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。

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.