SMYD1等第二生心区调控基因变异在右心发育不良综合征发生中的机制研究

Hypoplastic Right Heart Syndrome (HRHS) is one complex Congenital Heart Disease (CHD) characterized by hypoplastic right ventricle; Genetic network composed by transcription factors and signaling molecules regulate cardiac progenitor cells in Second Heart Field (SHF) for right ventricular development. SHF gene knockout animals have similar cardiac phenotype to human HRHS, however, the pathogenesis of HRHS in human is still unknown. According to our next generation sequencing for 17 candidate SHF genes in 16 HRHS patients and data analysis, we found low frequency and common variants in SHF gene both could increase the risk of HRHS. So our hypothesis is that SHF gene variants may take part in occurence of HRHS. Firstly, we are going to validate the relationship of SHF gene variants and HRHS in expansion samples; Secondly, we will investigate the effect of de novol mutation SMYD1 p.G16S in Mef2c-Smyd1-Hand2 pathway on occurrence of HRHS in cell level, then validate in human HRHS cardiac tissue; finally, we will study whether the common variants Foxc2: rs86600430 A＞G could increase risk of HRHS. In our study, we focus on the variants of SHF gene network which regulate right ventricle development, and investigate the pathogenesis of HRHS from 2 aspects: disease-causing mutation and common variants of SHF gene.

右心发育不良综合征（HRHS）是一类以右室发育不良为主要特征的复杂紫绀型先天性心脏病。转录因子和信号分子构成基因网络调控第二生心区（SHF）的心脏祖细胞发育成流出道和右室。SHF基因敲除动物表现明显的右室发育不良，但人HRHS发病机制仍不明。预实验对16个HRHS患儿17个SHF基因二代测序发现，SHF基因的低频变异和常见变异均可增加HRHS的发病风险。因此本课题假设SHF基因变异参与HRHS发生：1.在人群水平扩大样本验证SHF基因变异与HRHS发生的关系；2.在细胞水平探讨新发突变SMYD1 p.G16S影响Mef2c-Smyd1-Hand2通路而导致HRHS发生，并在人HRHS心肌组织中验证；3.在细胞和组织水平探讨常见变异Foxc2: rs86600430 A＞G增加HRHS的发病风险。本课题从致病突变和常见变异两个方面研究调控右室发育的SHF基因网络以探索HRHS基因发病机制。

右心发育不良综合征（HRHS）是一类以右室发育不良为主要特征的复杂紫绀型先天性心脏病。转录因子和信号分子构成基因网络调控第二生心区（SHF）的心脏祖细胞发育成流出道和右室。SHF基因敲除动物表现明显的右室发育不良，但人HRHS发病机制仍不明。本课题研究发现SHF基因变异参与HRHS发生：1. 通过对16例HRHS患者的DNA样本进行IonTorrent测序，并进行生物信息学分析，质谱分析及功能学实验，获得了23个有意义的新发和已知的SNP位点；2、发现6个位点可以显著增加HRHS的发病风险：Fgf10: rs75629618 C＞T, Fgf10: rs10461755 G＞A, Fgf10: rs75632187 A＞G, Fgf10: rs12518964 G＞A, Foxc2: rs34221221 A＞G, and Tbx20: rs59854940 C＞G；3、FOXC2，TBX20做为第二生心区的转录因子调控网络之一，参与了右室发育，其敲除FOXC2和TBX20的小鼠模型表现出明显的右心发育不良表型。通过基因型分析，FOXC2: rs34221221 A＞G中的G和TBX20: rs59854940 C＞G中的等位基因G位点能够显著增加HRHS的发病风险（OR=6.67, 95%CI=2.81-15.86, p=8.81×10 6；OR=3.64, 95%CI= 1.91 - 6.94, p= 3.75×10-5）。luciferase assay 提示位于promoter区的FOXC2: rs34221221 A＞G的突变型能够显著增加FOXC2 mRNA的转录水平，从而增加增加HRHS的易感风险；4、Mef2c→Smyd1→Hand2是一个对右室形成至关重要的SHF基因网络通路。其中敲除Hand2的小鼠中也表现出了严重的右室发育不良。在我们的测序数据挖掘出位于HAND2 promoter区的新发 SNP位点，HAND2 chr4：174451543 C>A，生物信息学分析发现YY1和FOXL1可能对该位点的调节影响HAND2的表达，进而发挥作用，因此后续luciferase assay提示HAND2 chr4：174451543 C>A突变型能够显著降低HAND2 mRNA表达水平，YY1和FOXL1结合在HAND2 chr4：174451543 C