Variations in serum erythropoietin and transferrin receptor during phlebotomy therapy of hereditary hemochromatosis: a case report

Serum levels of transferrin receptor and erythropoietin were determined in 2 patients with hereditary hemochromatosis undergoing phlebotomy therapy. The objective of the study was to determine changes in serum transferrin receptor and serum erythropoietin occurring during therapy, and to investigate if such changes could be useful to monitor the therapy. The study showed that serum transferrin receptor, and to a lesser extent serum erythropoietin, may be better parameters than serum ferritin as indicators of when phlebotomy should be discontinued. The most sensitive parameter, however, appeared to be the serum transferrin receptor/ferritin ratio.     

Clinical and pathologic findings in hemochromatosis type 3 due to a novel mutation in transferrin receptor 2 gene

BACKGROUND & AIMS: Although most patients with hereditary hemochromatosis are homozygous for a single mutation of the HFE gene on chromosome 6p, accumulating evidence indicates that the disease is genetically heterogeneous. Type 3 hemochromatosis, recently described in 4 families, is linked to mutations of the gene encoding transferrin receptor 2 on chromosome 7q22. Here we report data from a family carrying a new mutation of the transferrin receptor 2 gene. METHODS: Detailed clinical and histopathologic documentation was available for most family members. The entire coding sequence and exon/intron boundaries of the transferrin receptor 2 gene were analyzed by direct sequencing. RESULTS: A 12-nucleotide deletion in exon 16, causing the loss of 4 amino acids (AVAQ 594-597 del), was detected at the homozygous state in the 3 patients with histologically proven iron overload. The deletion segregated with the disease within the family and was not found in 100 healthy controls. Some clinical and pathologic characteristics, such as low penetrance in the premenopausal woman, and early iron deposition in periportal hepatocytes resembled those of classic, HFE-related hemochromatosis. CONCLUSIONS: Our data support the role of the transferrin receptor 2 gene in hemochromatosis type 3 as well as its critical involvement in the maintenance of iron homeostasis in humans.     

Human platelets express hemochromatosis protein (HFE) and transferrin receptor 2

OBJECTIVES: While body iron status may influence platelets, little information is available about platelet expression of proteins regulating iron homeostasis. HFE, the protein defective in hereditary hemochromatosis, and transferrin receptor 2 (TfR2) are two novel protein candidates that could be involved in mechanisms of iron transport across the platelet plasma membrane. METHODS: The expression and localization of HFE, TfR1 and TfR2 proteins in human platelets were examined using Western blotting and immunocytochemistry. RESULTS: Human platelets expressed HFE and TfR2, whereas no signal for TfR1 was found. The positive reactions for HFE and TfR2 were mainly confined to the platelet plasma membrane. CONCLUSIONS: Expression of HFE and TfR2 proteins in human platelets may indicate that the mutations in the corresponding genes could influence platelet count, size and/or activation. The presence of TfR2 and absence of TfR1 suggests that HFE may serve a different function in platelets compared with the other HFE-positive cell types, e.g. enterocytes, macrophages and syncytiotrophoblasts.     

Mutation analysis of the HFE gene associated with hereditary hemochromatosis in African Americans

Homozygosity for the mutation Cys282Tyr in the HFE gene has recently been identified as a cause of hereditary hemochromatosis, a disorder resulting in the inappropriate absorption of iron. Approximately 10% of Caucasians are heterozygous for this mutation; however, the gene frequency in African Americans is unknown. A study of a control population of African Americans was performed to determine the frequency of the Cys282Tyr and His63Asp alleles in this ethnic group. The carrier frequency for each mutant allele in our African American population was 3.0%. DNA studies of four African-American hemochromatosis patients did not identify any individuals with the Cys282Tyr allele. These findings suggest that if the Cys282Tyr mutation confers susceptibility to hemochromatosis in Caucasians (as suggested by recent studies) there is an alternative mechanism for hemochromatosis in the American black population. Am. J. Hematol. 58:213–217, 1998. © 1998 Wiley-Liss, Inc.     

Transferrin sialic acid contents of patients with hereditary hemochromatosis

Recent evidence suggests that asialotransferrin may enhance hepatic uptake of trivalent metal ions through the asialoglycoprotein-receptor pathway. In hereditary hemochromatosis, there is preferential uptake of iron by hepatocytes. We purified transferrin from two patients with this disease. Their transferrins had normal electrophoretic patterns and contained normal amounts of sialic acid. We conclude that the preferential uptake of iron by hepatocytes in hereditary hemochromatosis is not due to the presence of asialotransferrin in their plasma.     

Serum transferrin receptor in the megaloblastic anemia of cobalamin deficiency.

In order to further study the relation between transferrin receptor and erythropoiesis we examined serum receptor levels in megaloblastic anemia, which is the classic example of ineffective erythropoiesis. We studied 33 patients with unequivocal cobalamin deficiency, only 22 of whom were anemic. High serum transferrin receptor levels were found in 12 patients, all of whom were anemic and had high lactate dehydrogenase (LDH) levels; in contrast, only 10 of the 21 patients with normal receptor levels were anemic. Receptor correlated most strongly with LDH (r = 0.573, p < 0.001) and, inversely, with hemoglobin values (r = -0.560, p < 0.001); it also correlated with ferritin and total bilirubin levels, but not with cobalamin, MCV or erythropoietin. No association was found with the hemolytic component of megaloblastic anemia, represented indirectly by haptoglobin levels. Changes induced by cobalamin therapy were also examined in 13 patients. Transferrin receptors rose in all 6 patients who initially had high levels and in 2 of 3 patients who had borderline levels, but not in the 4 patients with initially normal levels. The receptor levels began to rise within 1-3 days, peaked at about 2 weeks and returned to normal at about the 5th wk. The findings indicate that serum transferrin receptor levels reflect the severity of the megaloblastic anemia. The elevated receptor levels rise further with cobalamin therapy, however, as effective erythropoiesis replaces ineffective erythropoiesis, and these persist until the increased erythropoiesis returns to normal.     

Monocyte transferrin-iron uptake in hereditary hemochromatosis

Transferrin-iron uptake by peripheral blood monocytes was studied in vitro to test the hypothesis that the relative paucity of mononuclear phagocyte iron loading in hereditary hemochromatosis results from a defect in uptake of iron from transferrin. Monocytes from nine control subjects and 17 patients with hemochromatosis were cultured in the presence of ⁵⁹Fe-labelled human transferrin. There was no difference in ⁵⁹Fe uptake between monocytes from control subjects and monocytes from patients with hemochromatosis who had been treated by phlebotomy and who had normal body iron stores. However, ⁵⁹Fe uptake by monocytes from iron-loaded patients with hemochromatosis was significantly reduced compared with either control subjects or treated hemochromatosis patients. It is likely that this was a secondary effect of iron loading since iron uptake by monocytes from treated hemochromatosis patients was normal. Assuming that monocytes in culture reflect mononuclear phagocyte iron metabolism in vivo, this study suggests that the relative paucity of mononuclear phagocyte iron loading in hemochromatosis is not related to an abnormality in transferrin-iron uptake by these cells.     

Soluble transferrin receptor in the study of fetal erythropoietic activity

In order to evaluate fetal erythropoiesis we measured red blood cells, hemoglobin, hematocrit, serum transferrin receptor (sTfR), and iron status parameters in fetuses undergoing percutaneous umbilical blood sampling, and in normal newborns at term. We found high levels of sTfR in fetuses and newborns as compared with normal adults (3,149 ± 181 vs. 1,881 ± 137 ng/ml, P < 0.00001). Concentrations of sTfR correlate with gestational age and red blood cell numbers (r = 0.441, P < 0.001; r = 0.366, P = 0.06). sTfR concentrations do not show correlation with iron status parameters. The increased sTfR concentration is consistent with the fact that fetal life is characterized by cell proliferation and tissue growth. sTfR concentration correlates with gestational age and numbers of red blood cells, and can therefore be considered a good indicator of fetal erythropoiesis. It is conceivable that, during intrauterine life, sTfR expression is independent from iron status. sTfR determination will help in reaching a better understanding of some aspects of fetal physiology, and will help elucidate the physiopathology of fetal hematological diseases. © 1996 Wiley-Liss, Inc.     

Serum transferrin receptor, ferritin, and reticulocyte maturity indices during the first year of life in 'large' preterm infants

BACKGROUND: Preterm infants are at risk of developing iron deficiency; among the iron status and hemopoiesis indices the serum transferrin receptor (sTfr) has been shown to be a useful indicator in assessing iron status, while immature reticulocyte production is regarded as an estimator of erythropoiesis. OBJECTIVE: To investigate age-related changes in iron status infants born 'moderately' preterm, with a gestational age (GA) of 32-36 wk, and identify associations between sTfr and other hematological and biochemical iron indices. DESIGN: Hospital-based prospective, longitudinal study in preterm infants. METHODS: Iron and erythropoiesis parameters were evaluated in 181 formula-fed preterm infants at 2 and 6 wk and 3, 6, 9, and 12 months chronological age. Hemoglobulin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), reticulocytes, serum iron (sFe), serum ferritin (sFer), sTfr, and reticulocyte subpopulations were measured. RESULTS: A total of 756 measurements were performed. After an initial decline, Hb rose from month 3 to 12 of life. SFe and sFer and immature reticulocyte count decreased from the second week to the third month and remained stable thereafter. STfr was lower up to 6 wk and stable from month 3 to 12. Iron deficiency anemia (IDA) was found in 5.5% of infants. In 76 measurements sFer was <12 microg/L, implying storage iron deficiency (SID). A negative correlation was observed between sTfr and other indices of iron status such as Hb, Hct, MCV, sFe, and sFer. Infants with sFer <12 microg/L had lower sTfr than those with sFer >12 microg/L. Reticulocyte production was positively associated with STfr, but this association was dependent on the chronological age of the infant. CONCLUSION: Iron depletion is common in formula-fed preterm (32-36 wk GA) infants between month 3 and 12 of life. STfr appears to be an indicator of iron status in preterm infants during the first year of life.     

Regulation of the hepatic transferrin receptor in hereditary hemochromatosis

The liver is the main site of iron accumulation and pathologic sequelae in hereditary hemochromatosis. Whether this is a result solely of inappropriately increased absorption of iron by the gastrointestinal tract or a more generalized regulatory failure of iron balance is unknown. Using immunohistochemical techniques, we have examined the effects of therapeutic changes in liver iron stores on the expression of the hepatic transferrin receptor in hereditary hemochromatosis. Ten patients with untreated hereditary hemochromatosis had no detectable staining for transferrin receptor in their liver biopsies. All had increased hepatic ferritin (mean = 19.9 micrograms per mg protein, range = 1 to 31.7 micrograms per mg protein) and hepatic iron levels (mean = 36.2 micrograms per mg protein, range = 3.6 to 69.9 micrograms per mg protein). In contrast, hepatocyte transferrin receptor was detected in seven patients in whom hepatic iron stores were markedly depleted by venesection (hepatic ferritin mean = 0.32 microgram per mg protein, range = 0.16 to 0.53 microgram per mg protein; hepatic iron mean = 0.98 microgram per mg protein, range = 0.3 to 2.1 micrograms per mg protein). Sequential data from one patient confirmed the reexpression of receptor in response to therapeutic iron depletion, whereas data from another patient studied during treatment illustrated a reciprocal relationship between liver tissue distribution of iron and expression of transferrin receptor. The finding that appropriate physiologic regulation of the hepatic transferrin receptor operates in hereditary hemochromatosis does not support the concept of a generalized defect in receptor-mediated uptake of transferrin-bound iron.     

Serum transferrin receptor as a marker of erythropoiesis suppression in patients on chronic transfusion

In the management of patients requiring chronic transfusion, various parameters may be used to evaluate the degree of erythroid marrow suppression. The aim of our study was to assess which of these parameters provide the most useful assessment of erythropoiesis. We studied 27 chronically transfused patients, 19 with sickle cell disease (SS patients) and 8 with thalassemia. Thirty-one nonchronically transfused SS patients and 74 healthy children served as controls. We measured serum transferrin receptor levels, reticulocyte counts, hemoglobin (Hb) concentrations and erythropoietin levels. The serum transferrin receptor levels were very elevated in control SS patients and remained significantly elevated in those on transfusion therapy, but were normal in thalassemia patients, indicating a more complete suppression of erythropoiesis. The reticulocyte counts were elevated in all SS patients, even when on chronic transfusion, but were in the normal range in patients with thalassemia. Erythropoietin levels were elevated in patients with thalassemia and in all the SS patients. Hb levels negatively correlated with serum transferrin receptor and erythropoietin in all SS patients. In the transfused SS patients, a higher HbS level correlated with higher reticulocyte counts, transferrin receptor, and erythropoietin levels. In thalassemia patients, erythropoiesis was more completely suppressed, as reflected both by normal reticulocyte counts and near-normal transferrin receptor levels. Though the reticulocyte counts were not significantly different in the transfused SS patients, the serum transferrin receptor levels were less elevated than in SS patients not on transfusion. The serum transferrin receptor level appears to be the most useful marker of marrow erythropoietic activity in chronically transfused SS patients. We recommend that reticulocyte counts be integrated with periodic measurements of serum transferrin receptor levels. Am. J. Hematol. 60:121–125, 1999. © 1999 Wiley-Liss, Inc.     

A time course of hepcidin response to iron challenge in patients with HFE and TFR2 hemochromatosis

Background

Inadequate hepcidin production leads to iron overload in nearly all types of hemochromatosis. We explored the acute response of hepcidin to iron challenge in 25 patients with HFE-hemochromatosis, in two with TFR2-hemochromatosis and in 13 controls. Sixteen patients (10 C282Y/C282Y homozygotes, 6 C282Y/H63D compound heterozygotes) had increased iron stores, while nine (6 C282Y/C282Y homozygotes, 3 C282Y/H63D compound heterozygotes) were studied after phlebotomy-induced normalization of iron stores.

Design and Methods

We analyzed serum iron, transferrin saturation, and serum hepcidin by both enzyme-linked immunosorbent assay and mass-spectrometry at baseline, and 4, 8, 12 and 24 hours after a single 65-mg dose of oral iron.

Results

Serum iron and transferrin saturation significantly increased at 4 hours and returned to baseline values at 8–12 hours in all groups, except in the iron-normalized patients who showed the highest and longest increase of both parameters. The level of hepcidin increased significantly at 4 hours and returned to baseline at 24 hours in controls and in the C282Y/H63D compound heterozygotes at diagnosis. The hepcidin response was smaller in C282Y-homozygotes than in controls, barely detectable in the patients with iron-depleted HFE-hemochromatosis and absent in those with TFR2-hemochromatosis.

Conclusions

Our results are consistent with a scenario in which TFR2 plays a prominent and HFE a contributory role in the hepcidin response to a dose of oral iron. In iron-normalized patients with HFE hemochromatosis, both the low baseline hepcidin level and the weak response to iron contribute to hyperabsorption of iron.     

High serum transferrin receptor level in anemia of chronic disorders indicates coexistent iron deficiency

Blood transferrin receptor (TR) level is largely determined by the quantum of erythropoiesis and by intracellular iron content of the cells of the erythroid lineage. Hence, a high serum TR level has been found to be useful in distinguishing iron deficiency anemia (IDA) from anemia of chronic disorders (ACD). In order to examine its potential role in the diagnosis of concomitant iron deficiency in ACD, we determined serum TR levels in 130 cases of ACD, in 25 cases of IDA, and in 40 normal adults. As expected, all patients of IDA had significantly higher serum TR levels compared to the normal subjects (4.2-19.2 microg/dL vs. 1.3-3.0 microg/dL) (P < 0.002). In 11/25 cases of IDA, the total iron-binding capacity (TIBC) was in the normal range although bone marrow iron store was absent and serum TR levels were high, thereby highlighting the superiority of TR level in the diagnosis of iron deficiency compared to TIBC. Although 54% (70/130) patients of ACD had normal or low serum TR levels (0.9-3.0 microg/dL) as expected, in 46% (60/130) of ACD patients, serum TR levels were high (3.2-11.0 microg/dL). Mean corpuscular volume, red cell distribution width, and transferrin saturation were significantly lower (P < 0.001) in the latter group of patients compared to the former, and these parameters resembled those in IDA patients. Also, serum iron was lower and TIBC was higher in this group of ACD patients compared to those with normal or low serum TR. All these features point to an "IDA-like" profile of ACD patients with high TR and support the possibility of co-existent iron deficiency in this subgroup of ACD patients. In light of these observations it would be prudent to treat ACD patients with high serum TR levels with iron replacement therapy.     

Juvenile hemochromatosis associated with pathogenic mutations of adult hemochromatosis genes

BACKGROUND AND AIMS: Juvenile hemochromatosis is a severe form of hereditary iron overload that has thus far been linked to pathogenic mutations of the gene coding for hemojuvelin (HJV), on chromosome 1, or, more rarely, that coding for hepcidin ( HAMP ), on chromosome 19. A milder adult-onset form is due to pathogenic mutations of HFE or, rarely, serum transferrin receptor 2. METHODS: We studied a pedigree with siblings affected by both juvenile and adult-onset hereditary hemochromatosis. Affected subjects underwent full clinical evaluation, as well as microsatellite and gene sequencing analysis. RESULTS: Two siblings (male and female, aged 24 and 25 years, respectively) were hospitalized for severe endocrinopathy and cardiomyopathy. At age 18 and 17 years, they had presented with impotence and amenorrhea, respectively, and increased serum iron levels. Hypogonadotropic hypogonadism was confirmed in both, and liver biopsy showed marked hepatic iron accumulation and micronodular cirrhosis. Iron levels were normalized after 24 months (female) and 36 months (male) of weekly phlebotomies. Microsatellite analysis showed no linkage with chromosome 1 and 19, and gene sequencing showed no hemojuvelin or hepcidin gene mutations. Instead, combined mutations of HFE (C282Y/H63D compound heterozygosity) and serum transferrin receptor 2 (Q317X homozygosity) were found. A 21-year-old brother with a milder phenotype resembling classic adult-onset hereditary hemochromatosis carried only the Q317X serum transferrin receptor 2 homozygote mutation. CONCLUSIONS: Juvenile hereditary hemochromatosis is not a distinct monogenic disorder invariably due to hemojuvelin or hepcidin mutations: it may be genetically linked to the adult-onset form of hereditary hemochromatosis.     

Serum erythropotietin and serum transferrin receptor levels in aplastic anaemia

Summary. Serum erythropoietin (EPO) and soluble transferrin receptor levels were serially measured in 74 patients with aplastic anaemia (AA). As control groups we investigated healthy controls (n = 24) and patients with iron-deficiency (n = 23) or haemolytic anaemia (n = 16). There was a significant negative correlation of log EPO on haematocrit both in AA patients and in the anaemic control group. However, for the same degree of anaemia, log EPO levels in AA were significantly higher than in iron-deficiency or haemolytic anaemia. EPO levels at diagnosis did not correlate with severity of aplastic anaemia, nor did they predict outcome after immunosuppression. During immunosuppressive treatment of AA with anti-thymocyte globulin and cyclosporine A, EPO levels were significantly lower compared with pre-treatment values without a corresponding change in haematocrit. This impaired EPO response to anaemia during immunosuppression might affect recovery of erythropoiesis. In AA patients, EPO levels declined with haemopoietic recovery. However, compared with normal controls, EPO levels in remission patients were still higher with respect to their haematocrit.
Results of this study argue against the model of a simple feedback regulation of EPO via hypoxic anaemia. Our data support the hypothesis that cytokines and the erythropoietic progenitor pool are involved in the regulation of EPO production. The results illustrate that serial measurements of EPO along with therapeutic interventions are necessary to identify patients who might benefit from treatment with exogenous recombmant human EPO.     

The mitochondrial nt 16189 polymorphism and hereditary hemochromatosis

It has been claimed that a noncoding mitochondrial polymorphism at nt 16189 is correlated with the penetrance of the homozygous state for the C282Y mutation of the HFE gene. We have genotyped homozygotes for the C282Y mutation and find no relationship between the ferritin levels and the inheritance of the mitochondrial polymorphism. Indeed, the small difference found is in the opposite direction of that reported previously.     

Accuracy of serum transferrin receptor levels in the diagnosis of iron deficiency among hospital patients in a population with a high prevalence of thalassaemia trait

The diagnosis of iron deficiency in hospital patients can be difficult in the presence of inflammation. A raised serum transferrin receptor (sTfR) level is useful as a marker of iron deficiency as it is unaffected by inflammation. However, diseases that cause an increase in erythropoietic activity can also result in a raised sTfR level. In South-East Asia, the prevalence of thalassaemia trait is high. As thalassaemia trait is associated with ineffective erythropoiesis and therefore an increase in the sTfR level, we studied the influence of thalassaemia trait on the diagnosis of iron deficiency in hospital patients. Among 431 patients with different combinations of iron deficiency, alpha- and beta-thalassaemia trait, we found that the sTfR level is an excellent diagnostic test for iron deficiency only in patients without thalassaemia trait. alpha-Thalassaemia trait worsened its diagnostic accuracy and beta-thalassaemia trait rendered it a non-diagnostic test. We conclude that in populations with a high prevalence of thalassaemia trait, the sTfR level is not useful in diagnosing iron deficiency unless the patient's thalassaemia status is known.     

Deferasirox in patients with iron overload secondary to hereditary hemochromatosis: results of a 1‐yr Phase 2 study

This open‐label, prospective, phase 2 study evaluated the safety and efficacy of deferasirox (10 ± 5 mg/kg/d) in patients with hereditary hemochromatosis (HH) and iron overload refractory to or intolerant of phlebotomy. Ten patients were enrolled and all completed the 12‐month treatment period. There were significant decreases from baseline to end of study (i.e., 12 months) in median serum ferritin (P < 0.001), mean transferrin saturation (P < 0.05), median liver iron concentration (P < 0.001), and mean alanine aminotransferase (P < 0.05). The median time to achieve serum ferritin reduction ≥50% compared to baseline was 7.53 months. The most common adverse events were mild, transient diarrhea (n = 5) and nausea (n = 2). No patient experienced an increase in serum creatinine that exceeded the upper limit of normal. These data confirm that deferasirox was well tolerated and effective in reducing iron burden in patients with hereditary hemochromatosis and could be a safe alternative to phlebotomy in selected patients.