Dysregulation of the Transforming Growth Factor Beta Pathway in Induced Pluripotent Stem Cells Generated from Patients with Diamond Blackfan Anemia [HuEx-1_0]. Homo sapiens

Diamond Blackfan Anemia (DBA) is an inherited bone marrow failure syndrome with clinical features of red cell aplasia and variable developmental abnormalities. Most affected patients have heterozygous loss of function mutations in ribosomal protein genes but the pathogenic mechanism is still unknown. We generated induced pluripotent stem cells from DBA patients carrying RPS19 or RPL5 mutations. Transcriptome analysis revealed the striking dysregulation of the transforming growth factor beta signaling pathway in DBA lines. Expression of TGF beta target genes, such as TGFBI, BAMBI, COL3A1 and SERPINE1 was significantly increased in the DBA iPSCs. We quantified intermediates in canonical and non-canonical TGF beta pathways and observed a significant increase in the levels of the non-canonical pathway mediator p-JNK in the DBA iPSCs. Moreover, when the mutant cells were corrected by ectopic expression of WT RPS19 or RPL5, levels of p-JNK returned to normal. Surprisingly, nuclear levels of SMAD4, a mediator of canonical TGF beta signaling, were decreased in DBA cells due to increased proteolytic turnover. We also observed the up-regulation of TGF beta 1R, TGF beta 2, CDKN1A and SERPINE1 mRNA, and the significant decrease of GATA1 mRNA in the primitive multilineage progenitors. In summary our observations identify for the first time a dysregulation of the TGF beta pathway in the pathobiology of DBA. Overall design: 6 Total human iPS samples were analyzed, including 2 wild type samples, 2 DBA samples with a RPS19 mutation, and 2 DBA samples with a RPL5 mutation. We generated the following pairwise comparisons using Partek Softare : RPS19

钻石黑范贫血（Diamond Blackfan Anemia, DBA）是一种遗传性骨髓衰竭综合征，临床表现为红细胞再生障碍，并伴随多变的发育异常。多数受累患者存在核糖体蛋白基因的杂合功能丧失突变，但其致病机制至今尚未明确。本研究从携带RPS19或RPL5突变的DBA患者体内诱导生成了诱导多能干细胞（induced pluripotent stem cells, iPSCs）。转录组分析显示，DBA细胞系中转化生长因子β（transforming growth factor beta, TGF-β）信号通路存在显著失调。TGF-β靶基因如TGFBI、BAMBI、COL3A1及SERPINE1的表达在DBA iPSCs中显著升高。我们对经典及非经典TGF-β通路的中间产物进行了定量检测，观察到DBA iPSCs中非经典通路介导因子磷酸化JNK（p-JNK）的水平显著上升。进一步研究发现，通过异位表达野生型RPS19或RPL5对突变细胞进行矫正后，p-JNK的水平可恢复至正常范围。令人意外的是，由于蛋白水解周转增强，经典TGF-β信号通路介导因子SMAD4的核内水平在DBA细胞中出现下降。我们同时观察到，原始多谱系祖细胞中TGF-β 1型受体、TGF-β 2型受体、CDKN1A及SERPINE1的mRNA表达上调，而GATA1的mRNA水平显著降低。综上，本研究首次证实TGF-β通路失调参与了DBA的病理生物学过程。实验设计概述：本研究共分析6份人类iPS样本，包括2份野生型样本、2份携带RPS19突变的DBA样本以及2份携带RPL5突变的DBA样本。我们使用Partek软件构建了如下两两比较组：RPS19