背景：主动脉夹层是一种危及生命的疾病，是导致心血管猝死的主要原因之一。尽管已经发现了一些与主动脉夹层相关的易感基因，但许多患者的遗传病因仍然未知。虽然单核苷酸多态性是目前遗传学研究中的主要工具，但基因拷贝数变异（Copy number variations，CNVs）在许多疾病的遗传学研究中也发挥着重要作用。然而，目前很少有研究探讨CNVs对主动脉夹层的贡献。因此，通过全基因组测序，本研究旨在探索CNVs在散发性胸主动脉夹层中的作用，以期进一步了解该疾病的遗传学机制。

方法：为了探索散发性主动脉夹层的新遗传机制，首先在发现队列中招募了100名散发性A型主动脉夹层患者和132名对照组进行全基因组测序，以寻找新的CNVs。为了验证这些新的CNVs是否与散发性A型主动脉夹层相关，纳入了三个独立队列，包括171例散发性A型主动脉夹层患者、157例散发性B型主动脉型夹层患者和886例健康对照，并通过多重连接探针扩增法来评估这些CNVs的致病性。通过免疫印迹检测主动脉夹层患者主动脉组织中基因的表达。为了进一步探究筛选到的CNVs涉及的基因是否参与了主动脉夹层的发生和进展，利用CRISPR/Cas9技术构建了基因敲除小鼠，并通过β氨基丙腈 （β-aminopropionitrile，BAPN）或PCSK9联合II型血管紧张素（Angiotensin II，AngII）诱导不同类型的主动脉夹层小鼠模型。收集小鼠主动脉进行苏木素-伊红染色和弹力蛋白Van Gieson染色评估主动脉夹层的病变程度。通过TUNEL染色法检测主动脉血管平滑肌细胞（Vascular smooth muscle cells，VSMCs）的凋亡水平。通过免疫荧光检测主动脉组织VSMCs凋亡和表型转换相关分子的表达水平。分离基因敲除或对照组小鼠的主动脉VSMCs，用BAPN（1mM）或AngII（1 μmol/L）+氧化低密度脂蛋白（Oxidized low-density lipoprotein，ox-LDL）（150 ng/ml）处理，通过流式细胞术检测细胞凋亡情况。通过免疫印迹检测凋亡相关蛋白和VSMCs表型转换相关蛋白的表达水平。RNA测序探究基因敲除后VSMCs基因表达谱的改变。提取敲除小鼠和对照组小鼠的VSMCs蛋白进行质谱分析，寻找基因的作用靶点。

结果：在发现队列中，鉴定出了4个与散发性主动脉夹层潜在致病相关的CNVs以及相关基因（DSCAM、APP、LINC00907和PROCR）。在三个独立的验证队列中，这4个候选基因均存在显著差异。特别地，与对照组相比，散发性主动脉夹层患者主动脉组织中APP的表达显著降低。进一步构建App基因敲除小鼠，并使用BAPN和PCSK9+AngII诱导主动脉夹层小鼠模型，结果显示App敲除小鼠的发病率和死亡率显著增高，且弹性纤维断裂更加严重。与对照组相比，App缺失促进了VSMCs凋亡标志物（cleaved caspase 3和cleaved PARP）和分泌表型标志物（MMP2、MMP9、I型胶原蛋白和III型胶原蛋白）的表达，同时收缩表型标志物（SM22α/transgelin、α-SMA、calponin-1和smoothelin）的表达水平降低。此外，凋亡细胞数量显著增多。用BAPN和AngII + ox-LDL处理原代小鼠主动脉VSMCs，同样的，App敲除组的原代小鼠主动脉VSMCs与对照组相比表现出更高的凋亡比例，并且凋亡相关蛋白和分泌表型相关蛋白的表达水平显著升高，而收缩表型相关蛋白的表达水平则显著降低。通过RNA测序分析App敲除后VSMCs基因表达谱的改变，发现与先前观察到的VSMCs凋亡和表型改变的结果一致。进一步的质谱分析和免疫印迹显示，Anxa1的表达水平在App敲除的平滑肌细胞中较对照组显著下调，这表明App可能通过调节Anxa1的表达来调控VSMCs的凋亡和表型转换。

结论：通过本研究，我们首次报道了APP相关的CNVs与主动脉夹层相关，并发现APP缺失会通过影响人和小鼠主动脉VSMCs的凋亡和表型转换，从而促进主动脉夹层的发生和发展。这一发现提示APP可能是主动脉夹层的遗传危险因素和潜在治疗靶点，为进一步探究主动脉夹层的遗传学机制和寻找新的治疗方法提供了新的方向和策略。

Background：Aortic dissection is a life-threatening disease and one of the leading causes of sudden cardiac death. Although some susceptibility genes related to aortic dissection have been identified, the genetic causes of the disease in many patients are still unknown. While single nucleotide polymorphisms are the main tool in current genetic studies, copy number variations (CNVs) also play an important role in the genetic research of many diseases. However, there is little research exploring the contribution of CNVs to aortic dissection. Therefore, through whole-genome sequencing, this study aims to explore the role of CNVs in sporadic thoracic aortic dissection, in order to further understand the genetic mechanism of the disease.

Methods：To explore new genetic mechanisms of sporadic aortic dissection, we first recruited 100 patients with sporadic type A aortic dissection and 132 controls for whole-genome sequencing in the discovery cohort to identify novel CNVs. To validate whether these novel CNVs were associated with sporadic type A aortic dissection, we included three independent cohorts comprising 171 patients with sporadic type A aortic dissection, 157 patients with sporadic type B aortic dissection, and 886 healthy controls, and assessed the pathogenicity of these CNVs using multiplex ligation-dependent probe amplification. We also used immunoblotting to detect gene expression in aortic tissue from patients with aortic dissection. To further investigate whether the genes involved in the selected CNVs were involved in the development of aortic dissection, we used CRISPR/Cas9 technology to generate gene knockout mice and induced different types of aortic dissection in these mice using BAPN or PCSK9+AngII. We collected the mice's aortas and assessed the degree of aortic dissection using hematoxylin-eosin staining and Elastin-Van Gieson's staining. TUNEL staining was used to measure the level of apoptosis in vascular smooth muscle cells (VSMCs) in the aortic wall, while immunofluorescence was utilized to examine the expression levels of molecules related to VSMCs apoptosis and phenotypical switch in the aortic tissue. To further investigate the involvement of screened CNV-associated genes in the occurrence and progression of aortic dissection, we isolated VSMCs from target gene knockout or control mice and treated them with BAPN (1 mM) or AngII (1 μmol/L) + ox-LDL (150 ng/ml), and assessed the degree of cell apoptosis using flow cytometry. The expression levels of proteins related to apoptosis and VSMCs phenotypical switch were also detected using immunoblotting. RNA sequencing was performed to explore changes in the gene expression profile of VSMCs after gene knockout, and VSMC proteins were extracted from knockout and control mice for mass spectrometry analysis to identify the target proteins of the genes.

Results：In the discovery cohort, we identified four CNVs and related genes (DSCAM, APP, LINC00907, PROCR) that may be potential pathogenic factors for aortic dissection. These four candidate genes showed significant differences in three independent validation cohorts. Notably, compared to the control group, the expression of APP in the aortic tissue of aortic dissection patients was significantly decreased. Furthermore, we constructed an App gene knockout mouse model and induced aortic dissection using BAPN or PCSK9+AngII. The results showed that the incidence and mortality of aortic dissection were significantly increased in the App knockout mice, and elastic fiber fractures were more severe. Compared to the control group, the loss of App promoted the expression of VSMC apoptosis markers (cleaved caspase 3 and cleaved PARP) and secretion phenotype markers (MMP2, MMP9, type I collagen, and type III collagen), while reducing the expression levels of contraction phenotype markers (SM22α/transgelin, α-SMA, calponin-1, and smoothelin). In addition, the number of apoptotic cells increased significantly. When primary VSMCs from the mice were treated with BAPN and AngII + ox-LDL, the primary VSMCs in the App knockout group showed a higher apoptotic ratio than the control group, and the expression levels of apoptotic-related proteins and secretion phenotype-related proteins were significantly increased, while the expression levels of contraction phenotype-related proteins were significantly reduced. RNA sequencing analysis of changes in the gene expression profile of VSMCs after App knockout showed consistent results with the observed VSMCs apoptosis and phenotypical switch. Further mass spectrometry analysis and immunoblotting showed that the expression level of Anxa1 was significantly downregulated in smooth muscle cells with App knockout, suggesting that App may regulate VSMCs apoptosis and phenotypical switch by regulating the expression of Anxa1.

Conclusions：Through this study, we have reported for the first time the association between APP-related CNVs and aortic dissection and found that the loss of APP promotes the occurrence and development of aortic dissection by affecting the apoptosis and phenotypical switch of VSMCs in both human and mouse aortas. This finding suggests that APP may be a genetic risk factor and potential therapeutic target for aortic dissection, providing new directions and strategies for further exploration of the genetic mechanisms and the development of novel treatments for this disease.