Marra, Roberta (2021) Dissecting the molecular genetics and pathogenesis of Hereditary Dyserythropoietic Anemias. [Tesi di dottorato]

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Hereditary anemias (HAs) embrace a heterogeneous group of chronic disorders with a highly variable clinical picture. Within HAs, congenital dyserythropoietic anemias (CDAs) are a large group of hypo-productive anemias that result from various kinds of abnormalities during late stages of erythropoiesis. Among them, CDAI is characterized by relative reticulocytopenia, and congenital anomalies. It is caused by biallelic mutations in CDAN1 and C15orf41. Differential diagnosis, classification, and patient stratification of CDAs and related HAs are often difficult, particularly between CDAI-II and enzymatic defects, such as pyruvate kinase deficiency (PKD). The classical diagnostic workflow for these conditions includes different lines of investigation, in which genetic testing by next generation sequencing (NGS) approaches has become the frontline system. Indeed, the primary aim of this study was to analyze a large cohort of HAs patients (n=244), by our (t)-NGS RedPanel, to identify the proper molecular diagnosis despite their clinical suspicion. Indeed, only 16.3% of patients originally suspected to suffer from CDA (14/86) showed a matched genotype. Conversely, 64% of patients (72/86) initially suspected for CDA were diagnosed as other HAs, mainly PKD. In agreement with this observation, the analysis of the main erythroid markers demonstrated that PKD patients showed a dyserythropoietic component that may underlie the frequent misdiagnosis with CDAI-II. Beyond achieving a definitive diagnosis, knowing the genetic basis of these patients is valuable also for guiding treatment. Indeed, in our cohort of patients, we identified a novel case of syndromic CDA due to a novel variant in CAD gene, leading to a specific treatment with uridine supplementation. Finally, we described three cases of CDAI, identifying two novel variants in the DNA binding domain of C15orf41, Y94S and P20T, and another one in the nuclease domain of the protein, H230P. Functional characterization of these variants showed that the H230P leads to reduced gene expression and protein levels, while Y94S and P20T do not affect C15orf41 expression. Moreover, Y94S and H230P variants accounted for impaired erythroid differentiation in K562 cells, and H230P mutant also exhibits an increased S-phase of the cell cycle. Nowadays, C15orf41 is still an uncharacterized gene, encoding a protein with an unknown function. Thus, we aimed to unravel novel insights on its physiological role. Indeed, we demonstrated that C15orf41 endogenous protein exhibits nuclear and cytosolic localization, being mostly in the nucleus. Our data showed that C15orf41 is a cell-cycle regulated protein, mostly expressed during G1/S phase, and that both the predicted isoforms of the protein are degraded by the ubiquitin-proteasome pathway. Finally, we demonstrated that gene expression of C15orf41 and CDAN1, the other causative gene of CDAI, is tightly correlated, suggesting a shared mechanism of regulation between the two genes. Overall, these studies pointed out the relevance of genetic testing for the achievement of a correct and definitive diagnosis of CDAs and the related HAs, for the treatment of these conditions, and for elucidating the underlying pathogenic mechanisms of such rare disorders.

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