目的 探讨先天性房室间隔畸形患者GATA-4基因的突变以及对所发现的突变点进行功能初步分析.方法 选择的50例汉族先天性房室间隔畸形患者,用聚合酶链反应方法扩增GATA-4基因6个外显子编码区和邻近序列后分别直接测序,并与100名同民族健康者比较;利用生物信息学方法预测点突变对GATA-4蛋白结构和功能影响;以野生型pcDNA3.1-GATA4为模板,用基因定点突变的方法构建突变型GATA-4真核表达载体,在体外和ANF报告质粒共转染Hela细胞,48 h后检测下游报告基因的活性.结果 发现GATA-4基因一个新的杂合子H436Y突变,在NCBI的SNP数据库中未见报道,且在100名对照者中也未发现;GATA-4基因编码第436位组氨酸在生物进化过程中处于高度保守;SIFT和PolyPhen程序预测H436Y突变可能影响GATA-4蛋白质的功能;突变型GATA-4 H436Y质粒激活下游报告基因活性明显下降,与野生型GATA-4质粒相比是(53.8±6.6)%,差异具有统计学意义(P<0.01).结论 GATA-4基因H436Y杂合子突变可能是中国汉族先天性房室间隔畸形致病原因之一.%Objective To perform the functional analysis of a novel H436Y mutation of GATA-4 gene identified in Han Chinese patients with congenital cardiac septal defects. Methods Using bioinformatics to predict if the H436Y mutation in the GATA-4 gene affects its protein function. H436Y mutation in the GATA-4 gene was generated by Quick Change Lightning site-directed mutagenesis kit and verified by DNA sequencing. GATA-4-wt or GATA-4-mut DNA was eotransfected into Heta cells with DNA for the luciferase reporter gene atrial natriuretie factor (ANF), and luciferase activity was measured by an LKB luminometer 48 h after transient transfeetion. Results Alignment of the GATA-4 amino acid sequence indicated that the histidine residue at position 436 was conserved, and H436Y mutation in the GATA-4 gene is expected to affect its protein function. The H436Y mutation significantly reduced the transcriptional activation of downstream reporter ANF when compared to wild-type GATA-4 (P<0. 01). Conclusion The mutation c. 1306C → T of the GATA-4 gene impaired the activation of the downstream target, suggesting that the H436Y mutation in the C-terminal region of the GATA-4 gene might prevent its biological function.
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