心房静止致病基因的定位克隆与功能研究

Atrial standstill (AS) is an extremely rare arrhythmia, characterized by the absence of electrical and mechanical activity in the atria. The genetic basis for familial AS is barely known. We identified a large AS family with six affected individuals in China, including two generations in whom structural heart disease or systemic disease was excluded. After reviewed prevous reference, this AS family were found the largest AS family until now. We also identified an rare physiological phenomenon, namely electrical mechanical dissociation, exist in the early stage of AS. In initial research, we successfully identified candidate genes in 17q1.1-17q1.2 through exome capture sequencing and linkage analysis. In the following study, we try to identify the true gene and mutation by sanger sequencing and bioinformatics. Next, we would explore the effect of mutation on myocyte physiology such as apotosis, electrophysiology and mechanical systole by gene clones, adenovirus infection, patch clamp in vitro. In vivo, direct knockout gene mice and transgenic mice were used to study heart development, heart function, and heart eletromechanic function, especially atrial function changes. We hope that this study would improve our understanding of AS and atrial arrhythmia.

心房静止(atrial standstil1，AS)是一种非常少见的心律失常，表现为心房电活动和机械活动的丧失，目前关于AS的病因及发病机制不明确。申请者2年前发现一AS家系，通过心电图，超声心动图等检查明确该家系各成员临床表型，发现该家系是目前已知最大AS家系，并首次发现AS发病早期存在 "心房电机械分离"罕见临床现象。前期研究采用全外显子测序法结合连锁分析技术，将致病基因定位在染色体17q1.1-17q1.2。申请者拟在后续工作中通过Sanger测序、生物信息学等技术明确致病基因及突变类型，并通过基因克隆、腺病毒感染、膜片钳等技术观察突变基因在体外对心肌细胞电生理、机械收缩等功能影响。体内研究，通过构建基因敲除和转基因小鼠，分别观察敲除基因小鼠、转基因小鼠心肌细胞、心房电机械功能影响。该研究成果将阐明AS以及房性心律失常的病因、发病机制具有重大意义。

背景和目标：心房静止（AS）是一种致死性心律失常，主要表现为心房电活动和机械活动的丧失。目前AS的致病机制不明。AS常与遗传有关，但目前致病机制也不清楚。本研究旨在探讨AS的致病基因和发病机制。.方法和结果：我们在临床发现一个大型AS家系，并对其进行了外显子组测序，通过生物信息学分析，定位了一个罕见的错义突变MYL4[c.31G> A（p.E11K）NM_002476和NM_001002841]。该突变与AS家系中疾病完全共分离[LOD= 5.3]。我们通过CRISPR/Cas9介导的基因组编辑技术构建了MYL4敲除大鼠模型及MYL4 p.E11K突变大鼠模型。突变大鼠既敲除大鼠显示明显的心房纤维化和电生理异常，能完全重复我们AS患者的临床表型。我们进一步研究发现心房纤维化主要是由于p.E11K突变导致了心房肌细胞的凋亡和促纤维化信号通路的激活。此外，我们还发现与野生型蛋白相比，MYL4突变蛋白减少肌球蛋白ATPase活性。.结论：这些结果令人信服地支持MYL4[c.31G> A（p.E11K）是AS的一个新的致病基因。该致病位点可以促进心房肌细胞的凋亡，从而明显增加心房纤维化。这些发现提示机械活动的长期功能障碍可能是心房纤维化和心房中电活动缺失的重要原因，为心房静止及心房心肌病的诊疗提供了新的理论基础和实验数据。