Acute Appendicitis: Transcript Profiling of blood identifies promising biomarkers and potential underlying processes

Background: The diagnosis of acute appendicitis can be surprisingly difficult without computed tomography, which carries significant radiation exposure. Genome-wide expression profiling was applied to whole blood RNA of acute appendicitis patients versus patients with other abdominal disorders, in order to identify biomarkers of appendicitis. From a large cohort of emergency patients, a discovery set of patients with surgically confirmed appendicitis, or abdominal pain from other causes, was identified. RNA from whole blood was profiled by microarrays, and RNA levels were filtered by a combined fold-change (>2) and p value (<0.05). A separate set of patients, including patients with respiratory infections, was used to validate a partial least squares discriminant (PLSD) prediction model. Results: Transcript profiling identified 37 differentially expressed genes (DEG) in appendicitis versus abdominal pain patients. The DEG list contained 3 major ontologies: infection-related, inflammation-related, and ribosomal processing. Appendicitis patients had lower level of neutrophil defensin mRNA (DEFA1,3), but higher levels of alkaline phosphatase (ALPL) and interleukin-8 receptor-ß (IL8RB), which was confirmed in a larger cohort of 60 patients using droplet digital PCR (ddPCR). Conclusions: Patients with acute appendicitis have detectable changes in the mRNA expression levels of factors related to neutrophil inate defense systems. The low defensin mRNA levels suggest that appendicitis patient's immune cells are not directly activated by pathogens, but are primed by diffusible factors in the microenvironment of the infection. The detected biomarkers are consistent with prior evidence that biofilm-forming bacteria in the appendix may be an important factor in appendicitis. 40 samples were grouped into Discovery and Validation sets based on their clinical dx and analyzed for differential gene expression within groups by conditions

背景：急性阑尾炎的诊断在不借助计算机断层扫描（computed tomography, CT）的情况下往往极具挑战性，而CT本身存在显著的辐射暴露风险。为筛选出阑尾炎相关生物标志物，本研究对急性阑尾炎患者与其他腹部疾病患者的全血RNA开展全基因组表达谱分析。研究人员从大规模急诊患者队列中，筛选出经手术确诊的阑尾炎患者，以及因其他病因引发腹痛的患者，组建发现集队列。采用微阵列技术对全血RNA进行表达谱分析，并通过联合筛选倍数变化（fold-change, FC）>2且P值<0.05的标准，对RNA表达水平进行过滤。另设包含呼吸道感染患者在内的独立患者队列，用于验证偏最小二乘判别（partial least squares discriminant, PLSD）预测模型。结果：转录组分析显示，与腹痛患者相比，阑尾炎患者体内存在37个差异表达基因（differentially expressed genes, DEG）。该差异表达基因集涵盖3大类主要本体功能：感染相关、炎症相关以及核糖体加工相关通路。阑尾炎患者的中性粒细胞防御素mRNA（DEFA1、DEFA3）表达水平较低，而碱性磷酸酶（ALPL）以及白细胞介素-8受体β（IL8RB）的表达水平更高，该结果在包含60名患者的更大规模队列中，通过液滴数字PCR（ddPCR）得到了验证。结论：急性阑尾炎患者体内与中性粒细胞固有免疫防御系统相关的因子mRNA表达水平存在可检测到的变化。防御素mRNA的低表达水平提示，阑尾炎患者的免疫细胞并非直接被病原体激活，而是被感染微环境中的弥散性因子致敏。本次检测到的生物标志物与既往研究结论相符，即阑尾内形成生物膜的细菌可能是引发阑尾炎的重要致病因素。本研究共纳入40份样本，依据临床诊断结果分为发现集与验证集，并按照实验条件对各组样本开展差异基因表达分析。