In vitro expansion of normal and Diamond Blackfan anemia-derived peripheral blood erythroid progenitors

The Affymetrix Human Gene 2.0 ST array was used to measure differential expression of RNA isolated from normal and Diamond Blackfan anemia (DBA) erythroid progenitors after ex vivo expansion of circulating, peripheral blood derived hematopoietic stem cells under erythroid growth conditions. The gene-level probe summaries reported in this series were computed using RMA as implemented in the Bioconductor package Oligo v1.36.1. Diamond Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by erythroid aplasia, usually without perturbation of other hematopoietic lineages. Approximately 65% of DBA patients with autosomal dominant inheritance have heterozygous mutations or deletions in ribosomal protein (RP) genes while <1% of patients with X-linked inheritance have been identified with mutations in the transcription factor, GATA1. Erythroid cells from patients with DBA have not been well characterized and the mechanisms underlying the erythroid specific effects of either RP or GATA1 associated DBA remain unclear. We have developed an in vitro culture system to expand peripheral blood CD34+ progenitor cells from patients with DBA and differentiate them into erythroid cells. Cells from patients with RP or GATA1 mutations showed decreased proliferation and delayed erythroid differentiation compared to controls. RNA transcript analyses of erythroid cells from controls and patients with RP or GATA1 mutations showed distinctive differences, with upregulation of heme biosynthesis genes prominently in RP-mediated DBA and failure to upregulate components of the translational apparatus in GATA1-mediated DBA. Our data show that dysregulation of translational function is a common feature of DBA caused by both RP and GATA1 mutations. 37 samples analyzed including 10 normal CD44+/CD235+, 8 normal CD44+/235-, 5 DBA CD44+/235+, and 14 DBA CD44+/CD235- samples.

本研究采用Affymetrix人类基因2.0 ST基因芯片（Affymetrix Human Gene 2.0 ST array），对在红系培养条件下体外扩增循环外周血来源造血干细胞后获得的正常及钻石黑范贫血（Diamond Blackfan anemia, DBA）红系祖细胞的分离RNA进行差异表达检测。本数据集的基因水平探针汇总值通过Bioconductor软件包Oligo v1.36.1中实现的RMA（稳健多阵列平均，Robust Multi-array Average）方法计算得到。钻石黑范贫血（Diamond Blackfan anemia, DBA）是一种先天性骨髓衰竭综合征，以红系再生障碍为特征，通常不累及其他造血谱系。约65%的常染色体显性遗传型DBA患者存在核糖体蛋白（ribosomal protein, RP）基因的杂合突变或缺失，而仅不到1%的X连锁遗传型DBA患者被发现存在转录因子GATA1的突变。目前对DBA患者的红系细胞特征研究尚不充分，RP或GATA1突变相关DBA的红系特异性致病机制仍未阐明。本研究建立了体外培养体系，用于扩增DBA患者外周血CD34阳性祖细胞并将其诱导分化为红系细胞。与对照组相比，RP或GATA1突变患者来源的细胞增殖能力下降，红系分化进程延迟。对对照组及RP或GATA1突变患者来源红系细胞的RNA转录本分析显示，两组存在显著差异：RP突变型DBA中血红素生物合成基因显著上调，而GATA1突变型DBA则无法上调翻译装置相关组分。本研究数据表明，翻译功能失调是RP及GATA1突变所致DBA的共同特征。本研究共分析37份样本，包括10份正常CD44+/CD235+样本、8份正常CD44+/235-样本、5份DBA CD44+/235+样本及14份DBA CD44+/CD235-样本。