NOS1AP基因突变上调心肌细胞NO触发不明原因心性猝死的分子机制研究

Unexplained sudden cardiac death (USCD) remains to be an important challenge for judicial identification because there are no clear pathogenic mechanism and well-established diagnostic criteria. We and others have shown that NOS1AP gene mutation is associated with USCD. More recently, we obtained initial evidence that inhibition of NOS1AP gene expression increases nitric oxide (NO) levels in cultured myocardial cells, leading to caveolin-1 (Cav-1) translocation and membrane potential change. These data lead to the formation of our central hypothesis that NOS1AP gene mutation up-regulates NO to trigger USCD. We have proposed the following specific aims to test our hypothesis: 1) to delineate the nature of NOS1AP gene mutation through large scale epidemiological screening; 2) to determine which mutation of NOS1AP gene plays a role in USCD by assessing the effects of NOS1AP gene mutation on Cav-1 translocation, membrane potential and potassium channel in vitro cultured cardiomyocytes as well as its effects on cardiac electrophysiology, cardiac conduction, heart rate and the rate of sudden cardiac death (SCD) in mice carrying NOS1AP gene mutation created by CRISPR/CAS9; 3) to determine whether NO plays a key role in NOS1AP gene mutation-induced USCD via decreasing NO levels in cultured cardiomyocytes as well as in mice carrying NOS1AP gene mutation and repeating the experiments in Aim 2. The completion of this study is expected to identify a new molecular marker that can be used to identify people at high risk for USCD and thus will help preventing sudden cardiac death as well as help with judicial identification for USCD via providing a reliable diagnostic biomarker.

不明原因心性猝死（USCD）缺乏明确的致病机制和诊断标准，是司法鉴定的热点难题。他人及我们前期研究发现NOS1AP基因突变与USCD相关，预实验显示抑制NOS1AP基因表达上调心肌细胞一氧化氮（NO）水平，导致窖蛋白Cav-1移位和细胞膜电位异常。因此，我们推测NOS1AP基因突变上调NO在触发USCD中起关键作用。本研究拟通过大样本流行病学调查筛选NOS1AP基因突变位点，采用CRISPR/CAS9定点突变NOS1AP基因，检测突变细胞Cav-1、膜电位、K+离子通道，以及突变小鼠心脏电生理，心脏传导、心律和心性猝死发生率等指标，确定导致USCD的关键NOS1AP基因突变位点。同时，降低NOS1AP基因突变细胞和小鼠NO水平，检测上述指标，确定NO在心性猝死中的关键作用。本课题预期筛选到USCD分子标志物，以便早期预警和干预高危人群，为预防心性猝死及司法鉴定USCD提供分子病理学依据。

不明原因心性猝死（USCD）在心脏性猝死（SCD）中占10~15%，因尸体解剖及毒物分析均阴性，容易被怀疑为暴力死亡而引发纠纷及上访。本课题组在前两项国家自然科学基金的基础上，发现部分USCD案例存在心脏传导系统异常。离子通道变异可以解释小部分USCD死亡原因，大部分USCD死亡原因仍不明。通过对USCD分组(伴有冠状动脉疾病组，不伴有冠状动脉疾病组）的实验研究发现，两组猝死中存在NOS1AP基因突变，且大部分变异同源。本研究进一步通过分子病理学筛查获得中国汉族人群心脏性猝死病例之CAV1及CAV3基因遗传变异的数据，初步发现：1.CAV基因变异可能是部分不明原因心性猝死的分子病因；同时证实了Cav-1主要表达于心肌细胞膜，其表达量显著降低可能是不明原因猝死区别于其他死因（冠心病猝死、非心因性死亡）的特殊分子表征。2.对中国人群中KCNQ1OT1基因编码区的STR多态性进行检测，发现其与冠心病猝死的易感性存在显著关联，提示KCNQ1OT1等位基因10-10是冠心病潜在的易感基因；3. Cav-1 和 KCNQ1存在相互作用参与SCD的发生。因USCD样本量较少，我们与各地高校、公检法系统合作，广泛收集标本。上述研究结果从CAV1基因变异角度预示USCD发病机制，为猝死案件的司法鉴定提供科学依据，也为冠心病及SCD防治工作提供基础性研究资料。本课题共发表论文11篇（其中SCI论文7篇，中文核心期刊4篇），并将陆续投稿；作为主要作者编写了GA/T170— 2019《法医学 猝死尸体检验规范》，该规范于2020年5月出版；作为副主编撰写了《高级法医学》教材。培养硕士生6名，博士研究生1名。项目投入经费58万，支出30.2519万，剩余经费27.7481万元。剩余经费计划用于本项目研究后续支出。