Identification of Hepatic-like Erythropoietin with Enhanced Activity As a New Cause of Hereditary and Acquired Erythrocytosis

 Background:

Hereditary and acquired erythrocytosis are characterized by increased red cell mass. They can develop secondary to disorders that cause tissue hypoxia or to an inappropriate increase in erythropoietin (EPO) production by interstitial kidney cells. EPO is the primary regulator of erythropoiesis during early fetal development and throughout adult life. Although EPO is produced in the fetal liver before birth, the major site of EPO production becomes the kidney shortly after birth.

In this work, we identified six unrelated families with hereditary erythrocytosis associated with circulating EPO levels within the normal range, and characterized this condition as a novel molecular and functional entity.

 Methods: Luciferase reporter constructs were placed under the control of the mutant EPO regulatory elements to study the impact on EPO gene expression. Induced pluripotent stem cells (iPSC) were derived from patients’ blood, which were differentiated into hepatocyte-like EPO-producing cells. EPO proteins from affected family members and healthy newborns were isolated from blood and analyzed by isoelectric focusing (IEF), then the activity of the purified EPO was quantified.

 Results:

Using next-generation sequencing and segregation analysis, we identified three novel non-coding heterozygous mutations in the promoter and intron 1 of the EPO gene in the six families. Experiments with reporter assays and iPSC-derived hepatocyte-like cells showed that mutations target previously uncharacterized regulatory elements of the EPO gene that exhibit high responsiveness to HIF-2α. The circulating EPO protein from all patients with hereditary erythrocytosis examined in this study exhibited a unique and distinctive IEF profile compared to EPO produced by the kidneys of healthy adults. An identical IEF profile was also found in premature neonates, in whom EPO is produced by the liver, as well as in patients with acquired erythrocytosis associated with liver diseases. Interestingly, this profile, which shows a shift of the main EPO isoforms to a more basic region, was related to glycosylation modifications. This suggests lower contents of sialic acids and other modifications, indicating a liver-type glycosylation pattern, which differs from the classic kidney-type glycosylation pattern observed in adulthood. Finally, our functional assays of EPO protein purified from patients’ plasma and umbilical cord blood revealed a statistically significant enhanced activity, as reflected by increased EPO receptor signaling in a human cell line, pointing to a potential gain-of-function for the liver-type glycosylation of EPO.

 Conclusions: We report a new entity of secondary erythrocytosis named Hepatic-like Erythropoietin Polycythemia (HEP), characterized by the production of liver-like EPO with an atypical glycosylation pattern and increased activity. This new form of EPO has been observed in all patients with hereditary EPO-mutated erythrocytosis, in cases of erythrocytosis associated with liver disease, and in neonates.

Interestingly, the shift from a more active liver-type EPO to kidney-type EPO in newborns may explain the significant decrease in hemoglobin levels typically observed in the first weeks of life.

Thus, it is essential to consider quantitative and qualitative EPO screening as part of the biological tests for the diagnosis of idiopathic and hereditary erythrocytosis.