Session: 101. Red Cells and Erythropoiesis, Excluding Iron: Poster I
Hematology Disease Topics & Pathways:
Fundamental Science, Research, Genetic Disorders, Hematopoiesis, Diseases, Biological Processes
Previous efforts have been made to understand pathogenesis in CDA-Ia using mouse models with constitutional erythroid-specific deletion of CDAN1; however, this model leads to failure of primitive erythropoiesis and death by mid-gestation (E12.5–E13.5) due to severe anemia, allowing limited in vivo studies to investigate the role of CDAN1 in erythropoiesis [1Noy-lotan S. et al, 2021 Front Physiol]. Alternatively, it may be more relevant to study CDA-Ia during definitive erythropoiesis, instead of primitive erythropoiesis, as a means to investigate the pathogenesis of Cdan1 deficiency in erythropoiesis and the therapeutic effect of IFN-α. Therefore, we hypothesized that a temporal and spatial approach to deleting CDAN1 may provide a mouse model which avoids the stressful period of fetal erythropoiesis, replicates the disease in patients with diminished CDAN1 function, and is amenable to IFN-α treatment. We generated our model from CDAN1flox/flox mice, originating from the CDAN1 knockout-first model [1], and bred with Gata1creERT2+ mice [2Yu L. et al Blood 2021]. To test our hypothesis, we induced deletion of CDAN1 in Gata1creERT2+; CDAN1flox/flox and control mice by tamoxifen treatment (i.p.) every other day, for a total of 10 injections. Two days after the final injection, we harvested the blood, hind limbs, and spleens for analysis. We performed CBCs and flow cytometry to assess terminal erythropoiesis in the BM and spleen. RT-qPCR and PCR were used to examine the recombination efficiency of CDAN1 at the RNA and DNA level, respectively. We found that Cdan1-deficient mice had splenomegaly and were anemic compared to control mice, with variable reticulocyte counts. We also found that the red cell distribution width (RDW) was increased with noticeable macrocytosis. Light microscopy of BM and spleen cytospins revealed binucleated erythroblasts and chromatin bridges, typical of CDA-Ia. TEM imaging on Cdan1-deficient mouse erythroblasts revealed a “Swiss cheese” appearance in the heterochromatin, the pathognomonic finding of CDA-Ia. Flow cytometry of whole BM and spleen showed reduced late erythroblasts in the BM but a marked increase in splenic stress erythropoiesis, leading only to a partial erythropoietic compensation of the resulting anemia in Cdan1-deficient mice. There was substantial deletion of CDAN1 mRNA expression in Ter119+ cells from the BM and spleen, although a complete recombination of the CDAN1 gene was not achieved. Overall, these data support the notion that this is a suitable mouse model on which to study the pathological mechanisms causing CDA-Ia, such as investigating the mechanisms of erythropoiesis defects caused by Cdan1-deficiency, the molecular pathology underlying the TEM phenotype in CDA-Ia erythroblasts, and the therapeutic effect of IFN-α.
Disclosures: Kalfa: Novo Nordisk: Research Funding; Agios Pharmaceuticals: Research Funding.
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