Table 1_Integrative multi-omics Mendelian randomization and functional validation identifies RNASET2 as a novel therapeutic target for autoimmune thyroiditis.xlsx

ObjectiveAutoimmune thyroiditis (AIT), a prevalent autoimmune disorder that frequently leads to hypothyroidism. A critical unmet need exists for disease-modifying therapies that target its underlying pathogenesis. This study aimed to identify and validate novel therapeutic targets for AIT. MethodsWe employed an integrative genomics approach, combining genome-wide association studies (GWAS) with molecular quantitative trait loci (QTL) analyses, including expression (eQTL), protein (pQTL), and DNA methylation QTL (mQTL), across two independent AIT cohorts for discovery and replication. We performed two-sample bidirectional Mendelian randomization (MR) with sensitivity analyses, followed by summary-data-based MR (SMR) and heterogeneity in dependent instruments (HEIDI) tests. Top candidates were further evaluated via phenome-wide association study (PheWAS) and computational drug screening. Guided by these findings, we quantified plasma levels of the top-priority candidate, Ribonuclease T2 (RNASET2), via ELISA in AIT patients and non-AIT controls. To functionally validate its therapeutic potential, we developed a novel three-dimension (3D) inflammatory thyrocyte spheroid model and evaluated potential therapeutic effects of recombinant RNASET2. Loss-of-function (small interfering RNA-mediated knockdown) and gain-of-function (recombinant protein RNASET2 rescue) experiments further supported RNASET2 as a therapeutic target. ResultsMulti-omics integration consistently nominated RNASET2 as a causal protective factor against AIT. Signals of pQTL and eQTL for RNASET2 were associated with decreased AIT risk, while three mQTLs were correlated with increased risk. PheWAS indicated minimal pleiotropic effects, supporting its therapeutic suitability. Computational drug screening nominated genistein, a soy isoflavone known to upregulate RNASET2 expression, as a repurposing candidate. Empirically, plasma RNASET2 levels were moderately elevated in AIT patients, potentially reflecting a compensatory anti-inflammatory response. Crucially, recombinant RNASET2 effectively mitigated inflammation and apoptosis in the thyrocyte spheroid model, confirming its functional protective role. Consistently, RNASET2 knockdown heightened susceptibility to inflammatory cell death and cytokine expression, a phenotype reversed by recombinant RNASET2 supplementation. ConclusionsBy integrating large-scale genomic analyses with functional validation, our study establishes RNASET2 as a promising therapeutic target for AIT. RNASET2 augmentation represents a potential disease-modifying strategy, providing a translational bridge from genetic discovery to clinical application.一、研究目标 自身免疫性甲状腺炎（Autoimmune thyroiditis, AIT）是一种常见的自身免疫性疾病，常可引发甲状腺功能减退症。目前仍存在尚未满足的重大临床需求，亟需靶向其潜在发病机制的疾病修饰治疗手段。本研究旨在识别并验证针对AIT的新型治疗靶点。 二、研究方法 我们采用整合基因组学策略，在两个独立的AIT队列中开展发现与验证研究，将全基因组关联研究（Genome-wide association studies, GWAS）与分子数量性状位点（Quantitative trait loci, QTL）分析相结合，涵盖表达QTL（expression QTL, eQTL）、蛋白质QTL（protein QTL, pQTL）以及DNA甲基化QTL（DNA methylation QTL, mQTL）。我们开展了带敏感性分析的双样本双向孟德尔随机化（two-sample bidirectional Mendelian randomization, MR）分析，随后进行了基于汇总数据的MR（Summary-data-based MR, SMR）以及异质性依赖工具检验（Heterogeneity in dependent instruments, HEIDI）。对筛选出的候选靶点，我们进一步通过表型组关联研究（Phenome-wide association study, PheWAS）以及计算机药物筛选进行评估。基于上述发现，我们通过酶联免疫吸附试验（Enzyme-linked immunosorbent assay, ELISA）定量检测了AIT患者与非AIT对照人群中优先级最高的候选靶点——核糖核酸酶T2（Ribonuclease T2, RNASET2）的血浆水平。为在功能层面验证其治疗潜力，我们构建了一种新型三维（3D）炎症性甲状腺细胞球体模型，并评估了重组RNASET2的潜在治疗效果。通过功能丧失实验（小干扰RNA介导的基因敲低）与功能获得实验（重组RNASET2挽救实验），进一步证实RNASET2可作为治疗靶点。 三、研究结果 多组学整合分析一致将RNASET2确定为对抗AIT的因果性保护因子。RNASET2的pQTL与eQTL信号与AIT风险降低相关，而三个mQTL则与风险升高相关。表型组关联研究显示其具有极小的多效性，支持其作为治疗靶点的适用性。计算机药物筛选筛选出染料木素（genistein）——一种已知可上调RNASET2表达的大豆异黄酮（soy isoflavone）——作为药物重定位候选药物。实验结果表明，AIT患者的血浆RNASET2水平呈中度升高，这可能反映了一种代偿性抗炎应答。至关重要的是，重组RNASET2可有效减轻甲状腺细胞球体模型中的炎症反应与细胞凋亡，证实了其功能性保护作用。与之一致的是，RNASET2基因敲低会增加细胞对炎症性死亡与细胞因子表达的易感性，而补充重组RNASET2可逆转这一表型。 四、研究结论 本研究通过整合大规模基因组分析与功能验证，确立了RNASET2作为AIT极具前景的治疗靶点。上调RNASET2的表达代表了一种潜在的疾病修饰治疗策略，为从遗传发现到临床应用搭建了转化桥梁。