染色体结构维护蛋白3突变影响纺锤体组装导致心脏发育异常的分子机制

Structural maintenance of chromosomes 3 (SMC3) is located on the bipolar spindle in cell mitosis and participates in DNA damage repair and maintenance of genomic stability. We identified four SMC3 mutations in patients with congenital heart disease (CHD) for the first time by whole exome sequencing. Immunohistochemistry showed that the expression of SMC3 was significantly decreased in cardiac diseased tissue of CHD patients. Significant malformations were found in the heart of the smc3 down-regulated zebrafish. Abnormal spindle morphology and distribution were observed in cell division phase transfected by SMC3 mutants. Based on the information above, we hypothesize that SMC3 mutations may cause cardiac deformities by affecting spindle function and want to finish the research works in this project as follows: 1. Constructing four smc3 point mutation zebrafish by CRISPR/Cas9 technology to determine their effect on cardiac development; 2. Obtaining cardiac conditional knockout Smc3 mice to observe the change of heart phenotypes by crossing Smc3fl/fl mice and Myh6-Cre mice; 3. Finding out the target molecules of SMC3 and observing the effect of various SMC3 mutants on these molecules and their downstream molecules. The implementation of the project will illustrate the molecular mechanism of SMC3 mutation leading to cardiac dysfunction and provide a new target for the prevention and treatment of CHD.

染色体结构维护蛋白3（SMC3）在细胞有丝分裂中定位于两极纺锤体，参与DNA损伤修复和维持基因组稳定。我们通过全外显子组测序首次发现先心病患者存在4种SMC3突变，免疫组化显示先心病患者心脏病变组织中SMC3表达明显减少；下调smc3的斑马鱼心脏出现明显畸形；转染SMC3突变体的细胞在分裂相存在纺锤体形态与分布异常。基于此我们推测SMC3突变可能通过影响纺锤体功能导致心脏发育畸形。项目拟完成：1.针对新发现的先心病SMC3突变，应用CRISPR/Cas9技术构建smc3点突变斑马鱼，分别确定它们对心脏发育的影响；2.将Smc3fl/fl小鼠与Myh6-Cre的小鼠交配，获得心脏特异敲除Smc3小鼠，观察心脏表型变化；3.寻找与SMC3结合的蛋白和靶基因，观察各种SMC3突变体对结合分子及下游分子的影响。项目的实施将明确SMC3突变导致心脏发育异常的分子机制，为先心病的防治提供新的靶点。

背景：先天性心脏病（Congenital heart diseases，CHD）是全球最为常见的出生缺陷，具有较高的遗传性。尽管目前已发现了部分易感基因，它们仍远不足以解释所有CHD的遗传病因。.方法：研究共纳入1,146名散发CHD患者。我们在56例CHD患者外周血的全外显子组测序结果中，鉴定出 CHD新的候选基因染色体结构维护蛋白质3（Structural maintenance of chromosomes 3,SMC3）。之后我们在另1,090例CHD患者中，针对SMC3的编码区、剪接位点区及调控区采用多重聚合酶链式反应（polymerase chain reaction，PCR）靶向测序的方法进行了变异检测；构建smc3下调的斑马鱼模型以及Smc3心脏特异性敲除小鼠模型，观察其在心脏发育中的作用；在心肌细胞系HL-1和AC16中检测SMC3缺陷对细胞增殖和纺锤体的影响，并采用RNA-seq、ChIP-qPCR和3C-HTGTS等技术探讨其分子机制。.结果：在1,146CHD患者中，我们发现35例患者携带了SMC3的4个编码区变异及28个非编码区变异。smc3下调的斑马鱼出现心脏严重畸形；心脏特异性Smc3纯合敲除小鼠完全胚胎致死并导致右心室发育不全（73.33%）以及永存动脉干（71.43%）；杂合敲除小鼠出现室间隔缺损合并主动脉骑跨（33.33%）。这些结果表明SMC3对心脏发育不可或缺。细胞研究显示，SMC缺陷可导致心肌细胞增殖抑制和多纺锤体极细胞比例增加。当SMC3下调时，差异表达基因在与心脏发育相关的通路中富集，其中心肌发育和冠状动脉形成相关基因ETS2表达减少。在心肌细胞中，SMC3缺失可以减弱ETS2启动子和超增强子之间的相互作用从而抑制其转录。在Ets2超级增强子敲除的小鼠也出现导致一系列心脏发育异常的表型，包括右心室发育不全、右室双出口以及房间隔缺损。.结论：我们首次鉴定SMC3是CHD新的易感基因，并阐明SMC3通过调控ETS2启动子和超级增强子之间的相互作用参与心脏发育。