Nostroso, Antonella (2025) Dissecting the molecular genetics and pathogenesis of dyserythropoiesis in Hereditary Anemias. [Tesi di dottorato]

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Abstract

Hereditary anemias (HAs) represent a large group of disorders caused by mutations in genes that regulate hemoglobin production, erythropoiesis, and the structure or metabolism of red blood cells (RBC). In this study, we investigated erythroid maturation defects in three different HA subtypes. Specifically, we focused on congenital dyserythropoietic anemias both type I and II (CDA I-II) and dehydrated hereditary stomatocytosis (DHS). CDA I and II, caused by mutations in CDAN1/CDIN1 or SEC23B respectively, are autosomal recessive disorders characterized by defective erythroid differentiation and ineffective erythropoiesis. In contrast, DHS is autosomal dominant anemia caused by gain- of-function mutations in PIEZO1, leading to abnormal intracellular cation content and altered cell volume. Despite their distinct pathogenesis, CDAs and DHS share hallmark features, including dyserythropoiesis and iron overload, which represent their main complications. Nowadays, the pathogenic mechanisms underlying these defects are not yet fully elucidated. Thus, the primary aim of this study was to investigate the mechanisms impairing erythroid differentiation using disease cellular models. By using HUDEP-2 erythroid progenitor cells, we demonstrated that loss-of-function mutations in SEC23B and CDIN1 block intermediate-to-late erythroid maturation, while gain-of- function variants in PIEZO1 enhance erythropoiesis by accelerating the accumulation of mature erythroblasts and promoting enucleation. Transcriptomic analyses revealed stage-specific alterations in shared biological pathways across CDA models, particularly in rRNA processing and ribosome biogenesis. Whereas the DHS model showed an imbalance in glycolytic flux, leading to energy deficits due to reduced ATP production. In parallel, the second aim of this study was to establish a genotype-phenotype correlation in patients with dual inheritance of SEC23B (CDA II) and PIEZO1 (DHS1) mutations. Among a large cohort of 581 HA patients referred for genetic testing, we identified patients carrying pathogenic variants in both genes, a condition termed dual inheritance. Patients with dual inheritance exhibited significantly higher hemoglobin levels, absolute reticulocyte counts, and bone marrow responsiveness index compared to those with isolated HA subtypes. Additionally, dual patients showed more severe iron overload, with 64% exceeding clinical ferritin thresholds. Accordingly, intermediate erythroferrone levels and markedly reduced hepcidin concentrations in dual-inheritance patients were observed. To further investigate these observations, we developed a hepatic cellular model combining SEC23B silencing and PIEZO1 gain-of-function mutation. Functional assays demonstrated a synergistic effect, including marked HAMP downregulation and disruption of the BMP/SMAD pathway compared to cellular models carrying isolated gene defects. This study sheds light on the molecular basis of erythroid dysfunction in hereditary dyserythropoietic anemias and reveals synergistic genetic effects on iron homeostasis, providing insights into improved diagnostics and therapies for hereditary anemias.

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