WDR62突变导致心脏发育异常的分子机制

Congenital heart disease (CHD) is the most common birth defects in the world, which is also the top one birth defect in China. However, the genetic etiology of CHD remains poorly understood. In our earlier study, genomic DNA samples of Tetralogy Of Fallot patients (one type of CHD) were used to be detected by whole-exome next generation sequencing and SNP/copy number variation chip. After analyzed by bioinformatics and verified by 1000 pair CHD and normal control samples, WD repeat domain 62 (WDR62) was selected as the CHD candidate gene and four novel mutations were found in some CHD patients. Down regulation of wdr62 in zebrafish showed that the heart malformations were found. And spindle in myocardial cell was abnormal after transfected by WDR62 mutation. Based on these pre-experiment, we proposed the project and want to do: ① To confirm the four novel mutations of WDR62 constructed by TALEN could influence the normal development of zebrafish heart; ② WDR62 could be found in cytoplasm. To find and make sure the interaction proteins of WDR62, related signaling pathway, as well as target genes in myocardial cell cytoplasm by immune co-precipitation, mass spectrometry, laser confocal, QPCR and so on; ③ WDR62 could also be found in nuclear. To observe whether the mutations of WDR62 could affect the histone modifications in myocardial cell nucleus, thus impact the expression of target genes for myocardial development by pull down, ChIP–Seq, live cell imaging and so on. Implementation of this project will determine whether WDR62 could play an important role in the development of heart and whether the mutations of WDR62 are the genetic causes of congenital heart disease.

先心病多年来位居出生缺陷首位，遗传和环境相互作用是该病的发病基础，但确切遗传变异所知甚少。课题组对先心病法洛氏四联症进行了全外显子组测序和基因芯片检测，然后在1000余对先心病和对照中验证，首次发现WDR62基因存在4个新突变，下调该基因后的斑马鱼表现出多种心脏异常表型，转染突变体的心肌细胞纺锤体出现异常。项目拟开展：①针对4种WDR62突变体，应用TALEN技术构建转基因斑马鱼，确定各种突变体对心脏发育的影响；②WDR62可定位于胞浆；应用免疫共沉淀、质谱、蛋白共定位等手段，寻找在心肌细胞浆中与WDR62相互作用的蛋白、信号通路及调控的心肌发育靶基因；③WDR62可入核；应用免疫印迹与沉淀、ChIP-Seq等方法，观察突变WDR62是否在心肌细胞核内影响组蛋白修饰并由此影响心肌发育靶基因的表达。项目实施将确定WDR62是否是影响心肌发育的关键基因，其突变是否为先心病的关键遗传学病因。

先天性心脏病（Congenital heart diseases，CHDs）是最为常见的出生缺陷，且遗传度较高。作为一种复杂的多基因遗传病，确切的遗传病因仍是未知的。课题组前期通过对散发的CHD患者进行全外显子测序，发现WDR62存在4个错义突变，wdr62下调会导致斑马鱼胚胎心脏发育异常。在本课题研究中，通过扩大样本对WDR62的编码区及调控区进行多重PCR靶向测序，我们共在1090例CHD患者中发现46例存在WDR62基因变异，突变频率达到4.2%. 之后通过斑马鱼和小鼠模型研究我们发现WDR62的下调或敲除会引起如环化不全、流出道变短、心肌致密化不全和室间隔缺损等与心肌增殖缺陷相关的表型。体外实验证明WDR62下调或突变会造成心肌细胞增殖减慢及纺锤体数量和定位异常。机制上，一方面，我们发现WDR62可以与多种有丝分裂相关蛋白结合，且首次发现WDR62可以与SMC3相互作用通过影响SMC3的定位影响纺锤体的组装。另一方面，通过对野生型及WDR62缺陷的胎鼠心脏组织及细胞系进行RNA-Seq，我们发现WDR62可以影响心脏腔室形成及细胞增殖相关基因的表达。我们的研究首次明确WDR62在心脏发育中发挥重要作用，是CHD的新易感基因，我们的研究成果将有助于CHD的产前诊断及预防。