Global gene expression profiling has been successfully applied to identify molecular signatures of hematopoietic stem cells [30
], as well as CD34+
cells, in aplastic anemia [34
]. We compared highly purified multipotential, erythroid, and myeloid bone marrow progenitors from diseased and control samples to investigate the molecular changes secondary to rps19 insufficiency in DBA. Our results are representative of at least DBA patients with RPS19
mutations who are in remission. Fold change analysis showed the highest number of significantly changed genes in DBA erythroid progenitors. These results correlate with clinical observation and in vitro studies of the disease, as clinically, the most prominent symptom of DBA is anemia, and bone marrow smears usually show an absence or insufficiency of erythroid precursors with normal myeloid and platelet lineages [1
]. Furthermore, in vitro colony assays have revealed deficiencies of BFU-E and CFU-E [35
], further indicating that erythroid cells are the most affected in DBA. Although our three patients were in remission at the time of BM aspirations, without any history of treatment for anemia for more than 10 years, their complete blood counts revealed evidence of persistent functional abnormalities of erythropoiesis (macrocytosis, low/borderline hemoglobin, and elevated eADA) with normal leukocyte and platelet counts (). Although, the percentages of both the CD34+
cells and the P, E, and M sorted populations in diseased BMs were very similar to control samples, the size of DBA erythroid colonies were smaller in DBA than in control individuals, as previously reported [13
]. In contrast, the CFU-GM and CFU-GEMM colonies appeared to be normal in the diseased samples. These observations and the fact that the most genes were changed in DBA erythroid populations indicate that the molecular defect is mostly expressed in, although not limited to [39
], the erythroid lineage. Models of DBA using siRNA-mediated knockdown of RPS19
also show a greater effect on erythroid progenitors, although the myeloid lineage is reduced as well [40
Interestingly, we found that transcription factor gene MYB
is sixfold underexpressed in diseased samples; furthermore, it is the only “erythroid” transcription factor altered in these cells. Yolk sac erythropoiesis in MYB
knockout mice is normal, but there is complete failure of erythropoiesis in fetal liver. Progenitors of other lineages, but not megakaryocytes, were also decreased, indicating that c-myb is required for early definitive cellular expansion [42
]. Furthermore, a knockdown allele of MYB
shows that suboptimal levels of c-myb favor macrophage and megakaryocyte differentiation, whereas higher levels are particularly important for erythropoiesis and lymphopoiesis [43
]. Our data suggest a pathway by which rps19 may be involved in erythroid proliferation.
Among the upregulated transcripts in diseased erythroid progenitors were several proapoptotic genes, including TNFRSF10B
(10-, 3-, 2.7-, and 12.8-fold, respectively), whereas the gene encoding apoptosis inhibitory protein, CFLAR,
was downregulated (3.77-fold) (; ). FAS has been shown to have an important role in regulation of apoptosis in early erythroid cells, whereas other anti-apoptotic genes are underexpressed [44
]. Importantly, in vitro studies previously showed that DBA erythroid progenitors were more susceptible to apoptotic death than normal erythroid progenitors after erythropoietin deprivation [45
It was recently shown in zebrafish that 11 ribosomal protein genes act as haploinsufficient tumor suppressors; the haploin-sufficiency for any of these genes caused malignant tumors of the peripheral nerve sheath [46
]. We found one of these genes, RPL36,
significantly underexpressed in DBA patients with RPS19
mutations. None of the studied patients showed any signs of malignancy to date; however, DBA is clearly associated with an increased risk of cancer [4
] in patients with or without RPS19
mutation (unpublished data). Although, it remains to be determined whether insufficiency of rps19 protein and disruption of its ribosomal or potential extraribosomal function contribute to neoplasm in humans, the secondary reduction of other RP genes may be a contributing factor in the increased risk of malignancy in DBA patients.
Our findings also indicate that some RP genes are closely coregulated in humans and that rps19 mutation results in down-regulation of the additional RP genes in both erythroid and nonerythroid cells in DBA patients.
In sum, these data suggest that RPS19
mutation and rps19 protein insufficiency in DBA patients may lead to impairment of ribosome biogenesis by the dysregulated stoichiometry of ribosomal components and subsequent reduction of protein translation capacity. This ribosomal abnormality may be particularly crucial for developing erythroid cells, whose survival and division require large amounts of protein synthesis. At the molecular level, erythroid progenitors seem to be most affected in DBA patients. However, it is also possible that specific targets, such as c-myb, are affected through an extraribosomal role of rps19. Since c-myb level is important for erythropoiesis [42
], the regulation and expression of this protein will be the subject of future studies.