Table_6_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）是一类遗传性骨髓（Bone Marrow, BM）衰竭综合征，以红系分化低下为核心特征。DBA主要由核糖体蛋白基因的突变引发，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生殖系突变可导致25%的DBA病例。另外，CECR1突变虽可引发与DBA相似的表型，却不属于核糖体病范畴。鉴于骨髓内红细胞生成受阻，本研究对DBA患者骨髓驻留细胞的三种不同细胞类型开展转录组谱分析，并与健康供体的对应细胞进行对比。研究人员从骨髓穿刺液中分离得到骨髓单个核细胞（Bone Marrow 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-红系祖细胞（Megakaryocyte-Erythroid Progenitor Cell, MEP）[CD71–CD34+ CD38hi]及早幼红细胞[CD71+ CD117+ CD38+]，随后通过转录组学技术完成分析。在所有受试细胞中，早幼红细胞的转录组谱差异最为显著。DBA患者早幼红细胞内，与细胞应激、免疫反应相关的基因表达上调，部分参与红系分化的转录因子表达亦发生异常改变。本研究同时证实，DBA患者早幼红细胞中核糖体蛋白的基因表达水平显著降低。此外，红细胞集落形成实验（Colony Formation Assay, CFU-E）为DBA患者的红系分化衰竭提供了功能学证据。基于所有兼具RPS19与CECR1突变表型的患者均存在共同转录组特征这一发现，提示炎性骨髓微环境可能参与DBA的发病过程。