Characterization of erythroferrone oligomerization and its impact on BMP antagonism.

Hepcidin, a peptide hormone that negatively regulates iron metabolism, is expressed by bone morphogenetic protein (BMP) signaling. Erythroferrone (ERFE) is an extracellular protein that binds and inhibits BMP ligands, thus positively regulating iron import by indirectly suppressing hepcidin. This allows for rapid erythrocyte regeneration after blood loss. ERFE belongs to the C1Q/TNF-related protein family and is suggested to adopt multiple oligomeric forms: a trimer, a hexamer, and a high molecular weight species. The molecular basis for how ERFE binds BMP ligands and how the different oligomeric states impact BMP inhibition are poorly understood. In this study, we demonstrated that ERFE activity is dependent on the presence of stable dimeric or trimeric ERFE and that larger species are dispensable for BMP inhibition. Additionally, we used an in silico approach to identify a helix, termed the ligand-binding domain, that was predicted to bind BMPs and occlude the type I receptor pocket. We provide evidence that the ligand-binding domain is crucial for activity through luciferase assays and surface plasmon resonance analysis. Our findings provide new insight into how ERFE oligomerization impacts BMP inhibition, while identifying critical molecular features of ERFE essential for binding BMP ligands.

Characterization of erythroferrone structural domains relevant to its iron-regulatory function.

Iron delivery to the plasma is closely coupled to erythropoiesis, the production of red blood cells, as this process consumes most of the circulating plasma iron. In response to hemorrhage and other erythropoietic stresses, increased erythropoietin stimulates the production of the hormone erythroferrone (ERFE) by erythrocyte precursors (erythroblasts) developing in erythropoietic tissues. ERFE acts on the liver to inhibit bone morphogenetic protein (BMP) signaling and thereby decrease hepcidin production. Decreased circulating hepcidin concentrations then allow the release of iron from stores and increase iron absorption from the diet. Guided by evolutionary analysis and Alphafold2 protein complex modeling, we used targeted ERFE mutations, deletions, and synthetic ERFE segments together with cell-based bioassays and surface plasmon resonance to probe the structural features required for bioactivity and BMP binding. We define the ERFE active domain and multiple structural features that act together to entrap BMP ligands. In particular, the hydrophobic helical segment 81 to 86 and specifically the highly conserved tryptophan W82 in the N-terminal region are essential for ERFE bioactivity and Alphafold2 modeling places W82 between two tryptophans in its ligands BMP2, BMP6, and the BMP2/6 heterodimer, an interaction similar to those that bind BMPs to their cognate receptors. Finally, we identify the cationic region 96-107 and the globular TNFα-like domain 186-354 as structural determinants of ERFE multimerization that increase the avidity of ERFE for BMP ligands. Collectively, our results provide further insight into the ERFE-mediated inhibition of BMP signaling in response to erythropoietic stress.

Erythroferrone and hepcidin levels in children with iron deficiency anemia.

BACKGROUND: Iron deficiency anemia remains a significant public health issue in developing countries. The regulation of iron metabolism is primarily controlled by hepcidin, a key regulatory protein. During erythropoiesis, erythroferrone (ERFE), a hormone produced by erythroblasts in response to erythropoietin (EPO) synthesis, mediates the suppression of hepcidin. In this study, it was aimed to determine the correlation between erythroferrone (ERFE) and hepcidin levels in children with iron deficiency anemia. METHODS: This is a case-control study conducted at Kirsehir Ahi Evran University Training and Research Hospital Pediatrics Clinic between 1 and 31 September 2020. The study included 26 healthy children and 26 children with iron deficiency anemia. In order to evaluate iron status,whole blood count, serum iron, total iron binding capacity (TIBC), and ferritin levels were analyzed. The study measured the levels of hepcidin and erythroferrone in the serum of children diagnosed with iron deficiency before and after one month of iron treatment, as well as in a control group, using the ELISA method. Correlation between whole blood count, initial ferritin, hepcidin, ERFE and ferritin in the iron deficiency group was evaluated. RESULTS: Compared with healthy controls, the iron-deficient group had significantly lower haemoglobin (p < 0.001), MCV (p = 0.001), MCH (p < 0.001), MCHC (p < 0.001), iron (p < 0.001), ferritin (p < 0.001) and hepcidin (p = 0.001). Ferritin and hepcidin levels increased while erythroferrone levels remained unchanged after iron deficiency treatment. There was no correlation between hepcidin and ferritin levels in treatment group. CONCLUSIONS: The study found a strong and positive correlation between ferritin and hepcidin levels in iron-deficient children, but not between ERFE levels, suggesting that hepcidin is largely regulated by iron deposition levels. In addition, there was an increase in ferritin and hepcidin levels after iron treatment. The study found no significant difference in erythroferrone levels between the iron-deficient group and the control group. It is thought that this may be due to the short duration of iron treatment given to the patients with iron deficiency anemia included in the study.

Elevated erythroferrone distinguishes erythrocytosis with inherited defects in oxygen-sensing pathway from primary familial and congenital polycythaemia.

Congenital erythrocytoses represent a heterogenous group of rare defects of erythropoiesis characterized by elevated erythrocyte mass. We performed molecular-genetic analysis of 21 Czech patients with congenital erythrocytosis and assessed the mutual link between chronic erythrocyte overproduction and iron homoeostasis. Causative mutations in erythropoietin receptor (EPOR), hypoxia-inducible factor 2 alpha (HIF2A) or Von Hippel-Lindau (VHL) genes were detected in nine patients, including a novel p.A421Cfs*4 EPOR and a homozygous intronic c.340+770T>C VHL mutation. The association and possible cooperation of five identified missense germline EPOR or Janus kinase 2 (JAK2) variants with other genetic/non-genetic factors in erythrocytosis manifestation may involve variants of Piezo-type mechanosensitive ion channel component 1 (PIEZO1) or Ten-eleven translocation 2 (TET2), but this requires further research. In two families, hepcidin levels appeared to prevent or promote phenotypic expression of the disease. No major contribution of heterozygous haemochromatosis gene (HFE) mutations to the erythrocytic phenotype or hepcidin levels was observed in our cohort. VHL- and HIF2A-mutant erythrocytosis showed increased erythroferrone and suppressed hepcidin, whereas no overproduction of erythroferrone was detected in other patients regardless of molecular defect, age or therapy. Understanding the interplay between iron metabolism and erythropoiesis in different subgroups of congenital erythrocytosis may improve current treatment options.

The effect of erythroferrone suppression by transfusion on the erythropoietin-erythroferrone-hepcidin axis in transfusion-dependent thalassaemia: A pre-post cohort study.

To track post-transfusion changes on the erythropoietin (EPO)-erythroferrone (ERFE)-hepcidin axis, we collected blood samples from 82 regularly transfused patients with ß-thalassaemia major (ß-TM) immediately before and 4-6 days after transfusion. The post-transfusion haemoglobin, hepcidin, and ferritin levels were increased, while the EPO, ERFE, and soluble transferrin receptor were suppressed. In addition, hepcidin change was inversely associated with erythropoietic change, which was confirmed by an increase in the hepcidin-to-ERFE ratio after transfusion. Age was the main predictor of serum ERFE, followed by EPO, transfusion frequencies, and ferritin. We found ERFE to be a highly sensitive indicator of erythroid activity in ß-TM and that the hepcidin-to-ERFE ratio after transfusion may be used as an appropriateness index of serum hepcidin regulation relative to the degree of erythropoiesis.

Managing the Dual Nature of Iron to Preserve Health.

Because of its peculiar redox properties, iron is an essential element in living organisms, being involved in crucial biochemical processes such as oxygen transport, energy production, DNA metabolism, and many others. However, its propensity to accept or donate electrons makes it potentially highly toxic when present in excess and inadequately buffered, as it can generate reactive oxygen species. For this reason, several mechanisms evolved to prevent both iron overload and iron deficiency. At the cellular level, iron regulatory proteins, sensors of intracellular iron levels, and post-transcriptional modifications regulate the expression and translation of genes encoding proteins that modulate the uptake, storage, utilization, and export of iron. At the systemic level, the liver controls body iron levels by producing hepcidin, a peptide hormone that reduces the amount of iron entering the bloodstream by blocking the function of ferroportin, the sole iron exporter in mammals. The regulation of hepcidin occurs through the integration of multiple signals, primarily iron, inflammation and infection, and erythropoiesis. These signals modulate hepcidin levels by accessory proteins such as the hemochromatosis proteins hemojuvelin, HFE, and transferrin receptor 2, the serine protease TMPRSS6, the proinflammatory cytokine IL6, and the erythroid regulator Erythroferrone. The deregulation of the hepcidin/ferroportin axis is the central pathogenic mechanism of diseases characterized by iron overload, such as hemochromatosis and iron-loading anemias, or by iron deficiency, such as IRIDA and anemia of inflammation. Understanding the basic mechanisms involved in the regulation of hepcidin will help in identifying new therapeutic targets to treat these disorders.

Iron Mining for Erythropoiesis.

Iron is necessary for essential processes in every cell of the body, but the erythropoietic compartment is a privileged iron consumer. In fact, as a necessary component of hemoglobin and myoglobin, iron assures oxygen distribution; therefore, a considerable amount of iron is required daily for hemoglobin synthesis and erythroid cell proliferation. Therefore, a tight link exists between iron metabolism and erythropoiesis. The liver-derived hormone hepcidin, which controls iron homeostasis via its interaction with the iron exporter ferroportin, coordinates erythropoietic activity and iron homeostasis. When erythropoiesis is enhanced, iron availability to the erythron is mainly ensured by inhibiting hepcidin expression, thereby increasing ferroportin-mediated iron export from both duodenal absorptive cells and reticuloendothelial cells that process old and/or damaged red blood cells. Erythroferrone, a factor produced and secreted by erythroid precursors in response to erythropoietin, has been identified and characterized as a suppressor of hepcidin synthesis to allow iron mobilization and facilitate erythropoiesis.

The secreted BMP antagonist ERFE is required for the development of a functional circulatory system in Xenopus.

The hormone Erythroferrone (ERFE) is a member of the C1q/TNF-related protein family that regulates iron homeostasis through the suppression of hamp. In a gain of function screen in Xenopus embryos, we identified ERFE as a potent secondary axis-inducing agent. Experiments in Xenopus embryos and ectodermal explants revealed that ERFE functions as a selective inhibitor of the BMP pathway and the conserved C1q domain is not required for this activity. Inhibition occurs at the extracelluar level, through the interaction of ERFE with the BMP ligand. During early Xenopus embryogenesis, erfe is first expressed in the ventral blood islands where initial erythropoiesis occurs and later in circulating blood cells. ERFE knockdown does not alter the expression of etv.2, aplnr and flt1 in tailbud stage embryos indicating endothelial cell specification is independent of ERFE. However, in tadpole embryos, defects of the vascular network and primitive blood circulation are observed as well as edema formation. RNAseq analysis of ERFE morphant embryos also revealed the inhibition of gja4 indicating disruption of dorsal aorta formation.

Erythroferrone structure, function, and physiology: Iron homeostasis and beyond.

Erythroferrone (ERFE) is the main erythroid regulator of hepcidin, the homeostatic hormone controlling plasma iron levels and total body iron. When the release of erythropoietin from the kidney stimulates the production of new red blood cells, it also increases the synthesis of ERFE in bone marrow erythroblasts. Increased ERFE then suppresses hepcidin synthesis, thereby mobilizing cellular iron stores for use in heme and hemoglobin synthesis. Recent mechanistic studies have shown that ERFE suppresses hepcidin transcription by inhibiting bone morphogenetic protein signaling in hepatocytes. In ineffective erythropoiesis, pathological overproduction of ERFE by an expanded population of erythroblasts suppresses hepcidin and causes iron overload, even in non-transfused patients. ERFE may be a useful biomarker of ineffective erythropoiesis and an attractive target for treating its systemic effects.

Amelioration of chronic kidney disease-associated anemia by vadadustat in mice is not dependent on erythroferrone.

Vadadustat is an investigational hypoxia-inducible factor prolyl hydroxylase inhibitor that increases endogenous erythropoietin production and has been shown to decrease hepcidin levels, ameliorate iron restriction, and increase hemoglobin concentrations in anemic patients with chronic kidney disease (CKD). In studies of physiological responses to other erythropoietic stimuli, erythropoietin induced erythroblast secretion of erythroferrone (ERFE), which acts on the liver to suppress hepcidin production and mobilize iron for erythropoiesis. We therefore investigated whether vadadustat effects on erythropoiesis and iron metabolism are dependent on ERFE. Wild type and ERFE knockout mice with and without CKD were treated with vadadustat or vehicle. In both wild type and ERFE knockout CKD models, vadadustat was similarly effective, as evidenced by normalized hemoglobin concentrations, increased expression of duodenal iron transporters, lower serum hepcidin levels, and decreased tissue iron concentrations. This is consistent with ERFE-independent increased iron mobilization. Vadadustat treatment also lowered serum urea nitrogen and creatinine concentrations and decreased expression of kidney fibrosis markers. Lastly, vadadustat affected fibroblast growth factor 23 (FGF23) profiles: in non-CKD mice, vadadustat increased plasma total FGF23 out of proportion to intact FGF23, consistent with the known effects of hypoxia-inducible factor-1α and erythropoietin on FGF23 production and metabolism. However, in the mice with CKD, vadadustat markedly decreased both total and intact FGF23, effects likely contributed to by the reduced loss of kidney function. Thus, in this CKD model, vadadustat ameliorated anemia independently of ERFE, improved kidney parameters, and decreased FGF23. How vadadustat affects CKD progression in humans warrants future studies.

Disordered serum erythroferrone and hepcidin levels as indicators of the spontaneous abortion occurrence during early pregnancy in humans.

Spontaneous abortion is a common, detrimental outcome of pregnancy, and can be induced by a variety of factors, including pathophysiological conditions and socioeconomic circumstances. Despite numerous studies examining the aetiology of spontaneous abortion, there is limited understanding of the disordered iron transportation between mother and fetus through the placenta. Recently, erythroferrone (ERFE) was recognized as a novel negative regulator of hepcidin that can elevate nutritional iron absorption and macrophagic iron egress for enhanced erythropoiesis. However, its diagnostic significance in different disease conditions associated with iron remains poorly understood. In the current study, we discovered disordered maternal iron homeostasis in women who had spontaneous abortions during early pregnancy, as characterized by increased serum iron and hepcidin levels, and conversely, reduced serum ERFE levels, compared to healthy control individuals and women with normal pregnancy. Comprehensive statistical analyses revealed the correlation between different variables and pregnancy status, signifying the pronounced diagnostic value of an increased ratio of serum hepcidin and ERFE (HE ratio) in recognizing adverse pregnancy status. In contrast to previous non-selective discrete surrogates, such as iron, hepcidin and ferritin, the HE ratio may otherwise stand for a novel and more representative hallmark for early spontaneous abortion.

High erythroferrone expression in CD71+ erythroid progenitors predicts superior survival in myelodysplastic syndromes.

Ineffective erythropoiesis and iron overload are common in myelodysplastic syndromes (MDS). Erythroferrone (ERFE) and growth/differentiation factor 15 (GDF15) are two regulators of iron homeostasis produced by erythroid progenitors. Elevated systemic levels of ERFE and GDF15 in MDS are associated with dysregulated iron metabolism and iron overload, which is especially pronounced in MDS with SF3B1 gene mutations. However, the role of ERFE and GDF15 in MDS pathogenesis and their influence on disease progression are largely unknown. Here, we analyzed the expression of ERFE and GDF15 in CD71+ erythroid progenitors of n = 111 MDS patients and assessed their effects on patient survival. The expression of ERFE and GDF15 in MDS was highly aberrant. Unexpectedly, ERFE expression in erythroprogenitors was highly relevant for MDS prognosis and independent of International Prognostic Scoring System (IPSS) stratification. Although ERFE expression was increased in patients with SF3B1 mutations, it predicted overall survival (OS) in both the SF3B1wt and SF3B1mut subgroups. Of note, ERFE overexpression predicted superior OS in the IPSS low/Int-1 subgroup and in patients with normal karyotype. Similar observations were made for GDF15, albeit not reaching statistical significance. In summary, our results revealed a strong association between ERFE expression and MDS outcome, suggesting a possible involvement of ERFE in molecular MDS pathogenesis.

Is the erythropoietin-erythroferrone-hepcidin axis intact in human neonates?

In a two-part process, we assessed elements of the principal hormonal pathway regulating iron homeostasis in human neonates. Part 1: Quantifying erythropoietin (Epo), erythroferrone (ERFE), hepcidin, and relevant serum and erythrocytic iron-related metrics in umbilical cord blood from term (n = 13) and preterm (n = 10) neonates, and from neonates born to mothers with diabetes and obesity (n = 13); Part 2: Quantifying serum Epo, ERFE, and hepcidin before and following darbepoetin administration. Part 1: We measured Epo, ERFE and hepcidin in all cord blood samples. Epo and ERFE levels did not differ between the three groups. Preterm neonates had the lowest hepcidin levels, while neonates born to diabetic women with a very high BMI had the lowest ferritin and RET-He levels. Part 2: Following darbepoetin dosing, ERFE levels generally increased (p < 0.05) and hepcidin levels generally fell (p < 0.05). Our observations suggest that the Epo/ERFE/hepcidin axis is intact in the newborn period.

Effects of altitude and recombinant human erythropoietin on iron metabolism: a randomized controlled trial.

Current markers of iron deficiency (ID), such as ferritin and hemoglobin, have shortcomings, and hepcidin and erythroferrone (ERFE) could be of clinical relevance in relation to early assessment of ID. Here, we evaluate whether exposure to altitude-induced hypoxia (2,320 m) alone, or in combination with recombinant human erythropoietin (rHuEPO) treatment, affects hepcidin and ERFE levels before alterations in routine ID biomarkers and stress erythropoiesis manifest. Two interventions were completed, each comprising a 4-wk baseline, a 4-wk intervention at either sea level or altitude, and a 4-wk follow-up. Participants (n = 39) were randomly assigned to 20 IU·kg body wt-1 rHuEPO or placebo injections every second day for 3 wk during the two intervention periods. Venous blood was collected weekly. Altitude increased ERFE (P ≤ 0.001) with no changes in hepcidin or routine iron biomarkers, making ERFE of clinical relevance as an early marker of moderate hypoxia. rHuEPO treatment at sea level induced a similar pattern of changes in ERFE (P < 0.05) and hepcidin levels (P < 0.05), demonstrating the impact of accelerated erythropoiesis and not of other hypoxia-induced mechanisms. Compared with altitude alone, concurrent rHuEPO treatment and altitude exposure induced additive changes in hepcidin (P < 0.05) and ERFE (P ≤ 0.001) parallel with increases in hematocrit (P < 0.001), demonstrating a relevant range of both hepcidin and ERFE. A poor but significant correlation between hepcidin and ERFE was found (R2 = 0.13, P < 0.001). The findings demonstrate that hepcidin and ERFE are more rapid biomarkers of changes in iron demands than routine iron markers. Finally, ERFE and hepcidin may be sensitive markers in an antidoping context.

Associations among erythropoietic, iron-related, and FGF23 parameters in pediatric kidney transplant recipients.

BACKGROUND: In pediatric kidney transplant recipients, anemia is common and oftentimes multifactorial. Hemoglobin concentrations may be affected by traditional factors, such as kidney function and iron status, as well as novel parameters, such as fibroblast growth factor 23 (FGF23). METHODS: Here, we evaluated associations among erythropoietic, iron-related, and FGF23 parameters in a cohort of pediatric kidney transplant recipients, hypothesizing that multiple factors are associated with hemoglobin concentrations. RESULTS: In a cross-sectional analysis of 59 pediatric kidney transplant recipients (median (interquartile range) age 16.3 (13.5, 18.6) years, median estimated glomerular filtration rate (eGFR) 67 (54, 87) ml/min/1.73 m2), the median age-related hemoglobin standard deviation score (SDS) was -2.1 (-3.3, -1.1). Hemoglobin SDS was positively associated with eGFR and calcium, and was inversely associated with erythropoietin (EPO), mycophenolate dose, and total, but not intact, FGF23. In multivariable analysis, total FGF23 remained inversely associated with hemoglobin SDS, independent of eGFR, iron parameters, EPO, and inflammatory markers, suggesting a novel FGF23-hemoglobin association in pediatric kidney transplant patients. In a subset of patients with repeat measurements, only delta hepcidin was inversely associated with delta hemoglobin SDS. Also, delta EPO positively correlated with delta erythroferrone (ERFE), and delta ERFE inversely correlated with delta hepcidin, suggesting a possible physiologic role for the EPO-ERFE-hepcidin axis in the setting of chronic kidney disease (CKD). CONCLUSION: Our study provides further insight into factors potentially associated with erythropoiesis in pediatric kidney transplant recipients. A higher resolution version of the Graphical abstract is available as Supplementary information.

Saccharated ferric oxide attenuates haematopoietic response induced by epoetin beta pegol in patients undergoing haemodialysis.

BACKGROUND: Decreased erythropoietin levels and impaired iron metabolism due to excessive hepcidin levels are responsible for renal anaemia in patients undergoing haemodialysis. Recently, erythroferrone (ERFE) has been identified as a factor that regulates hepcidin. In addition, fibroblast growth factor 23 (FGF23), which has been recognized as a phosphorus-regulating hormone, appears to be involved in haematopoietic regulation. Clarification of the detailed mechanism of haematopoiesis could lead to the improvement of renal anaemia treatment. METHODS: Epoetin beta pegol (CERA) was administered to patients undergoing haemodialysis at week 0, and the same amount of CERA with saccharated ferric oxide (SFO) was administered at week 4. The changes in haematopoiesis-related biomarkers, including ERFE, intact FGF23 (iFGF23), C-terminal FGF23 (cFGF23), and inflammatory markers, were examined. RESULTS: Administration of CERA increased ERFE levels, decreased hepcidin levels, and stimulated iron usage for haematopoiesis, leading to an increase in reticulocytes (Ret) and haemoglobin (Hb). Simultaneous administration of SFO with CERA (CERA + SFO) significantly attenuated the responses of ERFE, Ret, and Hb compared with CERA alone. Although iFGF23 levels were not affected by either CERA or CERA + SFO, cFGF23 was significantly elevated from baseline after CERA. Since cFGF23 levels were not affected by CERA + SFO, cFGF23 levels after CERA + SFO were significantly lower than those after CERA alone. The ratio of iFGF23 to cFGF23 (i/cFGF23 ratio) was significantly higher after CERA + SFO than that after CERA alone. In addition, high-sensitivity C-reactive protein (hsCRP) levels were significantly higher after CERA + SFO than after CERA alone. CONCLUSION: Administration of SFO suppressed haematopoietic responses induced by CERA. Elevation of i/cFGF23 ratio and hsCRP could account for the inhibitory effects of SFO on haematopoiesis. TRIAL REGISTRATION: This study was registered with the University Hospital Medical Information Network (ID UMIN000016552 ).

Effect of Erythropoietin on the Expression of Murine Transferrin Receptor 2.

Erythropoietin (EPO) downregulates hepcidin expression to increase the availability of iron; the downregulation of hepcidin is mediated by erythroferrone (ERFE) secreted by erythroblasts. Erythroblasts also express transferrin receptor 2 (TFR2); however, the possible role of TFR2 in hepcidin downregulation is unclear. The purpose of the study was to correlate liver expression of hepcidin with the expression of ERFE and TFR2 in murine bone marrow and spleen at 4, 16, 24, 48, 72 and 96 h following administration of a single dose of EPO. Splenic Fam132b expression increased 4 h after EPO injection; liver hepcidin mRNA was decreased at 16 h. In the spleen, expression of TFR2 and transferrin receptor (TFR1) proteins increased by an order of magnitude at 48 and 72 h after EPO treatment. The EPO-induced increase in splenic TFR2 and TFR1 was associated with an increase in the number of Tfr2- and Tfr1-expressing erythroblasts. Plasma exosomes prepared from EPO-treated mice displayed increased amount of TFR1 protein; however, no exosomal TFR2 was detected. Overall, the results confirm the importance of ERFE in stress erythropoiesis, support the role of TFR2 in erythroid cell development, and highlight possible differences in the removal of TFR2 and TFR1 from erythroid cell membranes.

Regulation of iron homeostasis through the erythroferrone-hepcidin axis in sickle cell disease.

Sickle cell disease (SCD) has a distinct pattern of transfusional iron overload (IO) when compared to transfusion-dependent ß-thalassaemia major (TDT). We conducted a single institution prospective study to evaluate plasma biomarkers of iron regulation and inflammation in patients with SCD with IO (SCD IO cases, n = 22) and without IO (SCD non-IO cases, n = 11), and non-SCD controls (n = 13). Hepcidin was found to be inappropriately low, as evidenced by a significantly higher median hepcidin/ferritin ratio in non-SCD controls compared to SCD IO cases (0·3 vs. 0·02, P < 0·0001) and SCD non-IO cases (0·3 vs. 0·02, P < 0·0001), suggesting that certain inhibitory mechanism (s) work to suppress hepcidin in SCD. As opposed to the SCD non-IO state, where hepcidin shows a strong significant positive correlation with ferritin (Spearman ρ = 0·7, P = 0·02), this correlation was lost when IO occurs (Spearman ρ = -0·2, P = 0·4). Although a direct non-linear correlation between erythroferrone (ERFE) and hepcidin did not reach statistical significance both in the IO (Spearman ρ = -0·4, P = 0·08) and non-IO state (Spearman ρ = -0·6, P = 0·07), patients with highest ERFE had low hepcidin levels, suggesting that ERFE contributes to hepcidin regulation in some patients. Our results suggest a multifactorial mechanism of hepcidin regulation in SCD.

Erythroferrone, the new iron regulator: evaluation of its levels in Egyptian patients with beta thalassemia.

Since iron overload is the commonest cause of morbidity and mortality in ß thalassemia major (ß-TM), it represents one major target in therapeutic management of the disease. The recently discovered erythroid regulator, erythroferrone (ERFE), governed by high levels of erythropoietin, was found to suppress hepcidin expression, thus increasing iron availability for developing erythroid progenitors. We aimed to investigate ERFE levels in Egyptian ß-TM patients as an attempt to understand its role in the prediction of iron overload states. Our study included 70 ß-TM patients, divided into two subgroups according to the degree of iron overload, and 30 sex and age-matched healthy subjects. ERFE gene expression was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR), and serum hepcidin was measured using enzyme-linked immunosorbent assay (ELISA) technique. Both ERFE gene expression levels and transferrin saturation (TS%) values were able to discriminate among cases with different degrees of iron overload, in contrast to hepcidin. TS% was acknowledged as the best predictor of iron overload (AUC 0.893) in comparison with serum hepcidin and ERFE gene levels (AUC 0.807 and 0.677, respectively), and ERFE gene expression was an independent predictor for the estimated TS%. In conclusion, we suggest that using the ERFE gene expression, combined with serum hepcidin estimation, can substantiate the role of estimated TS% as a promising tool in screening for iron overload in ß-TM patients.

Evaluation of Erythroferrone, Hepcidin, and Iron Overload Status in Iraqi Transfusion-Dependent ß-Thalassemia Major Patients.

Patients with ß-thalassemia major (ß-TM) show ineffective erythropoiesis and iron overload, which is the leading cause of mortality and organ injury. The present study aimed to investigate the relationships between two iron regulatory hormones, hepcidin and erythroferrone (ERFE) levels, and iron status parameters in Iraqi patients with ß-TM. Iron status parameters and hormones were measured in 60 patients and compared with 30 healthy controls. The results indicated significant changes in different iron status parameters, while ferritin with the ∼11-fold increase showed the most change. Significant reduction in hepcidin and an increase in ERFE levels were detected in patients when compared to the control group, while no direct correlation was identified with the other measured iron status parameters. The receiver operating characteristic (ROC) analysis showed that the z-score of the composite of ERFE + ferritin has a full diagnostic ability for ß-TM. In conclusion, our findings indicated the correlation between different iron status parameters and ferritin as the leading predictor of iron overload and two main iron regulatory hormones.