Identification of Exosomal microRNA signature by liquid biopsy in Hereditary Hemorrhagic Telangiectasia patients

Hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal dominant vascular dysplasia characterized by epistaxis, mucocutaneous telangiectases, and arteriovenous malformations (AVM) in visceral organs. In this study, we carried out a liquid biopsy consisting in the isolation of total RNA from plasma exosomes samples from HHT type 1 (HHT1 group) and type 2 (HHT2 group) patients, and healthy relatives (Control group). Upon gene expression data processing and normalization, a bioinformatics analysis was performed for the study of principal components, hierarchical clustering and pairwise comparisons between HHT samples and control group. Results were evaluated in a further validation cohort of HHT and healthy donors by real time PCR and the diagnosis value of exosomal miRNA determined by the ROC curves analysis. We found that exosomal miRNA expression signature clearly distinguishes among healthy and HHT samples and types. Thus, we identified a disease-associated molecular fingerprint of 35 miRNAs over-represented, being 8 specifics for HHT1 and 11 for HHT2; and 30 under-represented, including 9 distinctive for HHT1 and 9 for HHT2. These exosome-transported miRNAs have diagnosis value for HHT, and even allow to distinguish between HHT1 and HHT2. Overall design: Control group, healthy donors (n=10); HHT1 patients group (n=15); HHT2 patients group (n=15). Each sample was analyzed in triplicates.

遗传性出血性毛细血管扩张症（HHT）是一种罕见的常染色体显性遗传性血管发育异常疾病，以鼻出血、黏膜皮肤毛细血管扩张及内脏器官动静脉畸形（arteriovenous malformations, AVM）为主要临床特征。本研究针对1型遗传性出血性毛细血管扩张症（HHT1组）、2型遗传性出血性毛细血管扩张症（HHT2组）患者及健康亲属（对照组）的血浆外泌体样本开展液体活检，分离总RNA。完成基因表达数据处理与归一化后，我们进行了生物信息学分析，包括主成分分析、层次聚类以及HHT样本与对照组的组间两两比较。后续通过另一组HHT患者与健康供者的验证队列，采用实时定量PCR对研究结果进行验证，并通过受试者工作特征曲线（Receiver Operating Characteristic curves, ROC）分析评估外泌体miRNA的诊断价值。研究发现，外泌体miRNA表达谱可清晰区分健康样本与HHT样本，且能辨别不同亚型的HHT。本研究共筛选出35个表达上调的疾病相关分子特征miRNA，其中8个为HHT1亚型特异性标志、11个为HHT2亚型特异性标志；另有30个表达下调的miRNA，其中9个为HHT1亚型特有、9个为HHT2亚型特有。这些由外泌体转运的miRNA具备HHT的诊断价值，甚至可有效区分HHT1与HHT2亚型。本研究整体实验设计如下：对照组（健康供者，n=10）；HHT1患者组（n=15）；HHT2患者组（n=15）。所有样本均进行三次重复检测。