The pathophysiology of Diamond Blackfan Anemia (DBA) is not confined to erythropoiesis. Associated physical anomalies and growth retardation are common, and there is an increased risk of hemopoietic and non-hemopoietic cancers. Heterozygous mutations affecting RPS19, encoding ribosomal protein S19, are present in 26% of cases of DBA. The indistinguishable clinical phenotype of DBA patients with and without RPS19 mutations predicts that all DBA genes share a common function, or interdependence in a common pathway. If the known ribosomal role of RPS19 underlies the pathogenesis of DBA, as opposed to an as yet unknown erythroid specific extraribosomal role, then every cell type should be affected, by analogy with the Bst mouse, another mammalian example of a naturally occurring ribosomal protein haploinsufficiency. In addition, cells from DBA patients with and without RPS19 mutations should be similarly affected. EBV lymphoblastoid cell lines (LCLs) generated from peripheral blood lymphocytes from DBA patients were studied as an easily accessible source of non-erythroid DBA cells to test these predictions. As RPS19 haploinsufficiency may not affect ribosomal abundance in the steady state, cells were subjected to serum deprivation and refeeding to induce translational stress. Under these conditions, 6 DBA LCLs, including 2 with RPS19 mutations, showed impaired proliferation in comparison with control LCLs. A similar phenotype could be induced in control LCLs by low dose rapamycin or cycloheximide. On FACS analysis, two subpopulations of EBV LCLs can be distinguished. Staining with Pyronin Y or FITC showed the larger cells to have a tight RNA and total protein content, with no difference between normal and DBA LCLs, and unaffected by treatment with rapamycin or cycloheximide. The other population of smaller cells had a lower, more variable RNA and protein content. DBA LCLs or rapamycin treated control LCLs had a relatively lower proportion of the larger, actively proliferating cells. These results would be consistent with exit from cell cycle of DBA LCLs in response to translational stress. To determine whether the actively proliferating subpopulation of cells showed altered cell cycle kinetics, the cumulative entry into S phase of cells on refeeding was determined by the addition of BrdU at the time of serum refeeding. FACS analysis showed BrdU positive cells to be contained exclusively within the population of larger LCLs in control and DBA. The BrdU labeling index was reduced in DBA LCLs, and exacerbated by rapamycin. A reduction was also observed in rapamycin treated control LCLs. We have thus demonstrated an impaired growth phenotype and alteration in cell cycle kinetics in non-erythroid cells derived from patients with DBA with and without RPS19 mutations, supporting the hypothesis that ribosomal pathophysiology is the underlying common denominator in DBA. As there was no difference in the 28S:18S ratio in total cellular RNA extracted from control or 5 DBA LCLs, pulse-chase labeling with 3H-uridine was used to study nascent rRNA as an indicator of ribosomal biogenesis. Interestingly, in comparison with a control LCL, there was an apparent relative reduction in 18S nascent rRNA in one DBA LCL and in 28S rRNA in another, both without detectable RPS19 mutations, confirmed in repeat independent experiments. Further investigations are in progress to determine whether these findings reflect an epiphenomenon in the context of an immortalised cell line, or truly reflect abnormal ribosomal biogenesis in DBA.

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