先天性室间隔缺损中的一个GATA4基因新突变

A novel GATA4 mutation leading to congenital ventricular septal defect

目的 研究GATA4基因新突变导致先天性室间隔缺损（ventricular septal defect,VSD）的分子机制.方法 收集185例先天性VSD患者的临床资料和血标本,以200名健康者为对照.应用PCR扩增GATA4基因的全部外显子,采用双脱氧核苷链末端合成终止法对全部扩增片段进行测序以识别基因突变.克隆GATA4基因,通过定位诱变获得相应的突变体,应用脂质体将GATA4基因重组表达质粒及心房利钠肽基因启动子启动绿色荧光蛋白表达的报告载体转染HeLa细胞,应用逆转录-PCR研究GATA4基因突变对其编码的转录因子的活性的影响.结果 在1例VSD患者的GATA4基因发现1个新的杂合错义突变c.191G＞A,即第64位的密码子由GGA变为GAA,导致第64位的甘氨酸变为谷氨酸,即G64E突变.细胞表达分析显示GATA4突变G64E使转录因子的活性降低.结论 在先天性VSD患者发现GATA4新突变G64E,该突变可能通过抑制转录因子的活性而参与先天性VSD.

Objective To identify the GATA4 gene mutation of congenital ventricular septal defect （VSD） and study the molecular mechanism of a novel mutation. Methods The clinical data and blood samples from 185 unrelated subjects with congenital VSD were collected and evaluated together with 200 healthy individuals. The coding exons and the flanking intron regions of the GATA4 gene were amplified by PCR and sequenced using the di-deoxynucleotide chain termination approach. The GATA4 gene was cloned and the corresponding mutant was acquired by site directed mutagenesis. The recombinant plasmid expressing GATA4 and the reporter vector expressing enhanced green fluoresence protein （EGFP） driven by the promoter of atrial natrium peptide （ANP） gene were transfected into HeLa cells with Lipofectamine. The effect of mutated GATA4 gene on the transcriptional activity of encoded transcriptional factor was analyzed by reverse transcription （RT）-PCR. Results A novel heterozygous missense GATA4 mutation, c. 191G〉A was identified in 1 VSD patient. The mutation leads to glycine to glutamic acid change at amino acid residue 64 （G64E） in the GATA4 protein. Functional analysis showed that GATA4 G64E mutation decreased the transcriptional activity of GATA4 transcriptional factor. Conclusion A novel heterozygous missenseGATA4 mutation, G64E, was identified in 1 VSD patient. The mutation might cause VSD by impairing the transcriptional activity of GATA4 transcriptional factor.