目的:眼膜类天疱疮(OcMMP)是一种孤儿疾病,其特征是慢性自身免疫驱动的结膜炎症导致进行性瘢痕形成,使人衰弱的症状,和致盲后遗症。这项可行性研究旨在证明结膜遗传转录组学分析是询问OcMMP中致病信号通路的假定工具。
方法:使用NanoStringnCounter人类纤维化小组对从6名MMP患者(8只眼;4M/2F;中位年龄78[范围64-84]岁)和8名年龄匹配的对照参与者(15只眼;3M/5F;中位年龄69.5[范围69-88]岁)的结膜拭子提取的RNA进行结膜RNA分析。使用ROSALINDHyperScale结构分析来自770个基因的数据,并根据临床上可见的球结膜炎症水平进行分层。规范化,使用Benjamini-Hochberg方法计算了倍数变化(≥+1.5倍或≤-1.5倍)和p值调整(<0.05).
结果:在OcMMP与对照组之间观察到93个差异表达基因(DEGs),其中48个上调,45下调。前4个上调的DEGs代表纤维化(COL3A1,COL1A1,FN1和THBS1),而关键的低表达基因(SCIN,HMGS2,XCL1/2)指示眼表衰竭(杯状细胞丢失,角质化,对继发感染的脆弱性)。44个途径的整体显著性评分≥2,最显著的是那些与细胞外基质(ECM)重塑有关的,合成,和退化。这些途径在具有可见炎症的眼睛中加重。
结论:通过简单的结膜拭子获得的NanoString方法可识别OcMMP组中的促纤维化基因并区分发炎的眼睛。纵向采样和随后的调查干预将进一步的机械洞察力和新型生物标志物的发展,以监测疾病进展。
OBJECTIVE: Ocular Mucous Membrane Pemphigoid (OcMMP) is an orphan disease characterized by chronic autoimmune-driven conjunctival inflammation leading to progressive scarring, debilitating symptoms, and blinding sequelae. This feasibility study aims to demonstrate conjunctival genetic transcriptomic analyses as a putative tool for interrogation of pathogenic signaling pathways in OcMMP.
METHODS: Conjunctival RNA profiling using the NanoString nCounter Human Fibrosis panel was undertaken on RNA extracted from conjunctival swabs obtained from 6 MMP patients (8 eyes; 4 M/2F; median age 78 [range 64-84] years); and 8 age-matched control participants (15 eyes; 3 M/5F; median age 69.5 [range 69-88] years). Data from 770 genes were analyzed with ROSALIND HyperScale architecture and stratified according to the level of clinically visible bulbar conjunctival inflammation. Normalization, fold-changes (≥+1.5-fold or ≤ -1.5-fold) and p-values adjustment (<0.05) using the Benjamini-Hochberg method were calculated.
RESULTS: 93 differentially expressed genes (DEGs) were observed between OcMMP versus controls of which 48 were upregulated, and 45 downregulated. The top 4 upregulated DEGs represented fibrosis (COL3A1, COL1A1, FN1 and THBS1) while the key under-expressed genes (SCIN, HMGS2, XCL1/2) were indicative of ocular surface failure (goblet cell loss, keratinization, vulnerability to secondary infections). Forty-four pathways had a global significance score ≥2, the most significant being those related to extracellular matrix (ECM) remodeling, synthesis, and degradation. These pathways were accentuated in eyes with visible inflammation.
CONCLUSIONS: NanoString methodology acquired via a simple conjunctival swab identifies profibrotic genes in OcMMP group and differentiates inflamed eyes. Longitudinal sampling and following investigative intervention will further mechanistic insight and development of novel biomarkers to monitor disease progression.