家族性高血压易感基因GATA5的定位克隆及功能研究

Essential hypertension is the most common form of chronic disease in humans, and is associated with substantially increased incidence and mortality of cardiovascular diseases. Genetic defects are a leading cause of essential hypertension. In previous studies, the current research team mapped a novel locus for essential hypertension on human chromosome 20q13.3 by genome-wide scan with microsatellite markers, genotyping and linkage analysis. By sequence analysis of the candidate genes within the positional chromosomal region in the families with essential hypertension, missense mutations in the GATA5 gene were identified. Based on the pivotal roles of GATA5 in the cardiovascular development and regulation of blood pressure, the applicants make a scientific hypothesis that GATA5 is likely to be a novel gene susceptible to essential hypertension in humans. In order to test this hypothesis, the present program will firstly identify new GATA5 mutations predisposing to essential hypertension by sequencing the GATA5 gene in the patients with essential hypertension; secondly the identified GATA5 gene involved in the pathogenesis of essential hypertension will be cloned; thirdly, the functional characteristics of the mutant GATA5 proteins will be deciphered, including transcriptional activation of a target gene measured with a dual-luciferase reporter assay system and the ability to bind to a target gene promoter determined by electrophoretic mobility shift assay; fourthly the subcellular distribution and trafficking of the mutant GATA5 proteins will be observed by laser scanning confocal fluorescence microscopy; finally a mouse model with an equivalent GATA5 mutation knocked in will be generated by targeting of the GATA5 gene in embryonic stem cells and then be studied at levels of molecules, cells and model animals. These findings will not only reveal a novel molecular genetic mechanism underpinning essential hypertension, but also potentially discover a new drug target for the treatment of essential hypertension.

高血压是人类最常见的慢性疾病且大量增加心血管病的发病率和死亡率，遗传缺陷是其发生的重要原因。本课题组在前期研究中通过对高血压家系进行微卫星标记全基因组扫描、基因分型和连锁分析将1个新的高血压易感基因座定位于20q13.3，对该区域的候选基因GATA5进行测序分析发现了错义突变。基于GATA5在心血管发育和血压调节方面的关键作用，申请者提出“GATA5可能是一个新的人类高血压易感基因”这一科学假说。为证实该假说，本研究拟在高血压患者测序筛查GATA5以识别新的基因突变；克隆GATA5基因，应用双荧光报告基因分析和电泳迁移率分析等方法研究突变体的转录激活及其与靶基因启动子的结合力等功能特性，借助激光共聚焦显微镜分析突变体的亚细胞分布，运用干细胞基因打靶技术建立点突变敲入小鼠模型，在分子、细胞和整体水平揭示该基因突变的作用机制。该研究将不仅揭示高血压新的分子遗传机制，还可能发现新的药物治疗靶标。

本课题组在前期研究中通过对高血压家系进行微卫星标记全基因组扫描、基因分型和连锁分析将1个新的高血压易感基因座定位于20q13.3，对该区域的候选基因GATA5进行测序分析发现了错义突变：763C>T和677T>A。对于两种基因突变，我们采用CRISPR-Cas9技术在小鼠上实现这2种基因点突变，实验发现763C>T对应的R262W突变小鼠收缩压无明显改变。而677T>A对应的L233H纯合突变小鼠收缩压较对照组增高17.2%。在机制上，我们检测GATA5/L233H突变的小鼠主动脉组织发现，p-Akt/Akt 和p-eNOS/eNOS水平有着明显下降，提示GATA5/L233H突变诱导高血压的形成可能是通过减弱Akt/eNOS的磷酸化水平来影响NO的释放，进而影响血管的正常舒张，最终形成高血压。. 我们继续对GATA5/L233H突变小鼠进行研究, 发现GATA5/L233H突变在Ang II诱导的高血压模型中促进小鼠收缩压和舒张压的进一步升高，小鼠主动脉和肠系膜动脉血管出现不同程度的血管壁增厚和纤维化加重。GATA5/L233H突变能够加重Ang II诱导的小鼠心脏衰竭，研究发现在不接受Ang II刺激的情况下，GATA5/L233H突变组的EF值，FS值与对照组相比并无明显改变，但是在同等接受Ang II的刺激的情况下，GATA5/L233H突变组的EF值，FS值与相应的对照组相比有着明显下降，心室壁也有着明显的肥厚，心肌纤维化程度有着明显加重。同时我们检测多种参与调节血管舒缩的因子，在生理条件和Ang II刺激情况下GATA5/L233H突变均会导致肝脏组织的血管紧张素原Agt和肾脏组织的血管紧张素转化酶ACE的mRNA水平降低。这提示GATA5/L233H突变可能参与RAAS对于心血管病的影响。. 综上所叙，通过前期临床分析，测序，我们发现了GATA5的2个错义突变：763C>T和677T>A可能会参与血压的调控，继而我们发现其中一种突变677T>A，对应到小鼠层面的L233H突变会引起血压的升高，可能的机制是减弱Akt/eNOS的磷酸化水平。GATA5/L233H突变还参与Ang II诱导的高血压过程中血管壁的增厚和纤维化以及心肌肥厚纤维化的过程。

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