGEF16 wild-type and mutant plasmids were constructed and transfected into 293T cells. mRNA and protein levels were detectedquantitative real-time PCR (RT-qPCR) and Western blotting, respectively. Protein-protein interaction exploration was performedCytoscape software. Results · Two novel variants c.C236>T (A79V) and c.G619>C (G207R) were found in TAPVC patients and were not found in healthy controls. Compared with the wild type, the mutants ARHGEF16 were up-regulated in both mRNA and protein levels. Protein interaction analysis showed that ARHGEF16 and RAC1 were directly associated; RAC1 was up-regulated in HEK293 cells with ARHGEF16 over through RT-qPCR. Conclusion · The missense mutations of ARHGEF16 affect the mRNA and protein levels of ARHGEF16. Over of ARHGEF16 up-regulates the of RAC1, suggesting that it may participate in the development and formation of TAPVCregulating RAC1.
Key words: total anomalous pulmonary venous connection, ARHGEF16, missense mutations, RAC1, development
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