Background: A condition of an increased number of erythrocytes is termed erythrocytosis and coincides with elevated haemoglobin and/or haematocrit levels in the blood picture. The most common causes for erythrocytosis are acquired, while hereditary erythrocytosis is a rare condition with known causes in nine different genes (EPOR, VHL, EGLN1, EPAS1, EPO, HBB, HBA1, HBA2, and BPGM) that lead to eight different types of hereditary erythrocytosis (ECYT 1-8). Due to diverse aetiology, the diagnostics is problematic and the cause remains idiopathic in the majority of the cases. The molecular-genetic diagnostics of hereditary erythrocytosis was not yet established in Slovenia, hence many patients suspected of hereditary erythrocytosis remained without genetic cause and in some cases were also inappropriately treated.
Aims: The aim of this study was to find a genetic cause associated with the development of erythrocytosis in Slovenian patients suspected of hereditary erythrocytosis. We also evaluated the effect of the identified novel genetic variant on the development of erythrocytosis.
Methods: Selection of genes and regions for targeted genetic testing was performed with systematic revision of the literature and with analysis of molecular pathways, involved in erythropoiesis, using different in silico tools. A newly established national diagnostic algorithm identified 28 patients with symptoms of hereditary erythrocytosis, that were included in the study, including three families with two healthy family members. Samples were analyzed using a targeted next-generation sequencing (NGS). The selected novel genetic variant was further functionally assessed with localization of the variant on a 3D protein structure, protein expression analysis with quantitative western blotting, and protein activity analysis using a luciferase reporter assay.
Results: 39 genes with association with erythrocytosis and iron overload were selected for NGS analysis. Targeted NGS of 28 patients identified one known pathogenic variant in the EPAS1 gene, c.1609G>A (p.(Gly537Arg)), and four variants of unknown significance (VUS) in the EGLN1, JAK2, EPAS1, and SH2B3 genes. In one family we also identified two low-frequency variants (< 5%) in the EGLN1 and JAK2 genes. Additionally, we observed a high incidence of variant carriers for pathogenic variants in the HFE gene. VUS c.1072C>T (p.(Pro358Ser)) in the EGLN1 gene was observed in strong co-segregation with a disease in multiple family members, had high pathogenicity prediction scores, and was not reported in the literature and clinical database, therefore this variant was further functionally assessed. Localization on a 3D protein model showed that the variant is positioned in an active site of the protein and may affect protein activity. Protein quantification results showed statistically significant (P < 0.0001) reduced protein expression of EGLN1 with variant c.1072C>T (p.(Pro358Ser)) in comparison to the wild-type protein. Both results implicate reduced activity of the protein. However, the luciferase reporter assay did not confirm the effect of variant c.1072C>T (p.(Pro358Ser)) on the EGLN1 activity.
Conclusions: With targeted NGS we successfully confirmed the genetic cause for erythrocytosis in one patient, the first Slovenian patient with hereditary erythrocytosis (ECYT4). Additionally, we identified four VUS in the EGLN1, JAK2, EPAS1, and SH2B3 genes, among them VUS in the EGLN1 gene was selected for further functional assessment. Results of functional tests showed the position of the variant in an active site of EGLN1 protein and an effect on EGLN1 expression, but not on EGLN1 activity. However, further tests are necessary to review the effect of the variant on protein function and further transcription regulation of hypoxia-inducible targets.
|
