Biomarkers of ineffective erythropoiesis in patients with sickle cell anemia, at baseline and while treated with hydroxyurea, in comparison to patients with other iron-loading red cell disorders

Abstract Introduction. Ineffective erythropoiesis (IE) is defined by disproportionate expansion of the erythroid precursors relative to the red cell output. This imbalance significantly contributes to the pathology of several inherited erythroid disorders such as in β-thalassemia, congenital dyserythropoietic anemia (CDA), and as recently recognized, in sickle cell anemia (SCA). Increased expansion and activity of the bone marrow erythron leads to increased erythroferrone (ERFE) secretion that suppresses hepcidin production by hepatocytes, to increase iron absorption and mobilization and promote heme production for the heightened erythropoietic demand. With the increased erythroblast apoptosis/death characterizing IE, this overactivated ERFE–hepcidin axis drives tissue iron overload. Methods. We evaluated iron and IE biomarkers in patients with SCA with additional focus on the effects of hydroxyurea treatment. Key serum biomarkers measured by ELISA included ERFE, hepcidin, ferritin, growth differentiation factor 15 (GDF-15), and soluble transferrin receptor (sTfR). A total of 12 patients with SCA were evaluated over time, 11 before starting hydroxyurea and 10 after 1-10 years of treatment (18 total timepoints; 5 patients with paired samples at baseline and on hydroxyurea). In addition, 7 patients (17 total timepoints) with CDA-II, a typical example of IE, 8 patients with hereditary xerocytosis (HX) due to PIEZO1 carboxyterminal mutations with hemolysis but no anemia, and 15 healthy volunteers with no red cell disorder were also evaluated. Results. The hepcidin/ferritin ratio was significantly lower in SCA (both at baseline and on hydroxyurea), in CDA-II, and in HX compared to controls (p<0.0001), indicating suppressed hepcidin relative to the iron stores. Hydroxyurea treatment significantly improved this ratio in SCA patients (p=0.0219), suggesting amelioration of iron dysregulation possibly through reduced stress or inflammation. ERFE levels were significantly elevated in SCA at baseline, CDA-II, and HX (p=0.0014, p<0.0001, and p=0.001, respectively, vs controls), reflecting increased erythropoiesis. ERFE levels declined in SCA after hydroxyurea treatment; the reduction was not statistically significant between the two groups (p=0.0966), but paired samples had a significant decline (p=0.0463). GDF-15, another IE biomarker, was significantly elevated in SCA at baseline (p =0.0253) and on hydroxyurea (p=0.0073), as well as in CDA-II (p=<0.0001) compared to controls, but not in HX where erythropoiesis is increased but likely not ineffective. sTfR, a laboratory marker of increased erythropoietic activity and iron demand, was significantly elevated in SCA at both baseline (p<0.0001) and on hydroxyurea treatment (p=0.0025) compared to controls and was significantly lower in paired samples on hydroxyurea treatment (p=0.0014). Correlation analyses revealed a strong negative relationship between hemoglobin and sTfR as well as hemoglobin and ERFE levels in SCA (baseline) and CDA-II. Conclusion. In SCA and CDA-II, decreased hemoglobin levels are associated with IE. Achieving higher hemoglobin levels with hydroxyurea treatment in SCA and with chronic transfusions in CDA-II significantly lowers serum ERFE and improves IE. These findings also support the role of hydroxyurea in reducing erythropoietic stress and iron overload in SCA. HX and CDA-II are red cell disorders in which increased erythropoiesis and ERFE-mediated hepcidin suppression contribute to iron overload. Hepcidin suppression in HX has also been shown as an effect of direct inhibition of the BMP-SMADs pathway in hepatocytes by increased Ca2+ influx, due to overactive PIEZO1; even without history of transfusions, regular monitoring for iron overload in patients with HX is warranted. Patients with CDA-II frequently have profound IE and may require chronic transfusion in addition to iron chelation, with the goal to suppress IE-driven increased ERFE, and to decrease hepcidin suppression, in order to effectively treat iron overload. Finally, we demonstrate that SCA exhibits IE and iron dysregulation, with elevated ERFE and sTfR levels and inappropriately suppressed hepcidin. Hydroxyurea treatment in SCA appears to improve iron homeostasis by decreasing ineffective erythropoiesis, reducing ERFE and sTfR, and increasing the hepcidin/ferritin ratio, improving not only anemia but also the IE-driven iron overload.