P1470: identification of the pro20thr novel variant in cdin1 gene allowed exploring the biological processes impaired in congenital dyserythropoietic anemia type i

Topic: 28. Enzymopathies, membranopathies and other anemias Background: Congenital dyserythropoietic anemia type I (CDAI) is a rare anemia characterized by ineffective erythropoiesis. It is due to biallelic mutations either in CDAN1 or in the uncharacterized gene CDIN1. To date, only 10 CDIN1 pathogenic variants have been identified. We previously described two cases of CDIN1-related CDA I in which we identified two novel variants, c.281A>C, p.Tyr94Ser (Y94S) and c.689A>C, p.His230Pro (H230P), demonstrating that the nuclease domain variant, H230P, affects gene and protein stability, while the DNA-binding domain mutation, Y94S, preserves its expression. Nevertheless, both variants cause impaired erythroid differentiation in K562 mutant cells. To date, the diagnostic rate of hereditary red blood cell defects is about 80%, and the clinical heterogeneity and the overlapping phenotype of these conditions suggest that many CDA I patients are still unrevealed. Aims: Identify patients affected by CDAI and define biological processes impaired in CDIN1-related CDAI by differential expression analysis of K562 CDIN1-wildtype (WT) cells compared to Y94S-, H230P-, P20T-CDIN1 mutant cells. Methods: targeted-NGS (t-NGS); Whole Exome Sequencing (WES); stable clone development; erythroid differentiation; qRT-PCR; western blot; flow cytometry; RNA-sequencing. Results: We described two unrelated patients (P1 and P2, 8 and 7 y.o., respectively) both presenting mild slight-macrocytic hypo-productive anemia (Hb >10 g/dL; MCV between 90-100 f/L; bone marrow responsiveness index: P1=12, P2=74). Both patients presented hyper-cellularity of the BM and bi-/multi-nucleated normoblasts. P2 presented inter-nuclear bridges at BM smear. Sanger sequencing of CDAN1 of both patients showed no causative variants. Re-analysis of the cases by t-NGS or WES allowed us the identification of the same novel missense variant in the DNA-binding domain of CDIN1, c.58C>A, p.Pro20Thr, in homozygous state. In contrast to the effect observed for the previously identified variants, Y94S and H230P, K562 cells stably over-expressing CDIN1-P20T showed a slight increase of gene and protein expression compared to WT cells. Nevertheless all variants caused impaired erythroid differentiation after hemin treatment. To define the pathways impaired in all CDIN1 mutated cells, we performed RNA-sequencing of K562 CDIN1-WT, -H230P, -Y94S, and -P20T overexpressing cells. We observed on average 1130±175 genes differentially regulated (611±131 downregulated; 518±255 upregulated) in each mutant compared to the WT (genes with Log2FC ± 0.5 and -Log p-value > 1 were considered significant). We identified four main biological processes significantly downregulated in all mutants: regulation of transcription, cell proliferation, nucleosome assembly, and actin cytoskeleton organization which could represent the common denominator of CDIN1-related CDAI. Moreover, we identified 15 pathways (164 genes) commonly deregulated (up/down regulated) in all mutants. Among them, regulation of autophagy, response to hypoxia, and regulation of phosphatidylinositol 3-kinase signaling are currently investigated in a CDIN1 silenced HUDEP-2 cellular model. Summary/Conclusion: The identification of the novel variant c.58C>A, p.Pro20Thr in CDIN1 in two unrelated patients contributed to better defining CDAI pathophysiology and recurrence. Moreover, transcriptome analysis of three different CDIN1 variants (P20T, Y94S, and H230P) allowed the identification of the biological processes deregulated in CDIN1-related CDAI, opening new perspectives on the mechanisms that lead to impaired erythropoiesis in CDAI. Keywords: RNA-seq, Erythroid differentiation, Hemolytic anemia, Erythropoieisis