Abstract
Diamond-Blackfan Anemia (DBA) is associated with mutations in several ribosomal protein genes, including Ribosomal Protein S19 (RPS19), which is mutated in approximately 25% of patients. Most RPS19 mutations are deletions of all or part of the RPS19 gene and are predicted to cause DBA by a haploinsufficiency mechanism. However, approximately 30% of RPS19 mutations are missense mutations in the RPS19 coding sequence, which we hypothesize act through a dominant negative mechanism. To test for a dominant negative effect, we generated a transgenic mouse model expressing a common and penetrant mutation at codon 62 that replaces an Arginine with a Tryptophan (R62W). The constructs contain the ubiquitous actin promoter linked to the wild-type or R62W human RPS19 cDNA followed by the 3′ region of the Gamma globin gene to provide RNA stability and intron splicing to facilitate RNA transport to the cytoplasm. The constructs are flanked by chicken HS4 barrier elements to ensure transgene expression regardless of the location in the genome. Eight lines of wild-type RPS19 transgenic mice were fertile, expressed RPS19 in all tissues, and had normal hematology. Twelve RPS19R62W founder animals were generated, six of which died before they reached 2 months of age. Two of these animals were analyzed and found to have a macrocytic anemia. None of the other 6 founder animals transmitted the RPS19R62W transgene to F1 pups or d13.5 embryos, suggesting either that the RPS19R62W transgene was not present in the germ line and/or that expression of the RPS19R62W protein may cause early lethality. Supporting this hypothesis, embryonic stem cells (ES) expressing wild-type RPS19 were viable, while ES cells expressing RPS19R62W were not viable. To circumvent potential embryonic lethality, we generated conditional RPS19R62W transgenic mice with stop sequences flanked by lox P sites inserted between the promoter and the RPS19 gene. In the presence of Cre recombinase, lox P sites are combined, excising the sequences between them. Adult mice carrying the conditional RPS19R62W transgene and the interferon inducible Mx1-Cre gene were treated with poly (I:C) to induce excision of the stop sequence. Following poly (I:C) administration, hematocrits dropped significantly in RPS19R62W/Mx1-Cre animals compared to controls, but rebounded to normal within two weeks, due to incomplete stop sequence excision and expansion of unexcised cells in the bone marrow. Colony-forming cell assays indicate that RPS19R62W-expressing bone marrow contains 2 to 3 fold fewer BFU-E and CFU-E (p<0.05) and similar numbers of CFU-GM compared to wild-type animals. The decrease in erythroid progenitors was variable, indicating different levels of excision as well as penetrance. When RPS19R62W mice were crossed to Prion-Cre mice, which express Cre at the early embryonic stage, small, anemic d13.5 embryos and occasional small, adult animals with macrocytic anemia were observed. Day 13.5 RPS19R62W/Prion-Cre fetal livers had reduced overall numbers of erythroid cells, and reduced numbers of BFU-E and CFU-E. The decrease in erythroid progenitors was variable, especially in the line carrying 1 copy of the transgene compared to the line carrying 4 copies of the transgene. FACS analysis of d13.5 fetal liver and adult RPS19R62W/Prion-Cre erythroid cells revealed a relative accumulation of erythroid progenitor cells and a relative decrease in the number of terminally differentiating erythroid cells, suggesting that terminal erythroid differentiation is delayed. These findings are consistent with the reticulocytopenia observed in adult RPS19R62W/Prion-Cre mice. In summary we have successfully generated a mouse model of DBA caused by ectopic expression of mutant human RPS19R62W. The development of a severe anemia following conditional expression of mutant RPS19 suggests that the R62W missense mutation has a dominant negative effect that delays erythropoiesis causing an overall reduction in erythroid cells.
A dominant-negative SOX18 mutant disrupts multiple regulatory layers essential to transcription factor activity
