RNA-Sequencing to Predict Response to TNF-α inhibitors Reveals Possible Mechanism for Non-response in Smokers

<b>Background</b>: Several studies have employed microarray-based profiling to predict response to tumor necrosis factor-alpha inhibitors (TNFi) in rheumatoid arthritis (RA); yet efforts to validate these targets have failed to show predictive abilities acceptable for clinical practice. <b>Methods</b>: The eighty most extreme responders and non-responders to TNFi therapy were selected from the observational BiOCURA cohort. RNA-sequencing was performed on mRNA from peripheral blood mononuclear cells (PBMCs) collected before initiation of treatment. The expression of pathways as well as individual gene transcripts between responders and non-responders was investigated. Promising targets were technically replicated and validated in n=40 new patients using qPCR-assays. <b>Results</b>: Before therapy-initiation, non-responders had lower expression of pathways related to interferon and cytokine signaling, while also showing higher levels of two genes, GPR15 and SEMA6B (p=0.02). The two targets could be validated, however, additional analyses revealed that GPR15 and SEMA6B did not independently predict response, but were rather dose-dependent markers of smoking (p&lt;0.0001). <b>Conclusions</b>: The study did not identify new transcripts ready to use in clinical practice, yet GPR15 and SEMA6B were recognized as candidate explanatory markers for the reduced treatment success in RA smokers.

**背景**：已有多项研究采用基于微阵列的基因表达谱分析，预测类风湿关节炎（RA, Rheumatoid Arthritis）患者对肿瘤坏死因子-α抑制剂（TNFi, Tumor Necrosis Factor-alpha Inhibitors）的治疗应答；然而，验证上述靶点的相关研究均未能证明其具备临床可接受的预测效能。
**方法**：本研究从观察性队列BiOCURA中筛选出对TNFi治疗应答差异最为极端的80例患者，涵盖应答者与无应答者。于治疗起始前采集外周血单个核细胞（PBMCs, Peripheral Blood Mononuclear Cells）中的mRNA，开展RNA测序分析。对比分析应答者与无应答者的通路表达水平及单个基因转录本的表达差异。针对筛选得到的潜在靶点，本研究采用实时荧光定量PCR（qPCR）检测技术，在新增的40例患者队列中完成了技术重复与验证实验。
**结果**：治疗起始前，无应答者的干扰素及细胞因子信号通路表达水平更低，同时其GPR15与SEMA6B两个基因的表达水平更高（p=0.02）。尽管这两个靶点可被验证，但后续分析显示，GPR15与SEMA6B并不能独立预测治疗应答，而是吸烟剂量依赖性标志物（p<0.0001）。
**结论**：本研究未发现可直接应用于临床的新型转录本，但GPR15与SEMA6B可作为潜在解释性标志物，用于阐释类风湿关节炎吸烟者治疗成功率降低的内在机制。