Diamond Blackfan Anemia (DBA) is an inherited bone marrow 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 bone marrow, we investigated the transcriptomic profile of 3 different cell types of bone marrow resident cells of DBA patients and compared them with healthy donors. From bone marrow aspirates bone marrow mononuclear cells 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 bone marrow niche may have a role in DBA pathogenesis.

RNA was isolated with Single Cell RNA Purification Kit (Norgen Biotek Corp.) and stored at -80⁰C for further experiments. Total RNA samples of HSC, MEP and proerythroblasts were amplified with REPLI-g WTA Single Cell Kit (Qiagen). Transcriptome libraries were prepared with Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific) on Ion Chef Instrument (Thermo Fisher Scientific). Library quantitation was measured with Qubit dsDNA High Sensitive Quantitation Kit by using Qubit 2.0 Instrument (Thermo Fisher Scientific). Combined libraries were clonally amplified by emulsion PCR and Ion PI Hi-Q OT2 200 Kit was used. Next generation sequencing (NGS) reaction was performed with Ion PI Hi-Q Sequencing 200 Kit on Ion Proton Semiconductor Sequencer (Thermo Fisher Scientific).

Low reading transcripts were eliminated because the WTA whole transcriptome amplification kit was used before lib preparation. For quantitative transcriptomic analysis, plug-in codes in Ion Torrent server were used. After NGS reaction, raw reads were normalized according to the reads per million (RPM) method and mapped to genome assembly hg19 AmpliSeq Transcriptome version by TMAP (Torrent Mapping Alignment Program). All samples were studied in duplicate.