Codanin-1 Mutations Engineered in Human Erythroid Cells Allow Investigation of Mechanisms Underlying Congenital Dyserythropoietic Anemia

The generation of a functional erythrocyte from a committed progenitor requires significant changes in gene expression during a time of hemoglobin accumulation, rapid cell division, and nuclear condensation. Disruption of this process is associated with myelodysplastic syndromes and congenital anemias. Congenital Dyserythropoietic Anemia type I (CDA-I), is an autosomal recessive disease that presents with severe macrocytic anemia early in infancy or early childhood. Patients with CDA-I have erythroid hyperplasia in the bone marrow. The erythroblasts in CDA-I are frequently binucleate, have chromatin bridging, and defective chromatin condensation (Renella 2011). CDA-I is most commonly caused by mutations in the protein codanin 1 (CDAN1). The function of CDAN1 is poorly understood, but it is thought to regulate histone incorporation into nascent DNA during cellular replication (Ask 2012).

The study of CDA-1 has been limited by lack of in vitro models that recapitulate key features of the disease, and to date, the majority of studies on CDAN1 function have been done in non-erythroid cells. To model CDA-I we introduced a point mutation (PM) commonly observed in CDA-1 patients (R1042W) into HUDEP2 cells. In addition, we generated two HUDEP2 cell lines with heterozygous deletion of CDAN1 (del-R1042). All CDAN1 mutant cell lines had decreased viability in during both expansion and terminal maturation compared to control lines. Chromatin bridges and multinucleate cells were observed in all three mutant CDAN1 lines but were most prominent in the PM line. Intriguingly, global gene analysis demonstrated that all mutated lines had significantly elevated gamma-globin expression compared to controls, consistent with reports of elevated fetal globin expression in CDA-1 patients. Interestingly, the PM cell line had faster cell divisions than the del-R1042 or control lines, characterized by decreased doubling time and verified by quantifying dilution of a fluorescent dye. In contrast, the del-R1042 lines had slower cell doubling times than both the controls and the PM line. The PM line also had an increased median intensity of BrdU compared to controls and del-R1042 lines, suggesting an accelerated S-phase. KI67 staining of the PM line showed an increase percentage of mitotic cells. These data are consistent with the finding that multinucleate cells and chromatin bridges are more common in the PM line. Furthermore, electron microscopy suggests the PM cell line may have defects in heterochromatin formation. Together, these data imply a specific functional role for residue R1042, and suggest that within the context of R1042W, loss of the arginine residue and replacement thereof with a tryptophan may have different mechanistic consequences.

Collectively, our preliminary data suggests that CDAN1 is important in the regulation of DNA replication and organization in maturing erythroblasts. Specific mutations may confer a change of function which results in dysplastic erythroid cells that are sensitive to dysregulated cell cycle mechanics due to the high rate of cell division. We hypothesize R1042W substitution may accelerate cell division at the expense of appropriate checkpoints and result in dysplastic erythroid cells and mimic the clinical presentation of increased multinuclearity, decreased cell viability, and increased gamma globin expression. Most importantly, generation of models with specific patient mutations will provide further mechanistic insight into CDA-I pathology.