左室致密化不全心肌病新致病基因功能研究

Left ventricular non-compaction cardiomyopathy (LVNC) is a cardiomyopathy characterized by prominent left-ventricular trabeculations and deep recesses, which is thought to be due to an arrest of the regular process of intrauterine endomyocardial development. As a consequence, LVNC may manifest as congestive heart failure, arrhythmia, thromboembolic events, or sudden cardiac death. By applying whole exome sequencing, we identified the causal variants in the following novel cardiomyopathy genes linked to LVNC: FAM177A1, ASB2 and CHD8. These genetic studies are not only rapidly advancing the diagnosis of LVNC, but illuminating the potential molecular mechanism in pathogenesis of LVNC. FAM177A1 and ASB2 have been confirmed to be involved in cardiac development and pumping functions by our preliminary experiments. Targeted knockdown of FAM177A1 or ASB2 led to failing and dysmorphic hearts in zebrafish. Therefore, the goal of proposal is to investigate the function of these 3 novel LVNC causing genes. The functions of these genes will be studied by using different model systems and a variety of techniques. First, we will study the mutation frequency of 3 genes in a LVNC cohort in our center. Next, mice that have a deletion in FAM177A1 gene or ASB2 in heart will be generated to determine what effects the absence of this gene on disease pathogenesis in vivo. Then, proteins that interact with protein coded by FAM177A1, ASB2, and CHD8 will be identified using biochemical crosslinking techniques and potential signal pathway will be explored in the pathogenic mechanism of LVNC. Knowledge gained from these studies will reveal the roles of these genes in cardiac development and function, open promising new directions in the understanding, and ultimately improve the diagnosis and treatment of this disorder.

左室心肌致密化不全（LVNC）是左室心肌壁致密化心肌变薄，代之以数目众多的肌小梁和深陷交错小梁间隐窝为表现的心肌病。它被认为是心肌在胚胎期正常发育停滞所致。临床表现为心衰，心律失常，血栓事件及猝死。对LVNC家系进行全外显子测序，我们发现3个现有研究均未报道的心肌病致病基因：FAM177A1、ASB2和CHD8。预实验敲低斑马鱼FAM177A1或ASB2基因均导致心衰及心脏畸形。本项目拟研究3个新致病基因突变在LVNC发病机制中的作用。首先收集LVNC临床病例，检测新致病基因的突变率。然后构建FAM177A1或ASB2心脏条件性敲基因小鼠模型，观察胚胎心脏发育及心功能改变。体外培养心肌细胞，质谱分析与上述3个基因编码蛋白相作用的各种蛋白及其可能参与的信号通路。从体内外水平观察上述3基因突变是否参与LVNC发病及其可能信号通路。研究期望进一步完善LVNC病因学，提高疾病诊治水平。

左室心肌致密化不全（LVNC）是一类遗传性心肌病，表现出显著的遗传异质性。除了发现常见心肌病致病基因MYH7，MYBPC3外，我们还发现可能的致病基因包括FAM177A1，RMB20，LMNA，ASB2和CHD8。.本研究首先建立的临床LVNC队列，观察致病基因和临床表型间的关系。在肥厚型心肌病合并LVNC的队列中，观察到主要致病基因为MYH7和MYBPC3基因。随访观察发现该心肌病中MYH7基因突变的携带者心血管死亡显著增高。收集到10个LMNA基因突变的患者，发现三个患者呈现为LVNC的心肌病病表现，均携带LMNA的点突变，提示LMNA基因也是造成LVNC的致病基因。并且LMNA点突变比截断性突变携带者预后差。这一不寻常的发现提示在LMNA基因突变造成LVNC和心衰中显性负性突变效应可能发挥作用。所以，LVNC患者检出MYH7基因和LMNA基因突变提示临床预后不良。.之后，选取FAM177A1为研究对象探索其在LVNC中的作用。发现FAM177A1在主动脉缩窄（TAC）的大鼠心衰模型和心衰终末期患者心肌组织中转录和表达均升高，提示FAM177A1和心衰相关。而后，构建了FAM177A1基因敲除的大鼠模型和腺相关病毒在心脏中过表达FAM177A1小鼠模型。敲除FAM177A1，对大鼠心脏结构和功能并无影响。构建TAC心衰模型后，发现敲基因TAC大鼠的生存显著低于野生型TAC大鼠，提示FAM177A1可能是心衰保护因子。同时心脏过表达FAM177A1模型小鼠中，FAM177A1能够改善TAC导致的心肌肥厚。两个模型提示FAM177A1能够抑制心衰关键病理过程心肌肥厚的发生。采用质谱和免疫沉淀发现FAM177A1蛋白能和类胰岛素生长因子2受体(IGF2R)结合。细胞实验发现FAM177A1抑制IGF2R下游P38和JNK两个MAPK激酶的磷酸化抑制心肌肥厚发生。.最后研究选取RMB20作为候选基因，构建了RMB20的R637L突变转基因大鼠，目前正在繁殖中，未来将在该模型上进一步探究RMB20对心衰和LVNC的关系。.LVNC是目前认识不足心肌病，本研究在遗传水平阐明其主要的致病基因以及和临床预后关系。更重要发现FAM177A1突变和LVNC相关，且FAM177A1是能通过抑制IGF2R及其下游MAPK的激活，抑制心衰鼠的心肌肥厚重构，可能成为一个新的心衰治疗靶点。