川崎病是一种急性系统性血管炎，是目前发达国家（地区）儿童获得性心脏病的首要病因，给社会和家庭带来巨大负担。川崎病病因尚不明确，普遍认为是遗传、环境与免疫共同作用的结果。流行病学调查在一定程度上从遗传和环境两个角度阐明了川崎病发病的危险因素，而一碳代谢作为连接营养代谢状态、表观遗传修饰与免疫调节的核心通路，其代谢-免疫调控可能是川崎病新的发病因素。基于此，本研究开展以下三部分内容：1）上海地区2018-2022年川崎病流行病学调查；2）亲代围孕期一碳代谢物暴露与子代川崎病发生的关联分析；3）甲硫氨酸代谢在川崎病冠状动脉损伤中的作用和分子机制。通过结合流行病学研究方法、分子生物及组学技术全面探寻川崎病潜在发病因素及一碳代谢-免疫调节机制，为川崎病预防和早期干预提供科学依据。

第一部分     上海地区2018-2022年川崎病流行病学调查

研究目的

    自1998年以来，上海川崎病研究协作组每5年定期开展一次区域性流行病学调查，本研究为第五次调查。旨在分析2018年至2022年期间，上海地区川崎病的流行病学变化趋势及COVID-19疫情可能带来的影响。

研究对象与方法

    回顾性分析2018年1月1日至2022年12月31日期间在上海地区诊断为川崎病或皮肤粘膜淋巴结综合症（ICD-9-CM 446.1及ICD-10 M30.3）患儿的临床资料。数据主要来自申康医院发展中心建立的川崎病专病数据库，并通过问卷形式予以补充，共收集了上海地区48家提供儿科医疗服务医院的川崎病临床资料。主要研究结局为川崎病的发病率，次要结局包括川崎病一般临床特征及COVID-19疫情期间的变化情况。

研究结果

    共纳入5791例川崎病患儿，其中3549例为上海常住人口。2018-2022年上海地区5岁以下儿童川崎病发病率为61.07～94.84 /10万，平均为80.68 /10万，较上一轮调查有所下降。2020年和2022年上半年川崎病病例数降幅尤为明显，同时季节和月份分布发生改变，与COVID-19疫情防控实施时间重合，且通过就诊间隔时间分布及累计曲线进一步说明发病率下降是实际病例数量的减少。男女比例为1.58:1，发病年龄中位数为25.3月（IQR：14.1–46.1月）。3654例报告治疗相关信息中，3310例接受IVIG治疗，317例（9.58%）为IVIG不应答。4099例报告心脏超声检查结果，其中553例（13.49%）出现冠状动脉病变，191例（4.87%）为中、大型冠状动脉瘤。本次调查未见死亡病例。

小结

    自1998年开展川崎病流行病学调查以来，上海地区川崎病发病率首次呈下降趋势，与居家隔离、停工停学等严格的防控措施相重叠。这些防控措施可能阻断了潜在环境或感染性诱因的传播，从而减少川崎病的发病。

第二部分     亲代围孕期一碳代谢状态与子代川崎病发生的关联分析：

基于SPCC亲子队列的巢式病例对照研究

研究目的

    探讨父母双亲围孕期一碳代谢物水平与子代川崎病发生的风险。

研究对象与方法

    采用巢式病例对照研究。研究对象来自于2016年3月至2023年10月上海地区孕前亲子队列（SPCC），由孕前期和孕早期纳入的两部分人群组成。孕前期对象来自上海地区9个区和江苏省某县级市的孕前检查门诊，孕早期对象来自上海地区三甲产科专科医疗机构及两家区县妇幼保健院的早孕门诊。子代川崎病结局与第一部分上海地区第五次川崎病流行病学调查队列相匹配。纳入时现场收集血样，并收集人口学一般资料、膳食补充剂使用情况、孕期不良并发症等信息。血清同型半胱氨酸（Hcy）、叶酸和维生素B12的测量采用化学发光微粒子免疫法。一碳代谢相关代谢物包括氨基酸代谢物、胆碱代谢谱、叶酸代谢谱及水溶性维生素中19个靶标，采用超高效液相色谱-串联质谱联用法检测。通过Logistic回归分析围孕期一碳代谢物水平与子代川崎病的关系，计算OR值及95% CI作为关联的效应指标。

研究结果

    共计纳入353对围孕期母亲样本，包括53例子代川崎病（24例孕前期，28例孕早期）及300例子代健康（198例孕前期，100例孕早期），以及匹配其配偶185例孕前期父亲样本，包括12例子代川崎病，173例子代健康。孕前期及孕早期叶酸补充比例在川崎病组显著低于对照组（孕前期：10.34% vs 42.44%，P = 0.001；孕早期：51.72% vs 74.75%，P = 0.012），其余一般临床特征无显著差异。部分一碳代谢物在健康对照组与子代川崎病组之间分布存在显著差异，川崎病组母亲围孕期的胆碱水平［9.47 (7.22, 12.24) vs. 7.48 (5.92, 9.79) umol/L，P = 0.002］及S-腺苷同型半胱氨酸（SAH）水平［32.38 (24.17, 46.33) vs. 28.16 (22.03, 40.35) nmol/L，P = 0.046］显著高于对照组，而甲基丙二酸水平显著低于对照组［70.80 (58.67, 97.56) vs. 87.80 (67.00, 116.66) nmol/L，P = 0.003］，其余指标无统计学差异。在孕前期亚组中，胆碱浓度［9.76 (7.22, 15.02）vs. 7.25 (5.75, 9.25) μmol/L, P = 0.003］、甜菜碱浓度［54.86 (43.92, 62.00) vs. 42.47 (35.66, 52.80) μmol/L, P < 0.001］及甘氨酸浓度［313.77 (265.97, 427.00) vs. 264.43 (229.94, 312.16) μmol/L, P = 0.002］在川崎病组显著升高。相较之下，在孕早期亚组中，绝大多数代谢物在川崎病组与对照组间差异不显著。孕前期亚组关联分析中，半胱氨酸可能具有独立作用，其浓度每升高1 μmol/L，子代川崎病风险增加约5.1%（OR=1.051，95% CI: 1.011-1.093，P=0.013），在调整母亲年龄、BMI、血清叶酸、维生素B12与Hcy水平作为协变量后，甲硫氨酸亦达到统计学显著水平（OR=1.052，95% CI: 1.003-1.203，P=0.036）。孕早期父亲一碳代谢物水平在两组间总体差异均不显著。

小结

    孕前期母亲血清中半胱氨酸和甲硫氨酸水平升高与子代川崎病的发生显著相关，提示甲硫氨酸循环紊乱可能在川崎病早期发病机制中发挥重要作用，父源一碳代谢状态在川崎病发病中的作用可能相对有限。

第三部分     甲硫氨酸代谢在川崎病冠状动脉损伤中的作用及分子机制

研究目的

       明确甲硫氨酸代谢在川崎病冠状动脉损伤中的作用及其分子机制。

研究方法

       建立干酪乳杆菌菌壁提取物（LCWE）川崎病小鼠模型和肿瘤坏死因子-a（TNF-a）刺激人冠状动脉内皮细胞（HCAEC）的炎性细胞模型。通过超高效液相色谱-串联质谱联用法检测川崎病急性期患儿和LCWE小鼠血清中甲硫氨酸代谢相关靶标，分析甲硫氨酸代谢在炎性条件下的改变及二者间的相关性；通过Western Blot检测甲硫氨酸饥饿及TNF-a刺激HCAEC中组蛋白修饰表达水平，并通过外源性S-腺苷-甲硫氨酸（SAM）回补，筛选组蛋白修饰位点；通过免疫组化及多重免疫荧光技术检测组蛋白修饰位点H3K79me2在LCWE小鼠中表达水平及血管内皮细胞定位情况；使用H3K79me2抗体在甲硫氨酸饥饿及TNF-a刺激HCAEC细胞中进行ChIP-seq，并进一步探索H3K79me2调控炎性反应的分子机制。

研究结果

       成功建立LCWE川崎病小鼠模型及TNF-a刺激HCAEC炎性细胞模型。在川崎病急性期患儿及LCWE血管炎小鼠模型中普遍存在甲硫氨酸代谢紊乱特征，表现为甲硫氨酸及其代谢产物下降，甲基供体SAM及SAM/SAH比值升高。HCAEC细胞在炎症因子TNF-α刺激联合甲硫氨酸剥夺的条件下，组蛋白修饰位点H3K79me2表达水平显著升高，同时炎症相关蛋白表达上调。免疫组化结果表明，H3K79me2在LCWE小鼠的腹主动脉及冠状动脉中表达显著上调，且与内皮细胞标志物CD31共定位，支持其在血管损伤中的功能参与。ChIP-seq结果表明，在正常代谢条件下H3K79me2 在转录起始位点±3 kb 区域形成典型的富集“尖峰”，而在甲硫氨酸剥夺条件下异常富集于distal intergenic区域。GO和KEGG富集分析，结果显示其下游靶基因在多个与细胞应激与稳态维持密切相关的核心通路中显著富集。ChIP-seq数据结合关键靶基因分析发现H3K79me2在细胞焦亡通路的核心执行因子GSDMD启动子区域具有显著的富集特征，在甲硫氨酸缺乏状态下，该修饰峰大幅减弱，表明甲基供体的供应直接影响H3K79me2在功能位点的定位能力。

小结

    川崎病急性期及LCWE小鼠模型中普遍存在甲硫氨酸代谢紊乱特征，表现为代谢底物Met和tHcy耗竭与甲基供体SAM积聚，提示代谢-免疫之间存在潜在关联。HCAEC中H3K79me2在炎症状态下的表达水平和基因组分布呈现出代谢依赖性，对甲硫氨酸供给具有高度敏感性，并可调控焦亡相关基因GSDMD的表达。

Kawasaki disease (KD) is an acute systemic vasculitis and the most common cause of acquired heart disease in children in developed regions, leading to a significant burden on families and society. Although its etiology remains unclear, KD is widely believed to result from the combined influence of genetic susceptibility, environmental exposures, and immune dysregulation. Epidemiological studies have provided insights into genetic and environmental risk factors; however, one-carbon metabolism, as a central pathway linking maternal nutritional status, epigenetic modifications, and immune regulation, may represent a novel mechanism contributing to KD pathogenesis. Based on this hypothesis, the present study was conducted in three parts: (1) the fifth epidemiological survey of Kawasaki disease in Shanghai; (2) analysis of the association between parental periconceptional one-carbon metabolic exposure and KD risk in offspring using a nested case-control design; and (3) investigation into the molecular mechanisms of methionine metabolism in coronary artery lesions during KD. By integrating epidemiological methods with molecular biology and omics technologies, this study aimed to identify potential etiological factors and elucidate the metabolic–epigenetic–immune interactions underlying KD, thereby providing a scientific basis for the development of early prevention and intervention strategies.

Part I    Epidemiologic Features of Kawasaki Disease in Shanghai from 2018 through 2022

Background

Shanghai Kawasaki Disease Research Group has systematically quinquennial conducted four epidemiological surveys of KD since 1998 once every 5 years. This study was to analysis the epidemiologic trends of KD and the potential impacts of the COVID-19 in Shanghai from 2018 through 2022.

Methods

Medical records of KD patients diagnosed from January 2018 through December 2022 were retrospectively analyzed. Data was based on the Shenkang Kawasaki Disease Specific Database established by Shanghai Shenkang Hospital Development Center. Additional data were collected through questionnaires distributed to 48 hospitals providing pediatric medical care in Shanghai. The primary outcomes were the incidence of KD. The secondary outcomes included clinical features and changes observed during the COVID-19 pandemic.

Results

A total of 5791 cases were enrolled including 3,549 permanent residents of Shanghai. The incidence of KD was 61.07 to 94.84 per 100,000 children aged <5 years from 2018 to 2022. The incidence decreased compared to the last survey, with pronounced declines in the years of 2020 and 2022, periods that coincided with the implementation of COVID-19 containment measures. Male-to-female ratio was 1.58:1. Age at onset was 25.3 (14.1, 46.1) months. Out of 3654 cases with treatment information, 3310 cases were treated with intravenous immunoglobulin (IVIG), of which 317 cases (9.58%) were IVIG non-response. Out of 4099 echocardiogram reports, 553 cases (13.49%) developed coronary artery lesions, and 191 (4.87%) were medium to large coronary artery aneurysms. No death was reported in this survey.

Conclusions

For the first time, the incidence of KD in Shanghai has shown a decline trend. The containment measures implemented during the COVID-19 pandemic, such as citywide suspension of work and school closures, may have affected the incidence of KD.

Part II  Parental Periconceptional One-Carbon Metabolism and Offspring Risk of Kawasaki Disease: A Nested Case-Control Study Based on the Shanghai Preconception Cohort

Background

This study aimed to investigate the association between parental periconceptional one-carbon metabolic status and the risk of KD in offspring.

Methods

A nested case-control study was conducted based on the Shanghai Preconception Cohort (SPCC) from March 2016 to October 2023. Participants were recruited either before conception or during early pregnancy from preconception clinics and early pregnancy outpatient clinics across multiple districts in Shanghai and one county-level city in Jiangsu Province. The KD outcome in offspring was matched with cases from the fifth epidemiological survey of KD in Shanghai. Blood samples and questionnaire data were collected at enrollment. Serum levels of homocysteine (Hcy), folate, and vitamin B12 were measured by chemiluminescent microparticle immunoassay. Nineteen one-carbon metabolism-related biomarkers, including amino acids, choline and folate derivatives, and water-soluble vitamins, were quantified using ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS). Logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs).

Results

A total of 353 maternal samples were included, comprising 53 mothers of children with KD (24 preconception, 28 early pregnancy) and 300 mothers of healthy children (198 preconception, 100 early pregnancy), along with 185 matched paternal samples from the preconception group. The proportion of folic acid supplementation was significantly lower in the KD group than in controls, both preconceptionally (10.34% vs. 42.44%, P = 0.001) and in early pregnancy (51.72% vs. 74.75%, P = 0.012). Among one-carbon metabolites, maternal choline [9.47 (7.22–12.24) vs. 7.48 (5.92–9.79) μmol/L, P = 0.002] and S-adenosylhomocysteine [32.38 (24.17–46.33) vs. 28.16 (22.03–40.35) nmol/L, P = 0.046] levels were significantly elevated in the KD group, while methylmalonic acid levels were significantly reduced [70.80 (58.67–97.56) vs. 87.80 (67.00–116.66) nmol/L, P = 0.003]. In the preconception subgroup, choline, betaine, and glycine levels were significantly higher in the KD group; however, no significant differences were found in most metabolites during early pregnancy. Subgroup analysis revealed that maternal cysteine levels were positively associated with KD risk in offspring, with a 5.1% increase in risk per 1 μmol/L increase (OR = 1.051, 95% CI: 1.011–1.093, P = 0.013). After adjusting for maternal age, BMI, folate, vitamin B12, and Hcy, methionine also showed a significant association (OR = 1.052, 95% CI: 1.003–1.203, P = 0.036). No significant differences in paternal metabolite levels were observed between groups.

Conclusions

Elevated maternal serum cysteine and methionine levels during the preconception period were significantly associated with an increased risk of KD in offspring, suggesting a potential role of methionine cycle dysregulation in the early pathogenesis of KD. Paternal one-carbon metabolic status appeared to have limited influence on disease risk.

Part III The Role and Molecular Mechanisms of Methionine Metabolism in Coronary Artery Injury of Kawasaki Disease

Background

Dysregulated methionine metabolism may play a key role in inflammation and immune activation. However, its involvement in coronary artery injury in KD remains unclear. And this study aimed to elucidate the role of methionine metabolism and its epigenetic mechanisms in the development of coronary artery lesions in KD.

Methods

LCWE-induced mouse models and TNF-α–stimulated HCAEC inflammatory cell models were established. Methionine-related metabolites in serum from acute-phase KD patients and LCWE mice were measured using UHPLC-MS/MS. Histone methylation levels under methionine-deprived and TNF-α–stimulated conditions were assessed via Western blot, and specific histone marks were evaluated for reversibility by SAM supplementation. Immunohistochemistry and immunofluorescence were used to localize H3K79me2 expression in vascular tissues. ChIP-seq and RNA-seq were applied to determine genomic distribution and regulatory targets of H3K79me2 under inflammatory and metabolic stress.

Results

Both KD patients and LCWE mice exhibited disturbed methionine metabolism, characterized by decreased levels of methionine and tHcy, and elevated levels of SAM and SAM/SAH ratio. H3K79me2 expression was significantly increased in TNF-α–stimulated, methionine-deprived HCAECs and corresponded with enhanced inflammatory protein expression. In LCWE mouse tissues, H3K79me2 was markedly upregulated in coronary and abdominal aorta endothelium and colocalized with CD31+ cells. Under methionine deficiency, ChIP-seq revealed a loss of typical TSS-enriched H3K79me2 “peak” and an abnormal redistribution to distal intergenic regions. GO and KEGG analyses indicated enrichment of H3K79me2-regulated genes in autophagy, phospholipid metabolism, and DNA repair pathways. Importantly, H3K79me2 was enriched at the promoter of GSDMD, a key pyroptosis effector, and this enrichment diminished under methionine deprivation.

Conclusions

Methionine metabolic remodeling, characterized by precursor depletion and SAM accumulation, is a consistent feature of acute KD and vascular inflammation. H3K79me2, a metabolically sensitive histone mark, exhibits methionine-dependent redistribution and may regulate inflammatory gene expression such as GSDMD. These findings support a novel metabolic-epigenetic mechanism underlying KD-associated vascular injury.