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REVIEW ARTICLE
Year : 2021 | Volume
: 46
| Issue : 3 | Page : 133-142
Serum erythroferrone diagnostic value in patients with beta-thalassemia with iron overload
Aliaa Saeed1, Neven Nabil1, Walaa Elsalakawy1, Riham Metwali1, Ahmed Khattab2, Mary Gamal Naguib MD 1
1 Clinical Hematology and Bone Marrow Transplant Unit, Department of Internal Medicine, Ain Shams University, Cairo, Egypt
2 Department of Clinical Hematology, Zagazig General Hospital, El-Zagazig, Egypt
Correspondence Address:
Mary Gamal Naguib
Faculty of Medicine; Ain Shams University, Abbassia, Cairo, 11361
Egypt
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ejh.ejh_22_21
Introduction Patients with beta-thalassemia experience a major complication besides their anemia, which is the iron overload and its complications up to death. Erythroferrone (ERFE) and hepcidin are the major controlling factors for serum iron level, being inversely related to each other. Patients with iron overload are thought to have low serum hepcidin and high serum ERFE levels. So, serum ERFE is postulated to be involved in the pathogenesis of iron overload in patients with β-thalassemia. A cross-sectional study has been conducted, including 112 participants: 80 patients with β-thalassemia and 32 healthy age-matched and sex-matched controls. Serum ERFE, ferritin, and hepcidin were measured by enzyme-linked immunosorbent assay and compared among patients with β-thalassemia and healthy controls. Patients had significantly higher serum ferritin, ERFE, as well as lower serum hepcidin levels as compared with healthy age-matched and sex-matched controls, with P values less than 0.001, less than 0.001, and 0.045, respectively. Serum ERFE may serve as an important marker for iron overload and may represent a future possible therapeutic target using anti-ERFE to control iron overload.
Keywords: erythroferrone, ferritin, hepcidin
Thalassemia results from abnormality in one of the two forming globin chains of hemoglobin, that is, alpha and beta chains. Decreased production of beta chains, leading to the accumulation of free alpha chains inside red blood cells (RBCs), is the contributing factor in the pathogenesis of beta-thalassemia [1]. Unbound alpha chains precipitate and form reactive oxygen species which cause hemolysis of RBCs and decrease production of the functioning hemoglobin. The final result is ineffective erythropoiesis [2].
β-thalassemias are classified according to severity into three subtypes, that is, β-thalassemia major, intermediate, and minor, depending upon severity of anemia and the requirements of blood transfusion. β-thalassemia major is characterized by severe anemia and frequently required blood transfusion, whereas β-thalassemia intermediate has less severe anemia and requires occasional transfusion. β-thalassemia minor is the asymptomatic carrier form [3].
This ineffective erythropoiesis stimulates the release of many factors including hypoxia-inducible factor, growth and differentiating factor 15, twisted gastrulation protein homolog 1, and erythroferrone (ERFE). ERFE acts on hepatic hepcidin, which controls intestinal iron absorption and interferes with its release, leading to increased intestinal iron absorption, increased iron overload, the catastrophic iron deposition in parenchymal organs, and organs dysfunction up to organ failure [4],[5],[6],[7],[8],[9],[10].
Erythroferrone structureERFE is considered one of the regulators of erythroid progenitors which control iron homeostasis in responses to various types of anemia including β‐thalassemia and myelodysplastic syndromes [11].
ERFE is structurally considered one of the CTRP family hormones, which include 16 proteins, structurally related to each other formed of four domains: an N‐terminal responsible for protein secretion, a collagenous linker which connects the two larger domains and allows protein multimerization, a variable domain specified for each protein, and a C‐terminal [12].
ERFE is formed of 354 and 340 amino acids with 37.3 and 36.3 kDa predicted masses in human and mice, respectively [13].
Mechanism of action of erythroferrone and pathological role in B-thalassemia anemiaOld RBCs are phagocytosed by macrophages in liver and spleen at a regular rate. In case of anemia, resulted hypoxia stimulates renal production of erythropoietin [14],[15]. Elevated erythropoietin acts on erythroid progenitors and increases the number of erythroblasts and secreted ERFE, which acts by decreasing hepatic hepcidin synthesis in the liver and releasing the hepcidin-inhibiting effect on ferroprotein, allowing more intestinal iron absorption and more iron delivering into blood causing iron overload [7]. In chronic anemias with iron overload as in B-thalassemia, this iron overload is nontransferrin bound causing tissue injury via released reactive oxygen species. Iron-induced tissue injury affects the endocrinal glans, the heart, the liver together with increased infection risks [16].
In our study, we demonstrated the role of serum ERFE as a helpful laboratory marker of iron overload. We measured it in peripheral blood samples of patients with thalassemia in relation to healthy age-matched and sex-matched controls. We compared it with other markers of iron overload, that is, serum ferritin and serum hepcidin.
Patients and methodsPatients
This is a cross-sectional study with a total number of 112 participants recruited from the outpatient clinic of the Clinical Haematology and Oncology Unit, Internal Medicine Department, Ain Shams University, Cairo, Egypt. The study participants were divided into two groups: group I comprising 80 patients with beta-thalassemia and group II including 32 healthy age-matched and sex-matched controls.
Informed written consent was obtained from all the study participants. Furthermore, all the study procedures performed followed the ethical standards of the institutional and national research committee and in accordance with the 1964 Helsinki declaration and its later amendments.
Inclusion criteria
The following were the inclusion criteria:
Patients aged more than or equal to 18 years.
Patients with beta-thalassemia major and intermedia.
Exclusion criteria
The following were the exclusion criteria:
Patients with beta-thalassemia trait.
Patients with associated other types of hemoglobinopathies.
Other types of anemia.
MethodsAll the patients have been assessed regarding their original diagnosis, transfusion demands, history of splenectomy, and the iron overload manifestations. Moreover, history of the use and compliance to iron chelating agents have been obtained.
Patients were subjected to the assessment of their organ-specific complications, including endocrinal, hepatic, and cardiac-related problems using the standardized approaches delineated in the World Thalassemia Federation guidelines for transfusion-dependent and nontransfusion-dependent beta-thalassemia [17],[18].
Biomarkers for iron overload status assessment used included serum ferritin and hepcidin levels done by enzyme-linked immunosorbent assay (ELISA) assay.
The study biomarker, ERFE, has been measured in the serum samples by ELISA as follows: blood samples were collected in a plain tube with no additives and centrifuged at 2000–3000 RPM for 20 min. The serum was separated and stored at −80°C until analysis. ERFE was measured using commercially available ELISA kits (Biotin double antibody sandwich technology; Bioassay Technology, Shanghai Korain Biotech, Shanghai, China). Well plates were coated with ERFE monoclonal antibodies; a recombinant ERFE standard was serially diluted to obtain the standard curve. For sample injection, a blank well, standard solution, and a sample well were prepared, in which anti-ERFE antibody labeled with HRP was added and incubated at 37°C for 60 min followed by washing steps. The plate was further incubated at 37°C for 10 min after the addition of chromogenic solutions A and B. The reaction was stopped using stoppage solutions. Absorption was read at 450 nm using an ELISA plate reader (Biotek, Winooski city, Chittenden County, Vermont, United States), where the optical density for each sample was plotted on a standard curve to obtain its concentration.
Statistical analysis
Statistical presentation and analysis of the present study were conducted, using the mean, SD, Student t test, χ2, linear correlation coefficient, and analysis of variance tests by SPSS, V20 (Statistical Package for the Social Sciences, version 20, International Business Machines Corporation (IBM), New York, Armonk, USA) for Windows.
Unpaired Student t test was used to compare between two groups in quantitative data. We used also χ2 and analysis of variance tests.
Receiver operating characteristic (ROC) curve: ROC curve analysis.
Sensitivity: probability that the test results will be positive when the disease is present (true positive rate, expressed as a percentage).
Specificity: probability that the test results will be negative when the disease is absent (true negative rate, expressed as a percentage).
Positive predictive value (the probability that the disease is present when the test is positive).
Negative predictive value (the probability that the disease is present when the test is negative).
Accuracy: the ratio of the true positive and true negative on all patients.
Significance was set as follows:
P value more than 0.05: nonsignificant.
P value less than or equal to 0.05: significant.
P value less than 0.01: highly significant.
ResultsThis cross-sectional study included 112 participants, comprising 80 patients with β-thalassemia and 32 healthy age-matched and sex-matched controls.
[Table 1] demonstrates the demographic data of the study cohort. Patients with β-thalassemia had a mean age of 32.65±8.237 years, whereas controls had a mean age of 30.469±8.195 years. Cases included 53 (66.25%) females and 27 (33.75%) males, whereas controls included 20 (62.5%) females and 12 (37.5%) males. Both groups were comparable in terms of age and sex, with P value more than 0.05.
Transfusion dependence was observed in 64 (80%) cases of the cohort. Splenectomy was performed in 50 (62.5%) patients to relieve pressure symptoms or to treat hypersplenism manifested as increasing transfusion requirements.
Detailed clinical and laboratory assessment revealed the presence of endocrinal dysfunction. A total of 14 patients had impaired glucose homeostasis in the form of either impaired glucose tolerance (n=9) or clinically overt diabetes mellitus necessitating insulin therapy (n=5). Four patients had thyroid dysfunction making up 5% of the cohort, with three cases of hypothyroidism on levothyroxine replacement therapy and one case of subclinical hypothyroidism with expectant management. Gonadal dysfunction occurred in six (7.5%) patients. None of the studied population exhibited diabetes insipidus or adrenal dysfunction. Moreover, none had hepatic or cardiac clinical manifestations as a sequela of iron overload.
In terms of the use of iron chelation to combat iron overload, the majority of patients (77.5%) were set on treatment, whereas 18 patients were not put on chelation therapy. Of these 62 patients taking iron chelators, desferrioxamine, deferasirox, and deferiprone were used in 24 (30%), 24 (30%), and 14 (17.5%) patients, respectively ([Table 2] and [Table 3]).
Regarding iron overload biomarkers done, serum ferritin, hepcidin, and ERFE have been measured in the serum of patients and controls and compared. Cases exhibited a mean serum ferritin, hepcidin, and ERFE levels of 1965.488±1153.862, 143.625±101.426, and 475.875±336.586 ng/l, respectively, whereas controls exhibited mean values of 80.669±26.188, 188.438±116.241, and 149.375±120.267 ng/l for ferritin, hepcidin, and ERFE, respectively. Cases exhibited significantly higher ferritin and ERFE and lower hepcidin values in comparison with their age-matched and sex-matched controls, with P values of less than 0.001, 0.045, and less than 0.001, respectively ([Table 4] and [Figure 1],[Figure 2],,[Figure 3],[Figure 4],[Figure 5]).
Figure 1 Ferritin levels of cases and control. Cases showed higher serum ferritin level compared with control
Figure 2 Hepcidin levels of cases and controls. Cases showed lower hepcidin level compared with control.
Figure 3 Erythroferrone levels of cases and controls. Cases showed higher erythroferrone level compared with control.
Figure 4 Receiver operating characteristic (ROC) curve of serum ferritin. ROC curve has been plotted so as to characterize serum ferritin in cases and controls. A cutoff point was more than 123.2 ng/ml, with a sensitivity of 100 and a 100% specificity. Positive predictive value (PPV) is 100% whereas negative predictive value (NPV) is 100%, with accuracy of 100%.
Figure 5 Receiver operating characteristic (ROC) curve of serum hepcidin. ROC curve has been plotted so as to characterize serum hepcidin in cases and controls. A cutoff point was less than or equal to 170 ng/ml with a sensitivity of 72.5% and a 56.25% specificity. Positive predictive value (PPV) is 80.6% whereas negative predictive value (NPV) is 45%, with an accuracy of 59.9%.These three iron state biomarkers were plotted against the different clinical and laboratory features of the study cohort. Patients on iron chelators exhibited significantly higher serum ferritin than those not under treatment, with mean levels of 2212.597±1060.612 versus 1114.333±1077.454 ng/ml (P=0.001). However, serum ferritin was not associated with sex, history of splenectomy, or thyroid-stimulating hormone level, with P value more than 0.05. Correlating serum ferritin level with other numerical variables showed that it had a positive correlation with total calcium and parathyroid hormone. Otherwise, it exhibits no correlation with other parameters ([Table 5], [Table 8]).
As for serum hepcidin levels, it exhibited significant association with sex, being higher in males than in females (177.778±131.093 and 126.226±78.243 ng/ml, respectively), with a P value of 0.031 [Table 6]. Upon correlating it with quantitative variables, it had no correlation with the other two study biomarkers, that is, ferritin and ERFE. However, it demonstrates a significant negative correlation with FT4 and serum phosphorous levels, with P values of 0.033 and 0.021, respectively. This is well-illustrated in [Table 8].
With respect to the principal study biomarker, ERFE, it is significantly higher in patients on iron chelator therapy than those not on therapy. Moreover, it exhibits a positive correlation with age, hemoglobin level, red cell distribution width, and bilirubin levels, with P values of 0.0.31, 0.026, 0.048, and 0.001, respectively. Conversely, it displays a negative correlation with alpha-fetoprotein and PO4 levels, with P values of 0.035 and 0.002, respectively ([Table 7] and [Table 8]).
Table 8 Correlations between the quantitative variables of the patients and the levels of the markers of the studyNone of the three studied markers exhibited correlation with the other two markers, with P values more than 0.05 ([Table 8]).
A ROC curve has been plotted to identify a cutoff value for serum ERFE that segregates cases from controls. The cutoff level was set at 220 ng/l, with a sensitivity of 87.5%, specificity of 81.25%, positive predictive value of 92.1%, negative predictive value of 72.2%, and accuracy of 88.5% ([Table 9] and [Figure 6]).
Table 9 Agreement (sensitivity, specificity) for serum ferritin, hepcidin and serum erythroferrone to differentiate cases from controls
Figure 6 Receiver operating characteristic (ROC) curve of serum erythroferrone. ROC curve has been plotted so as to characterize serum erythroferrone in cases and controls. The cutoff level was set at more than 220 ng/l, with a sensitivity of 87.5 and 81.25% specificity, positive predictive value (PPV) of 92.1% and negative predictive value (NPV) of 72.2%, with an accuracy of 88.5%.The study cohort was again dissected as per their levels of ERFE. They were subdivided into patients with low serum ERFE levels and those with high ERFE levels as defined by the cutoff point of 220 ng/l as determined by the ROC curve.
Despite the fact that patients with high ERFE levels have higher serum ferritin and lower serum hepcidin levels in comparison with those with low ERFE levels, this did not culminate into statistical significance, with P values of 0.501 and 0.96, respectively ([Table 10]).
Table 10 Correlation between erythroferrone level to ferritin and hepcidin levels according to erythroferrone cut-off value Discussion β-thalassemia is still a major problem among benign hematological diseases. Despite its benign nature, patients with β-thalassemia experience major problems, on the top of them is iron overload and its complications. Liver, heart, and endocrinal organs are the main target organs affected by iron deposition, resulting in arrhythmias, heart failure, liver cirrhosis, hepatocellular carcinoma, diabetes, hypothyroidism, hypoparathyroidism, delayed puberty, and growth failure [19].
The main regulator for iron metabolism is the hepcidin hormone which is synthesized by the liver. Mutations in the HAMP, the hepcidin coding gene, is a major cause in hemochromatosis pathogenesis, suggesting the role of hepcidin in iron overload. Hepcidin decreases iron absorption in enterocytes, so hepcidin deficiency leads to increased iron absorption and iron overload [20]. ERFE is considered as a physiological and pathological regulator of erythropoiesis [21], which suppresses hepatic hepcidin production [22].
In this study, we studied different iron overload markers, serum ferritin and serum hepcidin, compared with serum ERFE in patients with beta-thalassemia. Our study shows the possibility of using serum ERFE as a reliable marker in patients with β-thalassemia to indicate iron overload. Moreover, it opens the gate to study the role of anti-ERFE in treating iron overload in patients with β-thalassemia.
A total of 112 participants were enrolled in the study, comprising 80 patients with beta-thalassemia and 32 healthy age-matched and sex-matched controls.
We found that patients with beta-thalassemia showed a higher serum ferritin level in comparison with controls, with values of 1965.488±1153.862 and 80.669±26.188 ng/ml, respectively, with a significant P value less than 0.001. Patients with beta-thalassemia demonstrated lower mean level of hepcidin in relation to controls, being 143.625±101.426 ng/ml versus 188.438±116.241 ng/ml, respectively, with a significant P value of 0.045. Regarding our study marker, ERFE levels, cases had a higher level than that of controls. Cases had a mean of 475.875±336.586 ng/l; however, controls had a mean of 149.375±120.267 ng/l, with a significant P value less than 0.001.
Patients with β-thalassemia showed high ERFE values, high ferritin, and low hepcidin levels. This may open the door to start using serum ERFE level as a reliable marker of iron overload in those patients. We can depend on these three markers to evaluate iron overload status in patients with beta-thalassemia.
So, iron overload can be explained by another cause rather than chronic hemolysis or chronic blood transfusion, which is high ERFE level, which is produced from erythroblast in patients with thalassemia and suppresses hepcidin, leading to increased iron absorption from intestine. This explains iron overload even in patients with thalassemia intermediate, with less transfusion requirements than patients with thalassemia major.
This is in agreement with Arezes et al. [23]. They studied the role of anti-ERFE antibody in mouse model of β-thalassemia [Hbb(th3/+) mice] and found that anti-ERFE antibody, which binds to N-terminal domain of ERFE, prevents EFRE-induced suppression of hepcidin synthesis by the liver, which helps to decrease iron overload in mouse model [23].
Moreover, in 2020, Mangaonkar et al. [24] assessed the hepcidin level in patients with sickle cell with iron overload versus healthy controls, and they found a lower hepcidin level in patients with sickle cell versus their controls. This is referred to their high ERFE level, which leads to hepcidin suppression by the liver in patients with sickle cell [24].
Kautz et al. [6], studied ERFE ablation in b-thalassemic mice. This leads to restoring hepcidin levels, decreasing intestinal iron absorption, and correction of iron overload, supporting the role of ERFE in iron overload via suppressing hepcidin level [6].
Looking thoroughly at the effects of iron overload on the study cohort, 14 of 80 patients had impaired glucose homeostasis, five had diabetes mellitus, and four patients (5% of cases) had hypothyroidism. Overall, 7.5% of our study patients had gonadal dysfunction (six patients). None of our patients exhibited hypoadrenalism or other forms of endocrinopathy. This matches with the results reported by Baldini et al. [25]. They studied retrospectively 70 patients with β-thalassemia intermedia, with mean follow-up of 20 years. Endocrinopathies were found in 15 (21%) patients. However, the prevalence of hypothyroidism in their study was 14%. Hypogonadism was encountered in three cases, whereas lower prevalence of diabetes mellitus and impaired glucose tolerance was observed in one and two cases, respectively. The authors of the study explained that the thyroid was the most frequently affected gland among their patients (10 cases, 14%); this percentage is in line with the high prevalence of autoimmune hypothyroidism in Italy where their study was held [25].
In our study, upon correlating ERFE level with the other study markers, it was not correlated with ferritin, with P values of 0.193, but for hepcidin, they are negatively related, however with no significant difference (P=0.761). This may be related to the presence of multiple factors affecting ferritin and hepcidin levels rather than ERFE.
A ROC curve has been plotted so as to characterize serum ferritin, hepcidin, and ERFE levels in cases and controls. As for ferritin, a cutoff point was more than 123.2 ng/ml; for hepcidin, a cutoff point was less than or equal to 170 ng/ml; and for ERFE, the cutoff level was set at more than 220 ng/l.
The study cohort was again dissected as per their levels of ERFE. They were subdivided into patients with low serum ERFE levels and those with high ERFE levels as defined by the cutoff point of 220 ng/l as determined by the ROC curve. High ERFE patients had lower hepcidin and higher ferritin levels compared with low ERFE patients with higher hepcidin and lower ferritin levels but without a significant (P=0.501 and 0.958, respectively).
This is in agreement with Mangaonkar et al. [24]; they found direct nonlinear correlation between ERFE levels and hepcidin levels in patients with sickle cell with iron overload but without a statistical significance. They suggested the presence of other factors regulating hepcidin levels rather than ERFE [24].
ConclusionSerum ERFE is considered as a potential marker for iron overload assessment, opening the future for the use of anti-ERFE therapy as a lifeboat for patients with chronic iron overload diseases.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
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