MESP2在心脏发育及先天性心脏病发生中作用机制的研究

Congenital heart disease (CHD) is one of the most seriously endangering congenital defects, the molecular mechanisms of which still need further elucidation. The MESP family genes are among the most inspiring transcription factors to play vital roles in embryonic development. Mesp1 is the earliest molecular marker expressed in the heart precursor cells, acting as a master regulator of multipotent cardiovascular progenitor cells migration and specification. And Mesp2 has been reported to play a major role on embryonic somitogenesis. Although a subset of previous studies have pointed that MESP2 may be essential in embryonic heart development, convincing evidence was still needed. In preliminary experiments, we identified the relatively high expression of MESP2 in human embryonic heart. Over-expressed MESP2 could activate the endogenous NKX2.5 expression in 293T cells just as MESP1. Meanwhile, two loss-of-function mutations of MESP2 were found in our CHD patients resulting in the repression of trans-activation on NKX2.5 endogenous expression. To further illustrate the regulatory role and mechanisms of MESP2 in heart development, the following experiments will be performed: (1) MESP2 mutation identification in more CHD patients and functional analysis of mutated proteins; (2) the establishment of MESP2 knock-out and mutation knock-in animal models to identify the regulation mechanisms of wild-type and mutant MESP2 proteins in heart development, as well as MESP2-MESP1 interaction. This study is expected to not only intensify our understanding of gene regulatory mechanisms underlying heart development and CHD pathogenesis, but also provide evidences for early diagnosis, genetic counseling and treatment for congenital heart disease.

先天性心脏病居出生缺陷首位，其发生机制尚远未阐明。MESP家族在胚胎发育过程中起重要作用，MESP1是心脏祖细胞最早的标记基因，调控生心区细胞迁移和分化；MESP2研究主要集中于体节发育。既往研究提示MESP2在心脏发育中起重要作用，但其作用机制未明确。本课题组前期研究显示MESP2在人类胚胎心脏发育过程中表达，能够反式激活NKX2.5的表达；先心病患儿中发现2例MESP2基因有害突变，突变体蛋白对NKX2.5启动子及其内源性表达的激活作用减弱。为进一步阐明MESP2在心脏发育及先心病发生中的作用，本课题首先筛查先心病中MESP2突变，分析突变对蛋白功能的影响；并建立MESP2基因敲除及突变敲入小鼠模型，分析野生型及突变型MESP2蛋白在心脏发育及先心病发生中的作用机制，并探索其与MESP1之间的相互作用。本研究将加深对心脏发育和先心病发生机制的认识，为先心病的早期诊断和治疗提供基础。

本课题对601名CTD患儿进行MESP2靶向基因测序，发现了4个可能致病的变异，这些变异在400名健康对照中并未检出。通过RT-qPCR、免疫荧光和免疫组化，我们首先证明了MESP2 / Mesp2在不同发育阶段的人和小鼠胚胎的OFT中表达。通过在HEK 293T、HL-1、JoMa1和原代胚胎神经嵴细胞上的一系列体外功能实验，我们发现G116R，Y307X和Q20L变异蛋白对MESP2下游靶基因MYOCD、GATA4、NKX2.5和CFC1转录激活的调节功能出现异常，并通过上调p21cip1或下调Cdk4来抑制CNCC增殖。因此，我们认为MESP2变异c.346G>C (p.G116R)、c.921C>G (p.Y307X) 和c.59A>T (p.Q20L)可能是CTD的致病原因。基于MESP2在早期心脏发育中的重要性以及变异对MESP2功能的影响，本次研究发现了在OFT发育过程中以MESP2为中心的调节网络，为CTD病因学的阐明提供了新的思路。此外，新致病变异的发现也将为遗传咨询和诊断提供一定的依据。

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