Novel EGLN1 variants identified in patients with erythrocytosis: a functional study

Erythrocytosis, a disorder with increased erythrocyte production, has a heterogeneous aetiology, including rare congenital types linked to dysregulation of the oxygen-sensing pathway. Variants in the EGLN1 gene, encoding the prolyl hydroxylase that regulates hypoxia-inducible factor (HIF) stability, are associated with familial erythrocytosis type 3 (ECYT3). In patients with idiopathic erythrocytosis we previously identified two novel EGLN1 variants, c.1072C>T (p.(Pro358Ser)) and c.1124A>G (p.(Glu375Gly)), classified as variants of uncertain significance. Herein, we performed in silico and in vitro analyses to assess their structural and functional effects, using the known pathogenic variant p.(His374Arg) as a positive control. AlphaFold3 predictions revealed minimal conformational changes in the protein core for all variants, while stability predictions suggested reduced protein stability. Functional assays in HEK293 cells demonstrated significantly decreased protein levels and stability for p.(Pro358Ser) and p.(Glu375Gly), comparable to p.(His374Arg). However, luciferase reporter assays showed that, unlike p.(His374Arg), the novel variants did not substantially impair EGLN1 enzymatic activity or activate HIF signalling. Our results suggest that the novel variants may contribute to erythrocytosis through destabilization of EGLN1, supporting further studies to elucidate their precise impact on hypoxia regulation. This study highlights the complexity of studying EGLN1 variants and the importance of functional evaluation for clinical interpretation.