先天性动脉导管未闭致病基因MESP2新突变的发现与功能分析

Identification and functional assay of a new MESP2 mutation responsible for congenital patent ductus arteriosus

目的:研究先天性动脉导管未闭(PDA)致病基因MESP2新突变。方法:选取2016年2月至2019年11月同济大学医学院附属同济医院儿科208例先天性心脏病(CHD)患儿和232名无CHD儿童,收集其血液标本及临床数据。常规纯化基因组DNA,测序分析转录因子基因MESP2以发现新的致病突变。克隆野生型MESP2基因并构建其真核表达质粒,通过定位诱变产生突变型MESP2表达质粒,转染HEK 293T细胞,通过报告基因分析揭示突变体的功能特性。结果:1例散发性先天性动脉导管未闭(PDA)患者检测出1个MESP2基因新突变,即NM_001039958.2:c.268G>T(p.Glu90*)突变。该突变不存在于对照组和其他CHD儿童中。报告基因分析显示突变型MESP2对靶基因NKX2.5的转录激活功能丧失。结论:MESP2基因缺陷可能是部分先天性PDA的分子病因,为先天性PDA的防治提供了新的分子靶标。

Objective:To investigate a new MESP2 mutation underpinning congenital patent ductus arteriosus(PDA).Methods:A cohort of 208 children inflicted with congenital heart disease(CHD)and a total of 232 control subjects with no CHD were recruited,and their blood specimens and clinical data were collected.Genomic DNA was routinely isolated from each study participant's blood leucocytes,which was utilized for sequencing analysis of the MESP2 gene to identify a new mutation contributing to CHD.The wild-type MESP2 gene was cloned,with its eucaryotic expression plasmid constructed.The mutant-type MESP2 expression plasmid was produced through site-directed mutagenesis.HEK 293T cells were transfected with various expression plasmids,and the functional characteristics of the mutant-type MESP2 were revealed by reporter gene analysis.Results:In a child suffering from sporadic congenital PDA,a new MESP2 mutation,NM_001039958.2:c.268G>T(p.Glu90*),was detected,which was not observed in the control group or other CHD children.Reporter gene analysis showed that the mutant MESP2 failed to transcriptionally activate its target gene NKX2.5.Conclusion:MESP2 defect is likely to be a molecular etiology giving rise to congenital PDA in a subset of patients,providing a novel molecular target for the medical prophylaxis and therapy of congenital PDA.