DOI: 10.5061/dryad.3bk3j9kjg
title: Proerythroblast cells of diamond-blackfan anemia patients with RPS19 and CECR1 mutations have similar transcriptomic signature

description: 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.
description: RNA was isolated with
 Single Cell RNA Purification Kit (Norgen Biotek Corp.) and stored at
 -80<sup>0</sup>C 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).
description: 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.