Dysregulation of the Transforming Growth Factor Beta Pathway in Induced Pluripotent Stem Cells Generated from Patients with Diamond Blackfan Anemia [HTA-2_0]

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. 8 Total samples were analyzed, including 4 wild type samples, 2 RPS19 mutant samples, 2 corrected RPS19 mutant samples. We generated the following pairwise comparisons using Partek Softare : RPS19 mutant<WT; RPS19 mutant<corrected RPS19 mutant. Genes with an FDR≤5% and a fold-change ≥2 were selected.