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中国循证儿科杂志

中国循证儿科杂志 ›› 2017, Vol. 12 ›› Issue (6): 457-462.

• 论著 • 上一篇    下一篇

ZFPM2基因的低频变异通过影响GATA4转录调控参与圆锥动脉干畸形的发生

郭晓1,钱琰琰2,肖德勇2,陈洪波2,马晓静1,黄国英1，2,王慧君2   

  1. 1 复旦大学生物医学研究院 上海，200032；2 复旦大学附属儿科医院，上海市出生缺陷重点实验室 上海，201102
  • 收稿日期:2017-11-29 修回日期:2017-11-22 出版日期:2017-12-25 发布日期:2017-12-25
  • 通讯作者: 黄国英，E-mail: gyhuang@shmu.edu.cn；王慧君，E-mail: huijunwang@fudan.edu.cn

Low frequency variant of ZFPM2 contributed to conotruncal abnormality via affecting GATA4 transcriptional regulation

GUO Xiao 1, QIAN Yan-yan 2, XIAO De-yong 2, CHEN Hong-bo 2, MA Xiao-jing1, HUANG Guo-ying1,2, WANG Hui-jun2   

  1. 1 Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China; 2 Shanghai Key Laboratory of Birth Defect, Children's Hospital of Fudan University, Shanghai 201102, China
  • Received:2017-11-29 Revised:2017-11-22 Online:2017-12-25 Published:2017-12-25
  • Contact: HUANG Guo-ying，E-mail: gyhuang@shmu.edu.cn; WANG Hui-jun, E-mail: huijunwang@fudan.edu.cn

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摘要： 摘要 目的：通过Sanger测序，检测圆锥动脉干畸形（CTA）患儿中ZFPM2基因的突变情况，并探讨其致病性及相关分子机制。方法： 对复旦大学附属儿科医院收治的非综合征型CTA患儿的心肌组织标本cDNA进行Sanger测序，检测ZFPM2基因的突变情况。构建ZFPM2、GATA4的表达载体和ANF启动子载体，并将ZFPM2突变型引入表达载体。采用免疫共沉淀（Co-IP）结合Western blotting的方法研究野生型或突变型ZFPM2与GATA4的相互作用。采用荧光素酶报告基因系统研究野生型或突变型ZFPM2对ANF启动子活性的影响。通过mRNA注射研究hZFPM2突变对斑马鱼心脏表型的影响。结果：①对107例非综合征型CTA患儿进行Sanger测序，男64例，年龄1月至14岁（中位年龄8个月）；包括单纯型法洛四联症（TOF）39例，TOF合并卵圆孔未闭（PFO）29例，TOF合并房间隔缺损（ASD）6例，TOF合并PFO和动脉导管未闭（PDA）8例，25例为复杂表型［如房室间隔缺损（AVSD）和左位上腔静脉（LSVC）等］。②在1例TOF合并PFO的患儿中，发现位于ZFPM2基因外显子4的1个杂合错义变异c.397A>Gp.M133V（rs77117583），它仅出现在东亚人群，突变频率为0.3‰，位于ZFPM2蛋白的N端转录抑制结构域，该结构域对于ZFPM2调控的GATA4依赖性转录是必需的。③HEK293T细胞和HL-1细胞中的Co-IP结果均表明ZFPM2发生M133V突变后与GATA4的结合增强。④荧光素酶报告基因实验：人胚肾细胞株HEK293T和小鼠心肌细胞株HL-1中的结果均表明，ZFPM2发生M133V突变后，其抑制GATA4对ANF的转录激活的作用增强。⑤mRNA注射斑马鱼胚胎后心脏发育畸形情况：未注射的对照组、WT组和M133V突变体组心脏畸形发生率分别为11.3%、15.0%和27.5%，M133V突变体组高于其他两组（P＜0.05）。心脏异常表现为：心脏的环化发生异常，心房和心室与正常心脏呈镜像。结论：在1例CTA患儿中检测到1个ZFPM2低频变异c.397A>G, p.M133V，它可能通过影响ZFPM2与GATA4的结合及GATA4转录调控来参与CTA的发生。

Abstract: AbstractObjective: To detect ZFPM2 mutations in conotruncal abnormality (CTA) patients by Sanger sequencing and study its pathogenicity and related molecular mechanism in CTA.Methods: Sanger sequencing was performed to detect ZFPM2 mutations in myocardial tissues of non-syndromic CTA patients in Children's Hospital of Fudan University. Plasmids were constructed, including expression constructs encoding GATA4, ZFPM2 wild type(WT) or variant and a reporter construct containing ANF promoter. The co-immunoprecipitation (Co-IP) and luciferase reporter gene assays were performed to study the change of interaction between ZFPM2 and GATA4 or the effect on ANF promoter activity in ZFPM2 WT or mutant. Injection of mRNA was performed to determine the effect of hZFPM2 mutant on cardiac phenotypes of zebrafish.Results: 107 non-syndromic CTA patients including 64 males and 43 females, aged from 1 to 168 months (median 8 months), were sequenced. Among them, there were 39 tetralogy of Fallot (TOF), 29 TOF with patent foramen ovale (PFO), 6 TOF with atrial septal defect (ASD), 8 TOF with PFO and patent ductus arteriosus (PDA), and other 25 multiple phenotypes, such as atrioventricular septal defects (AVSD) and left superior vena cava (LSVC). A heterozygous missense variant c.397A>G, p.M133V (rs77117583) was found in a CTA patient with TOF and PFO, which was located in the exon 4 and N-terminal transcriptional repression domain with 0.3‰ mutation frequency in Asian population. The Co-IP and luciferase reporter assays in HEK293T and HL-1 cells revealed that the M133V variant protein enhanced significantly the interaction between ZFPM2 with GATA4. And M133V mutant displayed a higher repression for GATA4-mediated transcriptional activation of ANF. Injection of hZFPM2 mRNA in zebrafish embryos demonstrated that the rate of cardiac abnormalities in M133V group(27.5%) was higher than control group without injection(11.3%) and WT group(15.0%), P＜0.05. The cardiac malformations were observed, including abnormal looping and the ventricle and atrium displayed the complete mirror image of normal.Conclusion: A low frequency variant of ZFPM2 c.397A>G, p.M133V was detected in a CTA patient and it was involved in the pathogenesis of CTA by affecting the combination with GATA4, and GATA4 transcriptional regulation.