Erythrocytosis is a clinical condition characterized by an increased red blood cell count and elevated hemoglobin levels. One of the causes of familial erythrocytosis is genetic variants in the EPAS1 gene, also known as hypoxia-inducible transcription factor 2-alpha (HIF2A), which is involved in the regulation of erythropoiesis. The aim of this master's thesis was to investigate the functional impact of the EPAS1 variant (NM_001430.5) c.2120A>C (p.Lys707Thr), currently classified as a variant of uncertain significance (VUS), and compare it with the pathogenic EPAS1 variant (NM_001430.5) c.1609G>A (p.Gly537Arg) and the wild type. The impact on transcriptional activity was analyzed using a luciferase reporter assay, the effect on EPAS1 protein synthesis was analyzed using western blot, while the effect on protein stability was assessed using a cycloheximide assay. Both assays were performed in the HEK293 cell line. The results demonstrated increased transcriptional activity of the hypoxia response element (HRE) promoter for both variants compared to the wild type. Furthermore, both the p.Gly537Arg and p.Lys707Thr variants exhibited lower stability of the EPAS1 protein relative to the wild type, despite higher expression. Our findings suggest that the p.Lys707Thr variant may enhance EPAS1 protein function, potentially through the disruption of the nuclear localization process, thereby contributing to the development of erythrocytosis. Further functional studies are required to definitively establish its pathogenicity.
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