单纯性圆锥动脉干畸形患儿GATA-6基因突变筛查及突变体蛋白功能研究

Screening and functional analysis of GATA-6 mutations in patients with nonsyndromic conotruncal defects

目的探讨转录因子GATA-6在单纯性圆锥动脉干畸形中的分子遗传机制,旨在为单纯性圆锥动脉干畸形的早期预防及遗传咨询提供支持。方法选取32例散发单纯性圆锥动脉干畸形患儿及100例健康者,应用聚合酶链反应(PCR)方法扩增转录因子GATA-6基因的7个外显子编码区及两侧部分内含子区,PCR产物纯化后ABI Genetic Analyzer 3100型DNA测序仪自动测序,所得结果与美国国立生物技术信息中心(NCBI)数据库中GATA-6基因序列进行比对,并且与健康对照组比较,观察转录因子GATA-6基因突变情况;以野生型pcDNA3.1-GATA-6为模板,用基因定点突变方法构建突变基因型真核表达载体GATA-6-G245R;野生型GATA-6、GATA-6-G245R和心房利钠因子荧光素酶(ANF luciferase)报告质粒共转染HEK 293T细胞,并以共转染的CMV-LacZ为内参,荧光素酶和半乳糖苷酶活性在转染24 h后用发光计测定并检测下游ANF-Luciferase报告基因的活性。结果在1例右心室双出口患儿中发现了转录因子GATA-6的错义突变,突变位于GATA-6基因第2外显子区c.733G>C,突变使第245位的甘氨酸(Gly)被精氨酸(Arg)取代(p.Gly245Arg,G245R)。pcDNA3.1(+)-GATA-6突变体真核表达质粒经酶切及正反2个方向测序验证构建成功;通过对luciferase荧光素酶报告基因活性的检测发现相比较野生型GATA-6和GATA-6-G245R突变基因转录活性下降。GATA-6-G245R与野生型GATA-6比较下降41.3%,二者比较差异有统计学意义(P<0.001)。结论转录因子GATA-6突变与单纯性圆锥动脉干畸形发生相关,转录因子GATA-6基因可能是单纯性心脏圆锥动脉干畸形患者的易感基因。

Objective To explore the molecular and genetic mechanism of transcription factor GATA-6 in nonsyndromic conotruncal defect(CTD)in order to provide evidence for early prevention and inheritance consultation of CTD.Methods A total of 32 cases of patients with nonsyndromic CTD and 100 healthy individuals were enrolled in the study.A total of 7 exons and bilateral partial intron-exon boundaries of GATA-6 were amplified by means of polymerase chain reaction(PCR).The PCR products were purified and directly sequenced by using an ABI Genetic Analyzer 3100 Automatic DNA sequence equipment.The acquired GATA-6 gene sequence was compared with standard gene sequence published in National Center for Biotechnology Information database,as well as the healthy control group to observe the GATA-6 gene mutations.The mutations were introduced into pcDNA3.1(+)by site-directed mutagenesis PCR on the basis of pcDNA3.1(+)-GATA-6 in order to generate the GATA6-G245R mutant constructs.Wild type GATA-6,GATA-6-G245R and atrial natriuretic factor-luciferase(ANF-luciferase)were cotransfected into HEK 293T cells in vitro,and the CMV-LacZ were cotransfected as internal reference.Luciferase and galactosidase activity were measured by using luminometer 24 h after transfection and detected in the downstream ANF-luciferase reporter gene.Results A heterozygous missense mutation in the GATA-6 gene was identified in a patient with double outlets of the right ventricle.The mutation was located in Gly245Arg(G245R)in exon 2 of GATA-6.The mutation of pcDNA3.1(+)-GATA-6 expression vectors were successfully constructed.Through the detection of luciferase reporter gene activity,it was found that GATA-6-G245R and wild-type GATA-6 decreased by 41.3%,and the comparison between them was statistically significant(P<0.001).Conclusions Transcription factor GATA-6 gene mutation is associated with the occurrence of nonsyndromic CTD.Transcription factor GATA-6 gene may be susceptible gene in human nonsyndromic CTD.