散发性先天性心脏病相关SMAD1基因新突变研究

Role of a new SMAD1 mutation in sporadic congenital heart disease

目的:研究散发性先天性心脏病相关SMAD1基因新突变。方法:入选202例散发性先天性心脏病患儿和238名健康者,收集其临床资料和血标本并常规抽提基因组DNA,测序分析SMAD1基因以发现致病新突变。克隆SMAD1基因,构建野生型SMAD1表达载体SMAD1-pcDNA3.1,通过定点诱变产生突变型SMAD1-pcDNA3.1,转染COS7细胞,应用双荧光报告基因分析试剂研究突变的功能特性。结果:在1例散发性先天性右心室双流出道合并室间隔缺损患儿中发现SMAD1基因新突变,即NM_005900.3:c.381T>A;p.(Cys127^(*))突变。该突变不存在于238名健康者中。报告基因分析表明突变型SMAD1对靶基因TBX20的转录激活作用丧失。结论:SMAD1基因功能丧失性突变可能是部分散发性先天性右心室双流出道合并室间隔缺损的分子病因,这对先天性心脏病的精准医学防治具有潜在意义。

Objective:To investigate the possible role of a new SMAD1 mutation in sporadic congenital heart disease.Methods:202 children with sporadic congenital heart disease and 238 healthy subjests were included in this study.Clinical data and blood samples were collected,and genomic DNA was isolated from the blood leucocytes of each study participant.Sequencing analysis of the SMAD1 gene was carried out to identify a new mutation underpinning sporadic congenital heart disease.The SMAD1 gene was cloned and its wild-type expression vector SMAD1-pcDNA3.1 was constructed.The mutant-type SMAD1-pcDNA3.1 was generated through site-directed mutagenesis.COS7 cells were transfected with expression vectors,and the functional characteristics of mutant-type SMAD1 were explored with dual-luciferase reporters.Results:A new SMAD1 mutation,NM_005900.3:c.381T>A;p.(Cys127^(*)),was identified in a child suffering from congenital double outlet of the right ventricle and ventricular septal defect.In contrast,it was not detected in healthy subjects.Reporter analyses demonstrated that Cys127^(*)-mutant SMAD1 lost the ability to transcriptionally activate its target gene TBX20.Conclusion:SMAD1 loss-of-function mutation is likely to be the molecular mechanism predisposing to sporadic congenital double outlet of the right ventricle and ventricular septal defect in a subset of patients,implying its potential implication for precise prophylaxis and treatment of congenital heart disease.