Inducible, Erythroid-Specific Knockout of Codanin-1 in Adult Mice Replicates Congenital Dyserythropoietic Anemia Type-Ia
Authors: Azucenas Corbin R , Voulgaridou Aikaterini , Stewart Maria , Ntoumaziou Athina , Elgammal Yasmin , Seu Katie , Kalfa Theodosia A. 
Published: December 2024

Congenital dyserythropoietic anemias (CDAs) are a heterogenous group of congenital blood diseases, characterized by ineffective erythropoiesis, hemolysis, secondary iron overload, and the presence of bi- or multinucleated erythroblasts in the bone marrow (BM). Several CDA types exist and they were originally classified by BM pathological findings. CDAs are now more accurately classified by their causative mutations when these are identifiable. CDA type-Ia is caused by biallelic pathogenic variants in the CDAN1 gene, which encodes Codanin-1 (Cdan1), a highly conserved protein. Currently, there are no known patients with complete loss of CDAN1, indicating that total loss of CDAN1 is incompatible with life. Patients with CDA-Ia frequently require transfusions in utero to survive and present with varying degrees of hemolytic anemia with relative reticulocytopenia (suggesting ineffective erythropoiesis). BM pathology reveals erythroid hyperplasia with 3-10% binucleated erythroblasts, the rare but characteristic finding of internuclear chromatin bridges between nearly-completely separated erythroblasts, and, under transmission electron microscopy (TEM), a “Swiss cheese” appearance in the nuclear heterochromatin of erythroid precursors. Blood transfusions and iron chelation are needed for many patients with CDA-Ia, while treatment with IFN-α is also an option. Most patients treated with IFN-α achieve transfusion independence and appear to have alleviated iron overload, suggesting that IFN-α may somehow interact with erythroblasts, erythroblast turn over, or another unknown mechanism.

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.