Defective Ribosomal Protein Gene Expression Alters Transcription, Translation and Oncogenic Pathways in Diamond-Blackfan Anemia.
Authors: Gazda Hanna T. , Kho Alvin T. , Sanoudou Despina , Zaucha Jan M. , Sieff Colin A. , Beggs Alan H. 
Published: April 2020

Abstract
Diamond-Blackfan anemia (DBA) is a broad developmental disease, characterized by anemia, bone marrow erythroblastopenia, physical anomalies, and an increased incidence of malignancy. Ribosomal protein S19 gene (RPS19) is mutated in approximately 25% of DBA probands. However, its role in the pathogenesis of DBA remains to be determined. Using global gene expression analysis (Affymetrix HG-U133A chips, &gt;22,000 probe sets) we compared highly purified multipotential (P) (CD34+CD71−CD45RA−) erythroid (E) (CD34+CD71hiCD45RA), and myeloid (M) (CD34+CD71lowCD45RA+) bone marrow progenitors from three RPS19 mutated and six control samples. For statistical analysis we applied Geometric Fold Change Analysis and Significance Analysis of Microarrays. As we have previously shown the highest number, 545, of significantly changed genes (≥2 fold up- or down-regulated), was identified in diseased vs control E progenitors, while only 106 and 72 genes were dysregulated in P and M progenitors, respectively. In addition to 10 ribosomal protein genes down-regulated in DBA samples, we found several genes involved in translation, including EIF5B, EIF2C2, EEF1D and EEF1E1 significantly under-expressed in diseased erythroid progenitors. Quantitative real-time PCR revealed the expression of 18S rRNA 3.5 to 7-fold up-regulated in the DBA P populations, 1.5–4-fold in the E populations, and unchanged in the M populations. We also found transcriptional control genes TAF9L, TAF12, TCF3, NFYA, ELYS, NFYB and CNOT8, greatly down-regulated mostly in the DBA E populations. In addition we found the erythroid transcription factor, c-Myb, 6-fold down-regulated in the DBA E populations. Importantly, we identified 29 cancer-related genes, oncogenes and tumor suppressor genes, including RAB2, RABL4, RAB20, RAB21, RB1 and PHB significantly dysregulated in the P, E or M DBA populations. We also studied the relationship between P/E, P/M and E/M populations separately in the diseased and control samples. This analysis revealed 3,846 genes ≥2-fold changed between diseased E and P populations (485 in control P/E) while the number of dysregulated genes between diseased P/M and E/M were 1,660 and 1,042, respectively, (controls 330 and 378, respectively). Our data show that at the molecular level, erythroid progenitors are the most affected in DBA. Identification of expression changes for multiple cancer-related genes suggests a molecular basis for the increased risk for malignancy in these patients. The results suggest that RPS19 mutation and RPS19 protein insufficiency in DBA patients leads to impairment of ribosomal biogenesis by dysregulated stoichiometry of ribosomal components and subsequent reduction of protein translation. However, it is also possible that specific targets such as c-MYB are affected through an extra-ribosomal role of RPS19. Since disruption of c-Myb is characterized by complete failure of fetal erythropoiesis, our data suggest a link between RPS19 mutations and reduced erythropoiesis in DBA.