第一部分：基于前瞻性抗磷脂综合征队列的临床表型及聚类分析研究: 目的：本研究旨在系统描述抗磷脂综合征（Antiphospholipid syndrome, APS）群体的临床表型，探索抗磷脂抗体谱的诊断效能和临床意义。同时通过聚类分析对抗磷脂抗体（Antiphospholipid antibodies, aPLs）持续阳性人群进行分群，以期为APS分层管理和预后评估提供依据。方法：本研究为一项单中心、前瞻性队列研究，序贯入组2012年至2023年在北京协和医院风湿免疫科门诊规律随诊的疑诊抗磷脂综合征的患者作为实验组，设立系统性红斑狼疮（Systemic lupus erythematosus，SLE）对照组和健康对照（Healthy control, HC）组，收集基线和随访信息，用国际认可的市售试剂盒检测包括抗β2糖蛋白I-结构域I抗体（Anti-β2-glycoprotein I Domain 1 antibodies, aβ2GPI-D1）、抗磷脂酰丝氨酸/凝血酶原抗体（Antiphosphatidylserine/prothrombin antibodies，aPS/PT）、抗膜联蛋白A5抗体（Anti-annexin A5 antibodies, aANX-A5）、抗磷脂酸抗体（Anti-phosphatidic acid antibodies, aPA）、抗磷脂酰肌醇抗体（Anti-phosphatidylinositol antibodies, aPI）在内的标准外抗磷脂抗体，描述并比较各组的临床表现、aPLs抗体分布情况，评估aPLs抗体谱的诊断效能以及与临床表型的相关性。对经典aPLs持续阳性的人群进行层析聚类分析和Kaplan-Meier生存分析，以比较各类人群的基线和预后差异。结果：本研究共纳入717人，其中实验组491例，SLE对照组137例，HC组89例。所有检测的aPLs中，aPS/PT-IgG/IgM为阳性率最高的标准外抗体，实验组的整体阳性率为50.7%，血清阴性APS（Seronegative antiphospholipid syndrome, SNAPS）患者的阳性率为27%。aβ2GPI和aCL与aPS/PT联合检测可将诊断准确度提升至94.4%。狼疮抗凝物（Lupus anticoagulant，LA）依旧是血栓事件的最强预测因子，而aβ2GPI-D1、aPS/PT、aPA、aPI均显示出与标准外表现、微血管病变的相关性。层次聚类分析确定了aPLs持续阳性的547名患者的四个亚类：第一类人群 （n=124, 22.7%）代表存在多种心血管高危因素的男性APS患者，第二类人群（n=139, 25.4%）对应妊娠型APS的已知表型，第三类人群（n=202, 36.9%）代表具有高危和高滴度经典aPLs、多种标准外抗体阳性且有标准外临床表现的患者，第四类人群（n=82, 15.0%）代表了以早产、LA和aPS/PT阳性为特点的女性继发APS患者。结论：标准外抗体，特别是aPS/PT-IgG/IgM可为APS诊断提供附加价值，并有助于识别SNAPS以及评估APS患者发生标准外表现的风险。经典aPLs阳性患者可被聚类为四种呈现不同表型和预后的群体。多种标准外表现同时携带多种标准外抗体可能提示合并SLE等其他自身免疫性疾病可能，这类患者除抗凝治疗外还需根据病情予以必要的免疫调节治疗。对于孤立LA阳性的继发APS患者，建议额外检测aPS/PT-IgG/IgM，并注意在女性妊娠期间密切监测，警惕妊娠高血压等并发症。此外，抗凝治疗应在充分筛查心血管危险因素的基础上进行。 第二部分：基于全外显子组测序探索抗磷脂综合征的遗传风险位点目的：本研究旨在基于全外显子测序技术，在大样本抗磷脂综合征（Antiphospholipid syndrome, APS）队列中探索与APS易感性和特定表型有关的遗传风险因素。方法：本研究纳入了2012年至2023年在北京协和医院风湿免疫科门诊规律随诊的确诊原发性APS的患者，对其外周血进行了全外显子组测序，以诺禾-中华数据库样本作为健康对照，基于低频有害突变筛选策略筛选位点，并通过Burden分析得到与患病相关的候选基因进行通路富集和表型关联，最终筛选关键基因进行实验验证。结果：共对299例患者进行了全外显子组测序，使用2827例诺禾-中华数据库样本作为健康对照，Burden分析共得到11710个在实验组存在低频有害突变的基因，其中1569个基因的低频有害突变在APS组富集（P＜0.05且OR＞1），有30个基因的富集程度可达到全外显子组显著水平（P＜4.27×10-6且OR＞1），42个基因的低频有害突变与APS特定表型有关联。Burden分析和已知基因与疾病的相关性分析均可富集到细胞外基质-受体相互作用通路和PI3K-Akt信号通路，其中SH2B衔接蛋白3（SH2B Adaptor Protein 3，SH2B3）基因与血栓和自身免疫相关，且参与PI3K-Akt信号通路。共发现该基因的9种低频有害突变（Burden分析P = 7.43×10-5），携带突变的14位患者中三种经典抗磷脂抗体阳性的比例较其他患者显著增加（71.4% vs 40.0%，P = 0.020）。验证阶段发现SH2B3 R562Q突变型患者外周血中性粒细胞SH2B3的转录、表达水平均较健康对照组显著降低，而使用APS患者来源的血清或IgG组分刺激健康对照外周血的中性粒细胞可降低其SH2B3的表达。结论：细胞外基质-受体相互作用通路和PI3K-Akt信号通路可能与APS发病相关，SH2B3基因突变可能是APS的遗传风险因素，抗磷脂抗体可能通过影响SH2B3的表达量发挥效应，但以上结论仍需要在细胞系和动物实验中进行验证。

Part I: A single-center, prospective cohort study of clinical phenotypes in antiphospholipid syndrome: Objective: We aimed to systematically describe the clinical phenotype of the antiphospholipid syndrome (APS) in Chinese population, and explore the diagnostic efficacy and clinical significance of antiphospholipid antibodies (aPLs). We also aimed to identify clusters among aPLs-positive patients in order to provide evidence for stratified management and prognosis assessment of APS. Methods: This was a single-center, prospective cohort study. APS-suspected patients presented to Peking Union Medical College Hospital from 2012 to 2023 were included as the experimental group, compared with systemic lupus erythematosus (SLE) control group and healthy control group. Baseline and follow-up information were collected. Anti-β2 glycoprotein I-domain I antibody (aβ2GPI-D1), antiphosphatidylserine/prothrombin antibodies (aPS/PT), anti-annexin A5 antibodies (aANX-A5), anti-phosphatidic acid antibodies (aPA) and anti-phosphatidylinositol antibodies (aPI) was detected with internationally-recognized commercial kits. The aPLs profile among groups, the diagnostic efficacy of aPLs and its correlation with clinical manifestations were assessed. Hierarchical cluster analysis and Kaplan-Meier survival analysis were performed to identify clusters among aPLs-positive patients and differences in baseline characteristics and prognosis.Results: A total of 717 people were included in this study, including 491 patients in the experimental group, 137 SLE patients, and 89 healthy controls. Anti-PS/PTIgG/IgM was the extra-criteria aPL with the highest detection rate in each group, with the overall positive rate of 50.7% in the experimental group and 27% in seronegative APS patients (SNAPS). The combination of aβ2GPI-IgG/IgM, aCL-IgG/IgM and aPS/PT-IgG/IgM provided a diagnosis accuracy of 94.4%. The combination of aβ2GPI and aCL with other extra-criteria aPLs also improved the diagnostic efficiency. LA was still the best predictor of thrombotic events, while aβ2GPI-D1, aPS/PT, aPA, and aPI showed correlations with extra-criteria manifestations and microangiopathy. Hierarchical cluster analysis identified four clusters among 547 aPLs-positive patients. Cluster 1 (n = 124, 22.7%) represented male patients with multiple cardiovascular risk factors. Cluster 2 (n = 139, 25.4%) represented the wellknown phenotype of obstetric APS. Cluster 3 (n = 202, 36.9%) represented patients with the high-risk and high-titer aPLs profile, with multiple extra-criteria aPLs and extra-criteria manifestations. Cluster 4 (n = 82, 15.0%) represented female patients with secondary APS, who were characterized by premature birth and positivity of lupus anticoagulant (LA) and aPS /PT-IgG/IgM.Conclusion: Extra-criteria antibodies, especially aPS/PT-IgG/IgM can provide additional value for the diagnosis of APS, the identification of SNAPS and prediction of extra-criteria manifestations. Four clusters can be identified in aPLs-positive patients with different phenotypes and prognosis. Extra-criteria manifestations with positivity of multiple extra-criteria aPLs may indicate underlying SLE, for which immunosuppressive therapy besides anticoagulation may be necessary. Anti-PS/PTIgG/IgM should be detected in secondary APS patients with isolated LA positivity, of whom attention should be paid to their pregnancy process for preventing gestational hypertension and other complications. Anticoagulant therapy should be carried out after adequate screening of cardiovascular risk factors. Part II: Genetic risk factors for antiphospholipid syndrome based on whole exome sequencing: Objective: We aimed to explore the genetic risk factors of APS susceptibility and clinical phenotypes. Methods: We included patients who were diagnosed with primary APS and regularly followed up in the Peking Union Medical College Hospital from 2012 to 2023. Whole exome sequencing (WES) was performed on their peripheral blood, and individuals from the Novo-Zhonghua database were included as healthy controls. Rare and potential deleterious variants were screened. Gene-based burden analysis was performed to identity candidate genes. Pathway enrichment and phenotypes correlation analysis was carried out with these genes. The hub gene was screened for experimental verification. Results: Two hundred and ninety-nine patients were included in the sequencing. A total of 2,827 individuals from the Novo-Zhonghua database were included as healthy controls. A total of 11,710 genes were identified by burden analysis, which had at least one case in patients with a potential deleterious genetic variant. The APS group was significantly enriched for variants of 1569 genes (P ＜ 0.05 and OR ＞ 1), of which 30 genes (P ＜ 4.27×10-6and OR ＞ 1) had an exome-wide significant enrichment. Forty-two genes were found associated with APS phenotypes.Extracellular matrix-receptor interaction pathway and PI3K-Akt signaling pathwaywere both enriched in burden analysis and the enrichment analysis of known APSrelated genes. The SH2B adapter protein 3 (SH2B3) gene (Burden P = 7.43×10-5) was related to both autoimmunity and thrombosis, and involved in PI3K-Akt signaling pathway. Fourteen patients carrying 9 distinct rare and deleterious mutations in SH2B3 was identified and showed higher proportion of triple positivity of classic aPLs than other patients (71.4% vs 40.0%, P = 0.020). Compared with health control, the transcription and expression levels of SH2B3 was lower in neutrophils from SH2B3 R562Q mutation carriers. Stimulation with serum or IgG components from APS patients led to a decrease in SH2B3 expression in neutrophils. Conclusion: Extracellular matrix-receptor interaction pathway and PI3K-Akt signaling pathway may be related to the pathogenesis of APS. SH2B3 represents a novel genetic locus associated with APS and triple positivity of classic aPLs.Antiphospholipid antibodies may exert its effect by affecting the expression of SH2B3. Cell lines experiments and in-vivo experiments should be carried out to validate the results.