Proerythroblast Cells of Diamond-Blackfan Anemia Patients With RPS19 and CECR1 Mutations Have Similar Transcriptomic Signature

Abstract

Diamond Blackfan Anemia (DBA) is an inherited bone marrow (BM) 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 BM, we investigated the transcriptomic profile of three different cell types of BM resident cells of DBA patients and compared them with healthy donors. From BM aspirates BM mononuclear cells (MNCs) were isolated and hematopoietic stem cells (HSC) [CD71^-CD34⁺ CD38^mo/lo], megakaryocyte-erythroid progenitor cells (MEP) [CD71^-CD34⁺ CD38^hi] 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 BM niche may have a role in DBA pathogenesis.

Keywords: CECR1; Diamond Blackfan Anemia; RPS19; proerythroblast; ribosomopathy; transcriptomics.

FIGURE 1

Erythroid colony formation assay. (A) DBA patient samples had diminished/lack of colony forming potential (B) and had significantly low number of erythroid colonies. HD: Healthy donor (^∗p: 0.0015; ^∗∗p: 0.0007; ^∗∗∗p: 0.0003).

FIGURE 2

Differentially expressed gene (DEG) numbers between DBA and healthy donors. For DEG analysis, all three DBA samples were grouped and compared with healthy donors.

FIGURE 3

(A) Pathway analysis of the upregulated genes, (B) significant pathways belong to biological process (BP) performed using g:Profiler, (C) differentially expressed genes belong to cellular stress in DBA proerythroblasts. BP: Biological Process, HDs: healthy donors.

FIGURE 4

Total ribosomal protein mRNA expression of different cell types. Different cell types analyzed with the same transcriptomic approach from the in-house dataset were evaluated. RPM: reads per million, NK: natural killer, BM-MSC: bone marrow mesenchymal stem cells. HSC: hematopoietic stem cell, MEP: Megakaryocyte–Erythroid Progenitor Cell, ns: non-significant. ^∗p value < 0.0001.

FIGURE 5

Differentially expressed transcription factors that are primarily related with erythroid lineage or other critical cellular responses. HDs: healthy donors.