Han Hua, Chen Yu, Liu Gang, Han Zengqiang, Zhao Zhou, Tang Yin
Department of Cardiovascular Surgery, Peking University People's Hospital, Beijing 100044, P.R. China.
Int J Mol Med. 2015 Jun;35(6):1545-53. doi: 10.3892/ijmm.2015.2178. Epub 2015 Apr 9.
Our previous study indicated that 8 patients from a family with a history of congenital heart disease had simple atrial septal defect (ASD) and carried the same mutation at codon 310 in the GATA4 gene. In the present study, to identify the functional defects caused by this mutation in an in vivo model, the transgene DNA constructs were microinjected into mice to generate a transgenic mouse model. The mice were genotyped using PCR and DNA sequencing. Protein expression was measured by western blot analysis. qPCR was used to determine the copy number of the transgenes. The heart tissue was fixed and sectioned by conventional procedures. The Vevo 2000 system was used to perform echocardiography on the mice. The expression of GATA4 target genes was measured using the real-time PCR system. The incidence of ASD in the heterozygous transgenic mice was found to be greater than that in the wild-type control mice (P<0.05). In addition, the expression of α-myosin heavy chain (α-MHC) in the heart tissues from the homozygous mice was lower than that in the heart tissues from their wild-type littermates (P<0.05). In conclusion, these results suggest that the introduction of GATA4 M310V negatively affects the normal expression of α-MHC. In accordance with previous findings on GATA4 mutation screening and in vitro experiments, this study confirms that GATA4 M310V mutation may lead to the development of the congenital heart defect, ASD.
我们之前的研究表明，来自一个有先天性心脏病家族史的家庭的8名患者患有单纯房间隔缺损（ASD），并且在GATA4基因的第310密码子处携带相同的突变。在本研究中，为了在体内模型中鉴定由该突变引起的功能缺陷，将转基因DNA构建体显微注射到小鼠体内以生成转基因小鼠模型。使用PCR和DNA测序对小鼠进行基因分型。通过蛋白质印迹分析测量蛋白质表达。使用qPCR确定转基因的拷贝数。心脏组织通过常规程序固定和切片。使用Vevo 2000系统对小鼠进行超声心动图检查。使用实时PCR系统测量GATA4靶基因的表达。发现杂合转基因小鼠中ASD的发生率高于野生型对照小鼠（P<0.05）。此外，纯合小鼠心脏组织中α-肌球蛋白重链（α-MHC）的表达低于其野生型同窝仔的心脏组织中的表达（P<0.05）。总之，这些结果表明GATA4 M310V的引入对α-MHC的正常表达产生负面影响。根据先前关于GATA4突变筛查和体外实验的结果，本研究证实GATA4 M310V突变可能导致先天性心脏缺陷ASD的发生。
