Erythrocytosis (ECYT) is a pathological condition characterized by an increased number of red blood cells and consequently higher concentrations of hemoglobin in the blood. A key role in regulating erythropoiesis is played by the hypoxia-inducible factor (HIF), whose stability and activity are regulated by prolyl hydroxylase 2 (PHD2), encoded by the EGLN1 gene. Certain genetic variants of EGLN1 reduce stability of EGLN1/PHD2 and are therefore associated with the development of erythrocytosis. The aim of this master’s thesis was to investigate the effect of two new genetic variants in the EGLN1 gene, c.1072C>T, p.(Pro358Ser) and c.1124A>G, p.(Glu375Gly), on the stability of the PHD2 protein. The stability of the EGLN1/PHD2 variants (p.(Glu375Gly) and p.(Pro358Ser)) was compared with the wild type (WT) as well as with a positive (p.(His374Arg)) and negative (p.(Gln157His)) control. The selected EGLN1 variants and controls were introduced into the HEK293 cell line, and after 24 hours, translation was halted by the addition of cycloheximide. Cells were lysed at various time points (T0, T3, T6, T10) following cycloheximide treatment, and protein expression was assessed via western blot analysis. The results showed that the WT and negative control did not affect EGLN1/PHD2 protein stability, whereas the other variants led to reduced stability. Statistically significant results were obtained for the p.(His374Arg) and p.(Pro358Ser) variants, while statistical significance was not achieved for p.(Glu375Gly), likely due to biological variability between replicates. Comparative analysis of the control variants confirmed the suitability of the selected method for functional analysis of EGLN1 gene variants. Reduced protein stability of the tested genetic variants indicates an impact on protein function, confirming that the new variant p.(Pro358Ser) potentially contributes to the development of ECYT.
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