Hereditary erythrocytosis is associated with a fetal-like EPO isoform

1. This case series identified three non-coding erythropoietin (EPO) variants that significantly increased gene expression under hypoxic conditions. 

2. The EPO variants induced altered glycosylation, modifying the protein’s isoelectric properties and enhancing its activity.

Evidence Rating Level: 3 (Average)

Study Rundown: Erythrocytosis, an excessive increase in circulating red blood cells, can be classified as primary or secondary. Secondary erythrocytosis often results from chronic hypoxia or inappropriate production of EPO, a hormone essential for red blood cell synthesis. Although genetic variants in the EPO gene have been linked to erythrocytosis, the mechanisms remain poorly understood. This study investigated six families with hereditary erythrocytosis who carried non-coding EPO variants and exhibited inappropriately normal serum EPO levels (5 to 25 IU/L). Researchers identified three specific variants: c.–252C→T in the promoter region and c.14-28T→C and c.14-26A→G in intron 1. Functional studies demonstrated that all three variants significantly increased EPO expression under hypoxic conditions, supporting their classification as pathogenic. Further analysis revealed that circulating EPO in affected individuals had a more basic isoelectric point than that of healthy controls, attributed to altered glycosylation. This profile resembled a hepatic-like EPO isoform, similar to fetal liver-derived EPO, which was also shown to have increased biological activity. These findings suggest that the variants not only promote overexpression of EPO but also result in a more potent, liver-like isoform, contributing to erythrocytosis. Although the study focused on only three variants, which may limit the generalizability of its conclusions, it provides novel insight into the pathogenesis of hereditary erythrocytosis. Specifically, it highlights a mechanism involving hypoxia-induced overexpression and altered post-translational modification of EPO driven by non-coding genetic variation.

Click here to read the study in NEJM

Relevant Reading: Erythrocytosis and Variants of EPO

In-Depth [case series]: In this case series, non-coding EPO genetic variants were identified and analyzed to investigate their role in the pathogenesis of erythrocytosis. Patients were included if they had erythrocytosis with moderate to severe elevations in hematologic values and normal serum EPO concentrations (5 to 25 IU/L). A total of 29 patients from six families were genetically analyzed, revealing three non-coding EPO variants: c.–252C→T (in the promoter region), and c.14–28T→C and c.14–26A→G (both in intron 1). No explicit exclusion criteria were provided. Functional analysis involved creating EPO promoter-driven luciferase constructs containing these variants. Luciferase assays were performed in HEK293 and Hep3B cells under both normoxic and hypoxic conditions (0.2% O₂ for 24 hours). Under hypoxia, reporter activity with the c.–252C→T variant was significantly increased compared to the wild-type (p<0.001), with upregulation dependent on the presence of a distal enhancer element. Additionally, c.–252C→T enhanced GATA4 binding (p<0.001), supporting its role in EPO upregulation. Luciferase assays also showed that inclusion of intron 1 reduced reporter activity in the presence of GATA3 (p=0.002), suggesting an inhibitory role of the intron. However, variants c.14–28T→C and c.14–26A→G diminished this repression, indicating disruption of the negative regulatory function. Induced pluripotent stem cells derived from patient samples were differentiated into hepatocyte-like cells. Under hypoxic conditions, these cells showed a 3.4- to 15.5-fold increase in EPO expression with the variants present (p<0.001). Isoelectric focusing revealed that patient-derived EPO had a more basic isoelectric point, reflecting altered glycosylation patterns. This profile was associated with increased biological activity, as measured by STAT5 phosphorylation, with significance observed at 1.2 IU/µL (p < 0.05). In summary, the study demonstrated that these non-coding EPO variants enhance gene expression, alter the isoelectric profile, and increase EPO biological activity, contributing to erythrocytosis.

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