P1475: mutations in the gap domain of racgap1 cause developmental and erythroid defects in zebrafish

Topic: 28. Enzymopathies, membranopathies and other anemias Background: We recently reported that RACGAP1 mutations are responsible for Congenital Dyserythropoietic Anemia (CDA) type IIIb, a rare autosomal recessive disease characterized by macrocytic anemia and giant multinucleated erythroblasts in the bone marrow. So far, three RACGAP1 mutations have been identified causing CDA IIIb in humans: Thr220Ala, Leu396Gln and Pro432Ser, the two latter affecting the GAP domain. It was previously described that complete abrogation of Racgap1 in Danio rerio (zebrafish) shows defects in cytokinesis and erythropoiesis with an anemic phenotype (ogre mutant). The effect of specific point mutations in the Racgap1 gene in zebrafish is not known. Aims: We generated two Racgap1 knock-in zebrafish models within its GAP domain (Pro415Ser and Pro440Ser, equivalent to human Pro407Ser and Pro432Ser, respectively) to study the pathogenicity of these variants in animal models. Methods: The knock-in mutants were generated with CRISPR technology. Single-cell zebrafish embryos were injected with protein Cas9, a dgRNA to generate a double-strand break and a ssODN for homologous recombination. Fish were allowed to grow for 3 months before screening experiments to identify the mutants. F1 heterozygous fish were crossed and the offspring was analyzed for developmental and erythroid defects. Results: The zebrafish Racgap1 Pro440Ser mutant is lethal, as shown by a negative selection of mutated fish. The homozygous zebrafish Racgap1 Pro415Ser mutant is viable; however, mutated zebrafish show development impairment and erythroid defects with a 10% of surviving individuals presenting a bloodless phenotype. Summary/Conclusion: Racgap1 mutations within the GAP domain in zebrafish are key for an appropriate global development and particularly for erythropoiesis. These results further confirm the pathogenicity of these variants in the erythroid context by D.rerio as an animal model.Left panels: WT zebrafish morphology (top) and of their circulating erythrocytes (bottom). Right panels: homozygous Racgap1 Pro415Ser zebrafish morphology (top) and of their circulating erythrocytes (bottom). Keywords: Anemia, Gene mutation, Zebra fish, Erythroid