DBA骨髓微环境的单细胞图谱解析及其对红系造血缺陷的影响机制研究

DBA is an inherited bone marrow failure syndrome mainly characterized by red cell dysplasia. However, previous studies hardly explored the pathology of DBA from the view of really pathological bone marrow microenvironment to find more valuable clues, which may be the main reason why its pathogenesis has not been fully clarified so far. Ribosomal protein (RPs) gene mutations are the main pathogenic factors of DBA. Our current results show that the pathways regarding inflammation, immunity, oxidative phosphorylation and adipogenesis in DBA bone marrow are significantly abnormally regulated, and different RPs mutations lead to evidently divergent transcriptomic profiling of bulk bone marrow cells in different DBA patients. Therefore, we hypothesize that there are the other abnormal cell types with unknown functions existed in DBA bone marrow in addition to the defective red blood cells. Differing from previous studies using bulk cells, we will perform single-cell transcriptome sequencing and molecular profiling for three DBA patients bone marrow cells with different RPS19 mutations in order to acquire the higher level resolution of molecular characteristics of DBA bone marrow cells under the real pathological conditions. RPS19 has been the most important pathogenic factor of DBA, which covers 25% of DBA patients. Furthermore, we will do a lot of functional experiments to explore the DBA pathology from three views including the studies on the influences of the bone marrow microenvironment in DBA, the origin of DBA defects as well as how RPS19 mutation specifically lead to red cell dysplasia in DBA. This research will provide very supportive theories for clinical treatment and pathological exploration for DBA with RPS19 mutations.

DBA是一种以红系造血衰竭为主要特点的骨髓衰竭综合征。然而，以往的研究并没有真正地从骨髓病理状态的微环境出发，去探寻更有用的解析红系造血缺陷的线索，这可能是当前其发病机理仍不完全明确的主要原因。核糖体蛋白（RPs）基因突变是DBA的主要致病因素。我们最近的研究表明：DBA骨髓中炎症、免疫、氧化磷酸化、脂肪生成等通路显著异常，不同RPs突变导致的转录组图谱差异较大，这可能表明除红系造血缺陷外，骨髓中还存在其他功能未知的异常的细胞组分。区别于以往利用群体细胞进行的分析，本研究拟以DBA最主要致病基因RPS19为代表，对其不同突变的DBA患者骨髓进行单细胞转录组测序与图谱绘制，从更高分辨率水平解析病理状态下骨髓单细胞的分子图谱特征，结合功能实验，从微环境的影响、DBA缺陷的溯源，以及RPS19突变特异性导致红系造血缺陷等方面对其导致DBA的病理机制进行探索，为临床治疗与机制解析提供重要理论基础。

DBA是一种以红系造血衰竭为主要特点的骨髓衰竭综合征。然而，以往的研究并没有真正地从骨髓病理状态的微环境出发，去探寻更有用的解析红系造血缺陷的线索，这可能是当前其发病机理仍不完全明确的主要原因。因此，对DBA特定致病基因病理机制的研究具有重要的理论与实践意义。RPS19是核糖体小亚基核糖体蛋白之一，是于1999年第一个被鉴定的DBA基因，在国外约有25%的DBA患者具有该基因突变，是突变率最高的致病基因。本项目主要通过对DBA患者骨髓微环境单细胞转录组测序数据分析，探究RPS19突变DBA患者的病理机制。.本项目收集了4例典型RPS19突变DBA患者和4例正常对照骨髓样本的单细胞转录组测序（自测2例，公共数据6例），这也是目前RPS19突变的全部单细胞测序数据。通过将这些数据进行去批次整合分析，首次绘制了RPS19突变的DBA骨髓微环境的单细胞转录组图谱，为进一步认识DBA疾病病理机制与骨髓微环境提供了重要数据。围绕项目主要研究目标和内容，利用生物信息学技术，我们有以下研究发现：（1）在单细胞水平发现在DBA患者的红系造血缺陷在造血干阶段就已经出现缺陷；（2）RPS19突变导致的核糖体亚基的累积具有谱系选择性，核糖体亚基的累积在红系发育谱系中最大，红系对突变导致的影响更加敏感，这种选择性最终调控了各个谱系的细胞命运；（3）通过DBA骨髓微环境全谱系分析，发现各个谱系的细胞高变差异基因主要是免疫炎性相关基因，通过对高变基因富集分析，发现免疫炎症相关通路显著富集；这些高变免疫炎性基因将有望成为新的DBA疾病治疗靶点。