儿童先天性心脏病TBX20基因突变及功能探讨

TBX20 Gene Mutation and Related Functional Implication in Children With Congenital Heart Disease

目的:对先天性心脏病(先心病)患儿进行TBX20基因突变研究,探讨TBX20基因与先心病发生机制之间的关系。方法:对203例中国汉族先心病患儿(病例组)进行TBX20基因编码区测序,同期300例门诊体检健康儿童为对照组,检测可能的先心病相关性基因突变。对新发现的突变应用蛋白质结构预测服务器I-TASSER进行同源建模,对TBX20基因野生型及突变后的蛋白质结构模型进行预测分析。并进一步构建TBX20野生型及突变型表达载体,分别转染到COS7细胞中,通过双荧光素酶报告基因检测它们在NKX2.5、GATA4协同作用下对下游靶基因心钠素(ANF)启动子转录激活作用的影响。结果:(1)病例组1例房间隔缺损患儿发现一个新的TBX20基因错义突变c.187G>A(A63T),DNA序列中第187位鸟嘌呤突变为腺嘌呤,导致氨基酸序列中第63位丙氨酸变为苏氨酸,在对照组中均未发现该变异。(2)对错义突变A63T进一步研究中,TBX20同源建模显示第63位的非极性、疏水性丙氨酸变为极性、亲水性的苏氨酸,与Gly65、Gly68间的氢键断裂,与Asp62、His64形成新的氢键,导致蛋白质构象发生变化。(3)体外细胞实验双荧光素酶报告基因检测结果显示A63T突变型TBX20对下游靶基因ANF启动子的激活作用较野生型增强(P<0.01)。结论:新发现的TBX20基因错义突变A63T导致蛋白质空间构象发生改变,可能通过上调ANF启动子活性而参与先心病的发病机制,拓宽了房间隔缺损的基因突变谱。

Objectives:TBX20 gene mutation was explored in patients with congenital heart disease(CHD),the potential relationship between TBX20 gene mutation and the pathogenesis of CHD was analyzed.Methods:TBX20 gene coding region was sequenced in 203 Chinese Han children with CHD(case group)and in 300healthy children(control group).For detected novel mutation,protein structure prediction server I-TASSER was used to model TBX20,and the structural models of wild type and mutant TBX20 were analyzed.Then the wild-type and mutant expression vectors of TBX20 were constructed and transfected into COS7 cells,respectively,and their effects on transcriptional activation of downstream gene atrial natriuretic factor(ANF)promoter under the synergistic action of NKX2.5 and GATA4 were detected by Dual-Luciferase Reporter Assay System.Results:(1)A novel TBX20 missense mutation c.187G>A(A63T)was found in 1 child with atrial septal defect in the case group.Guanine at position 187 in the DNA sequence was mutated to adenine,resulting in alanine at position 63 in the amino acid sequence to threonine.This mutation was absent in 300 controls.(2)In subsequent analysis on A63T,TBX20homology modeling showed that the non-polar and hydrophobic alanine at site 63 changed into polar and hydrophilic threonine,breaking hydrogen bonds with Gly65 and Gly68,and forming new hydrogen bonds with Asp62 and His64,which led to changes in protein conformation.(3)In vitro cell experiments,the Dual-Luciferase Reporter Assay System showed that A63T mutant TBX20 enhanced the activation of downstream gene ANF promoter compared with wild-type TBX20(P<0.01).Conclusions:The newly discovered missense mutation A63T of TBX20 gene could lead to changes in protein spatial conformation.It may be involved in the pathogenesis of CHD through up-regulating ANF promoter activity.This study broadened the gene mutation spectrum of atrial septal defect and provided new pathogenic evidence for the pathogenesis of CHD.