Table_5_Proerythroblast Cells of Diamond-Blackfan Anemia Patients With RPS19 and CECR1 Mutations Have Similar Transcriptomic Signature.XLSX

Diamond Blackfan Anemia (DBA) is an inherited bone marrow (BM) failure syndrome, characterized by a paucity of erythroid differentiation. DBA is mainly caused by the mutations in ribosomal protein genes, hence classified as ribosomopathy. However, in approximately 30% of patients, the molecular etiology cannot be discovered. RPS19 germline mutations caused 25% of the cases. On the other hand, CECR1 mutations also cause phenotypes similar to DBA but not being a ribosomopathy. Due to the blockade of erythropoiesis in the BM, we investigated the transcriptomic profile of three different cell types of BM resident cells of DBA patients and compared them with healthy donors. From BM aspirates BM mononuclear cells (MNCs) were isolated and hematopoietic stem cells (HSC) [CD71–CD34+ CD38mo/lo], megakaryocyte–erythroid progenitor cells (MEP) [CD71–CD34+ CD38hi] and Proerythroblasts [CD71+ CD117+ CD38+] were sorted and analyzed with a transcriptomic approach. Among all these cells, proerythroblasts had the most different transcriptomic profile. The genes associated with cellular stress/immune responses were increased and some of the transcription factors that play a role in erythroid differentiation had altered expression in DBA proerythroblasts. We also showed that gene expression levels of ribosomal proteins were decreased in DBA proerythroblasts. In addition to these, colony formation assay (CFU-E) provided functional evidence of the failure of erythroid differentiation in DBA patients. According to our findings that all patients resembling both RPS19 and CECR1 mutations have common transcriptomic signatures, it may be possible that inflammatory BM niche may have a role in DBA pathogenesis.

钻石黑范贫血（Diamond Blackfan Anemia, DBA）是一种遗传性骨髓（Bone Marrow, BM）衰竭综合征，以红细胞分化不足为核心特征。DBA多由核糖体蛋白基因发生突变所致，因此被归类为核糖体病（ribosomopathy），但约30%的患者无法明确其分子病因。其中，RPS19生殖系突变（germline mutations）可导致25%的DBA病例；另一方面，CECR1突变虽可引发与DBA相似的表型，却不属于核糖体病范畴。鉴于骨髓中红细胞生成（erythropoiesis）受阻，本研究对DBA患者骨髓驻留细胞的三类不同细胞群开展转录组谱（transcriptomic profile）分析，并与健康供体进行对比：研究人员从骨髓穿刺液中分离骨髓单个核细胞（BM mononuclear cells, MNCs），并分选得到造血干细胞（Hematopoietic Stem Cell, HSC）[CD71–CD34+ CD38mo/lo]、巨核细胞-红细胞祖细胞（Megakaryocyte-Erythroid Progenitor, MEP）[CD71–CD34+ CD38hi]及早幼红细胞（Proerythroblasts）[CD71+ CD117+ CD38+]，随后通过转录组学方法（transcriptomic approach）进行检测。在所有受试细胞群中，早幼红细胞的转录组谱差异最为显著：DBA患者的早幼红细胞中，与细胞应激/免疫应答相关的基因表达上调，部分参与红细胞分化的转录因子（transcription factors）表达亦发生改变；同时研究发现，DBA早幼红细胞内核糖体蛋白的基因表达水平呈下调趋势。此外，红细胞集落形成试验（Colony-Forming Unit-Erythroid, CFU-E）为DBA患者的红细胞分化障碍提供了功能学证据。基于所有兼具RPS19与CECR1突变特征的患者均存在共同转录组特征这一发现，提示炎症性骨髓微环境（inflammatory BM niche）可能在DBA的发病机制（pathogenesis）中发挥重要作用。