Severe Iron Overload Research Articles

Growth differentiation factor 15 in patients with congenital dyserythropoietic anaemia (CDA) type II

Congenital dyserythropoietic anaemias (CDAs) are heterogeneous, hereditary disorders hallmarked by ineffective erythropoiesis and tissue iron overload. Growth differentiation factor 15 (GDF15) was suggested to mediate iron overload in iron-loading anaemias, such as the thalassaemias and CDAI by suppressing hepcidin, the key regulator of iron absorption. Here, we show that serum GDF15 concentrations are elevated in subjects with CDAI and CDAII. Despite similar disease characteristics, CDAI patients present with significantly higher GDF15 concentrations compared to CDAII patients. Hepcidin concentrations are inappropriately low in CDAII patients considering the severe hepatic iron overload associated with this disorder. GDF15 significantly correlates with the degree of anaemia (Hb), the response of erythropoiesis (reticulocyte index) as well as with iron availability in the serum (transferrin saturation). The observation that GDF15 is elevated in CDAII patients is consistent with the proposal that GDF15 is among the erythroid factors down-regulating hepcidin and contributing to iron overload in conditions of dyserythropoiesis.

Iron-deficiency anemia from matriptase-2 inactivation is dependent on the presence of functional Bmp6

AbstractHepcidin is the master regulator of iron homeostasis. In the liver, iron-dependent hepcidin activation is regulated through Bmp6 and its membrane receptor hemojuvelin (Hjv), whereas, in response to iron deficiency, hepcidin repression seems to be controlled by a pathway involving the serine protease matriptase-2 (encoded by Tmprss6). To determine the relationship between Bmp6 and matriptase-2 pathways, Tmprss6−/− mice (characterized by increased hepcidin levels and anemia) and Bmp6−/− mice (exhibiting severe iron overload because of hepcidin deficiency) were intercrossed. We showed that loss of Bmp6 decreased hepcidin levels; increased hepatic iron; and, importantly, corrected hematologic abnormalities in Tmprss6−/− mice. This finding suggests that elevated hepcidin levels in patients with familial iron-refractory, iron-deficiency anemia are the result of excess signaling through the Bmp6/Hjv pathway.

Járnmaðurinn - sjúkratilfelli

The most common cause of microcytic anemia is iron deficiency. We report a 29 year old man with history of dyspnea, fatigue and severe microcytic anemia despite iron therapy for 3 years. Blood transfusions elevated the hemoglobin levels temporarily, but iv iron did not. Bone marrow showed sideroblastic anemia. The anemia resolved with pyridoxine treatment but severe iron overload necessitated multiple phlebotomies. Today the patient is asymptomatic on pyridoxine with a normal hemoglobin level.

Evaluation of cardiac iron load by cardiac magnetic resonance in thalassemia

To quantify myocardial iron stores by Cardiac Magnetic Resonance (CMR). Prospective cohort study. Thalassemia center in a teaching hospital. 60 transfusion dependant thalassemia major patients and 10 controls during 2008-2009. MRI T2* for cardiac iron load and cardiac functions was performed on a 1.5 Tesla Siemens Sonata machine using the thalassemia tools software. Ejection fraction (EF) was measured using standard CMR sequence and EF <56% considered as cardiac dysfunction. Quantification of iron deposition was categorized as T2* <10 milliseconds (ms) as high risk, 10-20 ms as intermediate risk and >20 ms as low risk. Simultaneous liver iron T2* values were categorized into normal i.e. >6.3 ms, mild iron overload 6.3-2.7 ms , moderate iron overload 2.7- 1.4 ms and severe iron overload <1.4 ms. Pretransfusion serum ferritin levels were simultaneously determined. Data was analyzed by paired and unpaired t test of mean. Of 60 patients, 50% had no cardiac siderosis; 33.3% had mild to moderate and while 16.7% had severe cardiac siderosis . In contrast, only 8.3% had normal liver iron values, 55.7% had mild to moderate and 36% had severe iron stores. The mean serum ferritin of all 60 cases was 3528.6 ± 1958.6 ng/mL. There was a statistically significant difference in the mean cardiac T2* of patients (23.45 ± 13.4 ms) as compared to controls (32.67 ± 2.68 ms) (P<0.01). Thalassemia patients had significantly higher cardiac iron stores as compared to controls. Serum ferritin and liver iron values did not correlate with cardiac iron values. Three of 10 patients <10 years showed evidence of myocardial siderosis.

Unrelated hematopoietic stem cell transplantation in a patient with congenital dyserythropoietic anemia and iron overload

CDA is a heterogeneous group of disorders that result in morphologically abnormal erythroid maturation and ineffective erythropoiesis. Curative therapy for CDA focuses on the use of HSCT using fully matched sibling donors. This is the first report of a Type II CDA patient with severe iron overload who was successfully treated with HSCT using a HLA-matched unrelated donor after aggressive chelation therapy. Given the challenges of HSCT in any patient with CDA and severe iron overload, the role of novel approaches to iron chelation and HSCT is discussed.

EARLY Detection of Cardiac and Hepatic Iron Overload by T2* Magnetic Resonance in Very Young Patients with Thalassemia Major in Oman

AbstractAbstract 4262 Background:Despite availability of iron chelation, iron-mediated cardiac toxicity remains the leading cause of death in thalassemia major patients. Although serum ferritin is widely used as a measure of iron overload, this has been challenged by recent magnetic resonance imaging (MRI) studies. Magnetic resonance using myocardial T2* is a highly sensitive, non-invasive and reproducible technique for detection of myocardial iron content. Materials and Methods:Seventy-four children are on follow-up at the Pediatric Thalassemia Day Care Center, Sultan Qaboos University Hospital, Muscat, Oman. Twenty-seven patients above the age of 7 years underwent T2* MRI procedure, and 9 of these patients had a follow-up T2* MRI at an interval of 1 year. MRI T2* was introduced at our institution in 2007 but was performed only on patients over the age of 12 years as it was thought that younger children would be unable to comply with the requirements of the MRI examination. Initially, we found that many of our patients failed to complete the procedure for T2* MRI (28.5% failure rate) mainly because of their inability to either hold their breath in expiration or due to movement during the procedure. But after training by physiotherapy we were successful in completing the procedure in children as young as 7 years, with no failures without the use of general anesthesia, as has been practiced by some centers. Results:Previous reports reveal no detectable cardiac iron in patients with thalassemia major less than 9.5 years of age. But we have detected in our patients severe and mild cardiac iron overload at the age of 7.5 years and 9.5 years respectively. At the time of the initial T2*MRI, the patient with severe cardiac iron overload was on chelation with Desferrioxamine with sub-optimal compliance, with a ferritin of 2605 ng/ml and a T2* MRI cardiac value of 9.3 ms. Repeat T2* MRI after 18 months (despite extensive counseling and optimization of chelation) revealed a cardiac T2* value of 4.8 ms at a ferritin level of 2796 ng/ml revealing that cardiac siderosis worsened despite the fairly constant ferritin level and the patient was shifted to combination chelation therapy. Also 44.5 % of our patients have moderate to severe hepatic iron overload. All these children were on regular 3–4 weekly follow-up for transfusion therapy with serial monitoring of ferritin levels guiding the chelation therapy. Of these, 62.9% (n=17) are on Deferiprone monotherapy at a mean dose 85.7 mg/kg, 33.3 % (n=9) are on combination chelation therapy with Deferiprone and Desferrioxamine, mean dose 95.6 mg/kg and 36.6 mg/kg respectively, and 14.2% (n=1) on Deferasirox at a dose of 40 mg/kg. Our results revealed inadequate iron chelation in some of our patients, most probably due to sub-optimal compliance that was not detected by serial ferritin monitoring (mean =1309 ng/ml). Moreover there was a poor correlation of ferritin to cardiac T2* and hepatic T2* values. Conclusions:With compliance to chelation therapy being a major issue in our patients, and failure of ferritin levels to predict the severity of cardiac and hepatic iron overload in some of the patients in a younger age group; we emphasize the importance of early and routine T2* MRI to detect organ iron overload for timely intervention with optimal iron chelation therapy in patients with thalassemia major. Disclosures:No relevant conflicts of interest to declare.

Highly Transfused MDS Patients Often Have Cardiac Iron Overload, as Shown by MRI Assessment

AbstractAbstract 2906 Background:Cardiac complications of transfusional iron overload are well documented in various inherited anemias. In regularly transfused MDS, the deleterious role of iron overload on cardiac disease is more disputed, due in particular to frequent concomitant causes of cardiac failure. Cardiac MRI T2* allows accurate and specific measurement of iron content. Methods:We prospectively evaluated in 4 centers of the GFM by standardized and transferable MRI methods both cardiac T2* according to Anderson (Eur Heart J. 2001Dec;22(23):2171-9) and liver iron content (LIC) according to Gandon (Lancet. 2004 Jan 31;363(9406):357-62), as well as cardiac function by routine echocardiography or MRI in regularly transfused MDS patients. Results:From Dec 2005 to March 2010, 73 patients (pts) were included (14 of them had more than one MRI evaluation over time): 38 M/35F, Median age 68 (24-86); WHO : RA=5, RARS=33, RMCD-RS=3, RMCD=1, RAEB1=9, RAEB2=5, RAEB-T/AML=1, 5q- syndrome=8 and unclassified=8; Karyotype: fav n=50, Int n=9, unfav n=4, failure n=10; IPSS: low n=29, Int-1 n=28, Int-2 n=5 and High n=1, unknown n=10. Median interval from MDS diagnosis and MRI T2* assessment was 49 months (range 0–324). Median serum ferritin at MRI assessment was 1750 ng/ml (range 282–7339) and 54/73 pts were on chelation therapy (CT) (median duration of CT prior to first MRI: 18 months, range 1–125). 37/73 pts had cardiac symptoms and 28 were on cardiac therapy. At first MRI T2* analysis, the median number of RBC units transfused was 68 (range 5–574). Median LIC was 330 micromoles/g/dw (range 40–908). Median Cardiac T2* was 27 ms (range 6–74). 14/73 pts had cardiac iron overload defined by MRI T2* ≤20 ms (19%) and among them 3/73 (4%) had severe cardiac iron overload (T2*≤ 10 ms). LVEF was below normal (55%) in 13/59 cases evaluated. A correlation was found between cardiac T2*and the number of RBC units transfused (Pearson correlation =-0.342, p=0.004) but not with LIC (p= 0.65) and serum ferritin (p=0.21). Cardiac overload was seen in 1/19 (5.5%) pts transfused <50 RBC units, 4/37(12.1%) pts transfused 50–150 units, 9/17 (52.9%) pts transfused >150 units (p= 0.0005). Those 3 pt subgroups also differed in median LIC (μmoles/g/dw) (<50 units= 250, 50–150 units=340, > 150 units=414) (p=0.044 Kruskall-Wallis' test), but not significantly in serum ferritin (p= 0.085).No significant correlation was found between decreased LVEF (< 55%) and cardiac T2* <20 ms (p=0.5), or T2*≤10 ms (p=0.23). In particular, 5/13 pts (38%) with LVEF <55% had T2*<20ms, vs versus 8/46 pts (17%) with LVEF >55% (p= 0.13). However, 1/14, 0/30 and 3/12 pts having received <50, 50–150 and > 150 RBC units had severe cardiac failure (ie LVEF≤35%)(p=0.012). 3/4 pts with severe cardiac failure had T2*< 20ms,compared to 8/54 pts without severe cardiac failure (p=0.023).14 pts had another cardiac MRI 6 to 34 months (median 18) after the first. All were on CT and had received a median of 60 and 214 PRBC units at first and last MRI, resp. Median Cardiac T2* was 21.6 ms (range 8.5–35.3) and. 28 ms (range 6.4–41) at last and at first assessment, respectively (p=0.3) Conclusions:Moderate and severe post transfusional cardiac iron overload was seen in 19% and 4% of regularly transfused MDS, respectively. The level of cardiac iron overload was well correlated to the number of RBC transfused. The impact of cardiac overload on LVEF was unclear except in pts with severe cardiac impairment (LVEF <35%), possibly suggesting that iron overload is only one of the factors responsible for cardiac disease in many of those elderly patients. Disclosures:No relevant conflicts of interest to declare.

Treatment with the Small Molecule Genistein Increases Hepcidin Expression In Zebrafish Embryos and Human Hepatocytes

AbstractAbstract 4245Hepcidin, a transcriptionally regulated peptide hormone, produced by hepatocytes in response to iron overload, decreases intestinal iron absorption and macrophage iron release via effects on ferroportin. Hepcidin is a potential drug target for patients with iron overload syndromes, because its levels are inappropriately low in these patients. Among patients with hereditary hemochromatosis, premenopausal women exhibit less severe iron overload than their male counterparts. While this has been attributed to menstrual blood loss, we hypothesized that estrogens may play a role in regulating Hepcidin expression. We tested estradiol and the phytoestrogens, genistein, apigenin, daizdein, and genistin, for their effects on hepcidin expression in zebrafish embryos. In addition to its estrogenic effects, genistein is known to inhibit multiple protein tyrosine kinases. We found that genistein treatment from 28–52 hours post-fertilization in zebrafish embryos enhanced hepcidin transcript levels relative to liver size, as assessed by whole mount in situ hybridization, while estradiol and the other phytoestrogens did not produce a similar effect. To evaluate whether these effects were conserved in human hepatocytes, we treated HepG2 cells with genistein for 24 hours. We found that genistein treatment was associated with a significant increase in Hepcidin transcript levels, which was abrogated by co-treatment with the bone morphogenic protein (BMP) signaling antagonist dorsomorphin, but not by co-treatment with the estrogen receptor antagonist ICI 182,780. In conclusion, genistein promotes Hepcidin expression in hepatocytes in a BMP-dependent, but estrogen receptor independent manner. Future experiments will address the signaling pathway by which genistein exerts its effect on Hepcidin expression. Disclosures:No relevant conflicts of interest to declare.

Pulmonary Hypertension Is Uncommon In Well-Transfused Thalassemia Major Patients

AbstractAbstract 4273 Introduction:Pulmonary hypertension is a common and serious cardiovascular complication in patients with thalassemia intermedia and sickle cell disease. Circulating platelet and erythrocyte fragments, as well as cell-free hemoglobin from intravascular hemolysis, may contribute to nitric oxide depletion, intimal proliferation, and pulmonary vascular remodeling. Splenectomy has been also been associated with pulmonary hypertension in some studies. Ineffective erythropoiesis, via release of PLGF and elevation of other proinflammitory cytokines, has also been associated with pulmonary hypertension. However, the risk for pulmonary hypertension in thalassemia major patients remains controversial, with prevalence estimates ranging from 10%-60%. We report echocardiography results from 80 thalassemia major patients enrolled in the Early Detection of Iron Cardiomyopathy Trial (EDICT) at Children's Hospital Los Angeles. Methods:Patients were enrolled from August 2004 until May 2009 in a combined cross-sectional and observational trial probing for early predictors of cardiac dysfunction. Patient visits were scheduled within one week of transfusion. All patients underwent echocardiography and cardiac MRI analysis within 4 months of each other; most were performed during the same clinical visit (median time between scans one day). Comprehensive assessments of pulmonary artery pressure (tricuspid and pulmonary regurgitation velocities), systolic function, and diastolic function were performed using two dimensional imaging, M-mode, and routine and tissue Doppler. All images were collected by experienced echocardiography technicians and analyzed by the principal investigator. Three to five beat averages were used to improve measurement stability. Tricuspid regurgitation (TR) jet was only reported if a full envelope was recognized; at our institution, the upper limit of normal for TR jet is 2.7 m/s. Results:Patient demographics are summarized in Table 1. Patients were gender-balanced and well distributed between 11 and 47 years of age. Patients who had been splenectomized (N=34) tended to be older. Iron overload was severe, with a mean liver iron concentration of 12.4 ± 14.2 mg/g dry weight. Roughly half of the patients had detectable cardiac iron and 9% had overt left ventricular dysfunction (LVEF < 56%). TR jet was detectable in 62/80 patients. Only one patient exhibited pulmonary artery hypertension (TR jet 2.9 m/s), however this patient also had severe cardiac iron overload and overt left ventricular systolic and diastolic dysfunction (LVEF 42.7%, E/E' 10). Two patients had TR jets of 2.6 ms and three had TR jets of 2.5 m/s. Pulmonary insufficiency jets were normal in all patients.TR velocity did not correlate with age, cardiac index, cardiac iron or liver iron, but demonstrated a weak (r=0.29, p=0.02) association with left ventricular diastolic dysfunction (E/E′). Discussion:The EDICT patient cohort suggests a low risk for pulmonary hypertension in well-transfused thalassemia major patients. The single elevated TR jet was explained by iron cardiomyopathy and normalized (2.2 m/s) after two years of aggressive chelation therapy. TR velocities at the upper limits of normal (2.5-2.7 m/s) were observed in five patients; these have been associated with poor outcomes in some sickle cell disease cohorts, but not in thalassemia major. Long-term surveillance remains critical as pulmonary hypertension risk may increase with age. Disclosures:Harmatz:Novartis: Research Funding. Coates:Novartis: Research Funding, Speakers Bureau. Wood:Novartis: Research Funding; Ferrokin Biosciences: Consultancy.

Combination of Two Orally Active Iron Chelating Agents: Efficacy and Safety In a Clinical Setting

AbstractAbstract 2064For patients with transfusion dependent anemias, in particular thalassemia major, as part of a compassionate –use program that was approved by the hospital's IRB, we report the efficacy and safety outcomes of combining therapy with deferasirox (DFX) and deferiprone (DFP). The combination of DFP and deferoxamine (DFO)has been shown to be effective in reducing cardiac iron overload but some patients are unable or unwilling to use DFO. DFP alone has also been reported to be cardioprotective. Based on concerns for their welfare in the presence of excessive cardiac iron load, a reduced left ventricular ejection fraction and either severe allergy or intolerance to DFO, 4 adult patients with thalassemia major have been treated with the combination of DFX and DFP for between 6 and 60 months (mean 18 months). All four patients were initially treated with DFO or DFX but DFP at 75–100 mg/kg/day was initiated because of severe cardiac iron overload. DFX at 15–40 mg/kg/day was added based upon high liver iron concentrations(LIC). Efficacy was evaluated by monthly ferritin levels and semiannual cardiac T2* and LIC estimates (using MRI R2 and R2*). All 4 patients had at least two MRI assessments. Table 1shows the changes in ferritin, cardiac T2*, LVEF and mean LIC. Cardiac T2* improved from 5.8 ±1.5 to 7.0 ± 1.5 ms., (p=0.15). If the T2* is recalculated as cardiac iron concentration then the change was from 4.1± 1.3 to 3.3± 1.1 mg/g dry weight (p=0.09). One patient who has been receiving the treatment for 6 months has shown a 1% per month deterioration in T2*. The patients who have been on the combination for 12, 24 and 60 months, have had reductions of 2.7%, 0.5% and 1.5% per month respectively. LVEF improved overall from the baseline value of 52.8% to 58.9% (p=0.02). Ferritin fell from a mean of 5826 to 5544 ng/L (p=0.86). LIC increased from a mean of 20.7 to 28.1 mg/g dry weight (p=0.36). Table 2 shows the baseline and minimum absolute neutrophil counts and baseline and final ALT (IU/L). No drug-related neutropenia (ANC of <1.5 ×109/L), agranulocytosis or arthralgia were observed. No patients demonstrated significant proteinuria and mean creatinine levels were unchanged. ALT's showed fluctuations that were compatible with the degree of LIC. It is important to note that compliance was extremely variable in that one patient (P3), only took DFX once monthly with many lapses in her DFP treatment as well. The other patients also had significant lapses in their compliance as well as long vacation periods during which they did not receive treatment. These results indicate that the combined use of the two oral chelators (DFP & DFX) prevented further cardiac iron loading with a tendency for its reduction, significant improvement in left ventricular ejection fraction, maintenance of ferritin levels and LIC. It is also possible that the DFP cardioprotective effect may be activated even in the presence of poor compliance with it. The combination was well tolerated and easier to manage. It is more acceptable for life-long chelation and positively influenced patients' quality of life. It seems likely that maximum doses of both medications need to be prescribed for better outcomes and longer periods of follow up are essential. In particular, a prospective randomized study in patients with excessive cardiac iron will be necessary before this combination could be considered standard therapy.Table 2Showing baseline absolute and lowest neutrophil count (ANC ×109/L), baseline and latest ALT (IU/L).IdentifierBaseline ANCLowest ANCBaseline ALTLatest ALTP13.42.29164P24.82.12395P33.82.87269P44.74.513364 Disclosures:Berdoukas:ApoPharma Inc.: Consultancy. Wood:Novartis Inc.: Research Funding; ApoPharma: Honoraria, Membership on an entity's Board of Directors or advisory committees. Coates:Novartis: Research Funding, Speakers Bureau.

Congenital Erythropoietic Porphyria Due to Co-Inheritance of GATA1 and UROS Gene Mutations

AbstractAbstract 3208The Congenital Erythropoietic Porphyria (CEP) is a rare form of Chronic Porphyria characterized by severe photosensivity. Secondary infections of cutaneous lesions may lead to scarring, deformities and disfigurement of the light-exposed parts of the body such as hands, ears, nose, and eyelids. Erythrodontia, osteodystrophia, combining osteolysis and osteoporosis, hypercellular bone marrow, hemolytic anemia, hypersplenism, splenomegaly and red-coloured urine are present in almost all patients. Generally, CEP is an autosomic recessive disease caused by mutations in homozygosis or compound heterozygosis in UROS gene coding for the fourth enzyme of the heme biosynthesis pathway. At present a new form of CEP with an X-linked inheritance associated with R216W mutation in the GATA 1 gene has been described only in one French-English family by Philips JD et al. GATA1 is a key zinc finger transcription factor that coordinates hematopoietic cell differentiation. We described a Turkish family where a severe CEP phenotype segregates with thalassemia and thrombocytopenia. The propositus is an 4 year old boy who presented photosensitive bullous dermatosis, splenomegaly, short stature and pallor. His mother was chronically anemic (Hb 10.9 g/dL) and thrombocytopenic (96×109/L) known as chronic immune thrombocytopenic purpura, whereas the father and the brother were asymptomatic. At birth, the boy had hypochromic, microcytic anemia, thrombocytopenia and thrombasthenia. Reticulocyte counts slightly increased. At 2 months he was transfused, splenomegaly and heart murmur were detected on physical examination. At this point his Hb level was 6,9 g/dl, WBC 7,400×109/L, platelets 64–106×109/L, MCV 70 flL, RDW 28%. The patient's peripheral-blood smear revealed microcytic, hypochromic red cells, target cells, basophilic stippling, and rare nucleated red cells, findings compatible with thalassemia but DNA analysis for beta and alpha thalassemia was normal. A bone marrow aspirate revealed a hypercellular marrow and erythroid hyperplasia. Dyserythropoiesis was noted with nuclear budding, nuclear bridging, and occasional multinucleation. Megakaryocytes were decreased in number, Hb electrophoresis revealed increased levels of fetal hemoglobin (HbA2 2%; Hb F 68%). At 3 years liver MRI detected iron overload. Ferritin level was 329ng/dL. Because of some skin lesions on his body he was then tested with urine and plasma analysis and the results were consistent with the diagnosis of congenital erythropoietic porphyria. Urine total porphyrins were 5414 nmol/mmol creatinin (N<35) with an excess of isomer I of both Uroporphyrins and Coproporhyrins. Total plasma porhyrins were 312 nmol/L (N<10). We analyzed the UROS gene and we identified a new mutation c.338A>T (D113V) in heterozygosis in the propositus, in his father and in his brother. On the basis of hematological status we decided to analyze also GATA1 gene and the mutation c.646C>T (R216W) has been detected in the child and on one allele of his mother who presents a balanced pattern of X-inactivation. The patient is similar to the case of association of CEP with an hematologic phenotype of beta-thalassemia intermedia. Markedly elevated Hb F levels, and thrombocytopenia result from mutations in the transacting factor GATA-1 presented by Philips JD et al. This patient additionally has a severe liver iron overload. A newborn sibling died at birth because of sepsis and neonatal hepatitis. Liver iron overload was also detected in the sibling's autopsy. All components of family were heterozygous for the H63D mutation in HFE gene but no other HAMP, HJV and SLC40A1 mutations were found. The patient started a regular transfusion program and Deferasirox was given for hemosiderosis after Desferroxamine uncompliance. This is the first report of the association of GATA1 and UROS gene mutations that could explain the very severe hematological phenotype. Disclosures:No relevant conflicts of interest to declare.

Serum Ferritin and Cardiac/Liver Magnetic Resonance Imaging In Evaluating Iron Overload for Patients with Bone Marrow Failure Conditions Undergoing Non-Myeloablative HSCT

AbstractAbstract 1331Several groups have identified iron overload, in terms of raised pre-transplant serum ferritin levels, as an independent adverse prognostic factor for patients undergoing myeloablative HSCT. While serum ferritin has been used as a common marker in clinical studies to evaluate the impact of iron overload following allogeneic transplantation, there are limitations to its use with it being an acute phase reactant, as well as its lack of specificity for predicting end-organ toxicities. Patients undergoing HSCT for bone marrow failure (BMF) syndromes usually have a significant red cell transfusion history, and although the majority of these patients receive non-myeloablative HSCT regimens, it is unclear as to the impact of iron overload in these patients on subsequent transplant outcomes.In order to address these questions, we performed a prospective study evaluating the pre-transplant serum ferritin together with concurrent T2* cardiac magnetic resonance imaging (MRI) and R2 liver MRI in 18 patients with BMF syndromes undergoing allogeneic HSCT. The diagnosis of the patients included MDS (RCMD/hypoplastic MDS) =10, acquired aplastic anaemia =7, fanconi anaemia =1. The median age of the patients at transplantation was 42 years, and all patients received a T-cell depleted non-myeloablative HSCT. All patients were transfusion dependent pre-HSCT, with a median number of red cell transfusions of 45 (range: 8–115). Pre-HSCT ferritin was performed within 2 weeks of HSCT, and the results were correlated with albumin and C-reactive protein to reduce the impact of ferritin as an acute phase reactant. T2* and R2 MRI were similarly performed within 2 weeks of HSCT.The median pre-HSCT ferritin was significantly raised at 2119 ug/l(range: 559–12235). In contrast, the T2* cardiac MRI was normal for all but one patient who had evidence of mild cardiac iron overload. All patients had a corresponding cardiac echocardiogram performed with an ejection fraction within normal limits. For the liver T2* MRI, 7 patients had evidence of none or mild hepatic iron overload, while 11 patients had moderate to severe iron overload. There was no correlation between pre-HSCT transfusion burden and serum ferritin levels. Furthermore, there was no correlation between either the transfusion burden or serum ferritin, and the T2*MRI readings.In terms of HSCT outcome, the median time to neutrophil engraftment was 14 days. 2 patients had primary graft failure and only 1 patient died within 100 days due to an intra-cerebral haemorrhage. No patients had any clinical features of hepatic veno-occlusive disease (VOD), and 5 patients had evidence of grade I-II acute grade versus-host-disease. Data were also collected on the incidence of bacterial, fungal and viral infections post-HSCT for the cohort. There was however no significant association between transfusion burden, serum ferritin or T2* imaging and any of the HSCT outcomes (engraftment/day 100 TRM, GvHD, VOD or infections).In the context of heavily transfused BMF patients receiving allogeneic HSCT, serum ferritin does not correlate with end-organ deposition of iron. Despite the high transfusion burden in our cohort of patients, cardiac deposition of iron appears minimal while hepatic iron deposition is significant in a large proportion of patients. Reassuringly, a raised iron overload by either of the above mentioned parameters had no effect on HSCT outcomes. Our findings highlight the limitations of using serum ferritin as a marker of iron overload pre-HSCT. The role of active pre-HSCT chelation of BMF patients receiving non-myeloablative HSCT regimens remains unclear, and further studies are warranted. Disclosures:No relevant conflicts of interest to declare.

Continued improvement in myocardial T2* over two years of deferasirox therapy in -thalassemia major patients with cardiac iron overload

The efficacy of cardiac iron chelation in transfusion-dependent patients has been demonstrated in one-year prospective trials. Since normalization of cardiac T2* takes several years, the efficacy and safety of deferasirox was assessed for two years in patients with β-thalassemia major in the cardiac sub-study of the EPIC trial. Eligible patients with myocardial T2* greater than 5 to less than 20 ms received deferasirox, with the primary endpoint being the change in T2* from baseline to two years. Baseline myocardial T2* was severe (> 5 to < 10 ms) in 39 patients, and moderate-to-mild (10 to < 20 ms) in 62 patients. Mean deferasirox dose was 33.1 ± 3.7 mg/kg/d in the one-year core study increasing to 36.1 ± 7.7 mg/kg/d during the second year of treatment. Geometric mean myocardial T2* increased from a baseline of 11.2 to 14.8 ms at two years (P < 0.001). In patients with moderate-to-mild baseline T2*, an increase was seen from 14.7 to 20.1 ms, with normalization (≥ 20 ms) in 56.7% of patients. In those with severe cardiac iron overload at baseline, 42.9% improved to the moderate-to-mild group. The incidence of drug-related adverse events did not increase during the extension relative to the core study and included (≥ 5%) increased serum creatinine, rash and increased alanine aminotransferase. Continuous treatment with deferasirox for two years with a target dose of 40 mg/kg/d continued to remove iron from the heart in patients with β-thalassemia major and mild, moderate and severe cardiac siderosis. (Clinicaltrials.gov identifier: NCT 00171821).

Longer survival associated with HLA‐A*03, B*14 among 212 hemochromatosis probands with HFE C282Y homozygosity and HLA‐A and ‐B typing and haplotyping1

Human leukocyte antigen (HLA) haplotypes may influence iron phenotypes in patients with HFE hemochromatosis and could affect survival. We tabulated general characteristics of HLA-A and -B types and haplotypes of HFE C282Y/C282Y probands diagnosed in medical care and analyzed these data to identify HLA survival modifiers. There were 212 probands (130 men, 82 women). Mean follow-up was 12.0 ± 6.4 yr (0.1-41.2 yr; 34 deaths). HLA-A*03 was more prevalent in men (76.9% vs. 61.0% women; P = 0.0129); 35.4% of men and 29.3% of women had A*03, B*07; and 7.7% of men and 8.5% of women had A*03, B*14. Twenty-three probands had cirrhosis; none had A*03, B*14. Positivity for A*03 or A*03, B*07 was not a significant predictor or modifier of survival. In multiple regression analyses, A*03, B*14 predicted longer survival (P = 0.0004). Kaplan-Meier analysis confirmed longer survival in probands with A*03, B*14 (P = 0.0199, log-rank test). After excluding the 23 non-A*03, B*14 probands with cirrhosis, survival of probands with A*03, B*14 was still greater than that of probands without A*03, B*14 (P = 0.0254; log-rank test). Twenty-four years after diagnosis, cumulative survival of probands with and without A*03, B*14 was 100% and 58%, respectively. The percentage of deaths due to iron overload was lower in probands with A*03, B*14 (0% vs. 21.9%; P = 0.0392). In hemochromatosis probands with HFE C282Y/C282Y, survival was longer in those with HLA-A*03, B*14. Earlier age at diagnosis and less severe iron overload in probands with A*03, B*14 could explain this difference.

Bone loss caused by iron overload in a murine model: importance of oxidative stress

AbstractOsteoporosis is a frequent problem in disorders characterized by iron overload, such as the thalassemias and hereditary hemochromatosis. The exact role of iron in the development of osteoporosis in these disorders is not established. To define the effect of iron excess in bone, we generated an iron-overloaded mouse by injecting iron dextran at 2 doses into C57/BL6 mice for 2 months. Compared with the placebo group, iron-overloaded mice exhibited dose-dependent increased tissue iron content, changes in bone composition, and trabecular and cortical thinning of bone accompanied by increased bone resorption. Iron-overloaded mice had increased reactive oxygen species and elevated serum tumor necrosis factor-α and interleukin-6 concentrations that correlated with severity of iron overload. Treatment of iron-overloaded mice with the antioxidant N-acetyl-L-cysteine prevented the development of trabecular but not cortical bone abnormalities. This is the first study to demonstrate that iron overload in mice results in increased bone resorption and oxidative stress, leading to changes in bone microarchitecture and material properties and thus bone loss.

Conserved proximal promoter elements control repulsive guidance molecule c/hemojuvelin (Hfe2) gene transcription in skeletal muscle

Repulsive guidance molecule c (RGMc; gene symbol: Hfe2) plays a critical role in iron metabolism. Inactivating mutations cause juvenile hemochromatosis, a severe iron overload disorder. Understanding mechanisms controlling RGMc biosynthesis has been hampered by minimal information about the RGMc gene. Here we define the structure, examine the evolution, and establish mechanisms of regulation of the mouse RGMc gene. RGMc is a 4-exon gene that undergoes alternative RNA splicing to yield 3 mRNAs with 5′ different untranslated regions. Gene transcription is induced during myoblast differentiation, producing all 3 mRNAs. We identify 3 critical promoter elements responsible for transcriptional activation in skeletal muscle, comprising paired E-boxes, a putative Stat and/or Ets element, and a MEF2 site, and muscle transcription factors myogenin and MEF2C stimulate RGMc promoter function in non-muscle cells. As these elements are conserved in RGMc genes from multiple species, our results suggest that RGMc has been a muscle-enriched gene throughout its evolutionary history.

Severe iron overload and hyporegenerative anemia in a case with rhesus hemolytic disease: therapeutic approach to rare complications.

A 33 weeks' gestation, a baby with rhesus hemolytic disease (RHD), who had received intrauterine transfusions twice, developed cholestatic hepatic disease and late hyporegenerative anemia. Her serum ferritin and bilirubin levels increased to 8842 ng/ml and 17.9 mg/dl, respectively. Liver biopsy showed cholestasis and severe iron overload. Treatment with recombinant erythropoietin (rHuEPO) decreased the transfusion need, and intravenous deferoxamine resulted in a marked decreased in serum ferritin levels and normalization of liver function. In patients who have undergone intrauterine transfusions due to RHD, hyperferritinemia and late hyporegenerative anemia should be kept in mind. Chelation therapy in cases with symptomatic hyperferritinemia and rHuEPO treatment in cases with severe hyporegenerative anemia should be considered.

Hepatic but not brain iron is rapidly chelated by deferasirox in aceruloplasminemia due to a novel gene mutation

Background & AimsAceruloplasminemia is a rare autosomal recessive neurodegenerative disease associated with brain and liver iron accumulation which typically presents with movement disorders, retinal degeneration, and diabetes mellitus. Ceruloplasmin is a multi-copper ferroxidase that is secreted into plasma and facilitates cellular iron export and iron binding to transferrin.ResultsA novel homozygous ceruloplasmin gene mutation, c.2554+1G>T, was identified as the cause of aceruloplasminemia in three affected siblings. Two siblings presented with movement disorders and diabetes. Complementary DNA sequencing showed that this mutation causes skipping of exon 14 and deletion of amino acids 809–852 while preserving the open reading frame. Western blotting of liver extracts and sera of affected patients showed retention of the abnormal protein in the liver. Aceruloplasminemia was associated with severe brain and liver iron overload, where hepatic mRNA expression of the iron hormone hepcidin was increased, corresponding to the degree of iron overload. Hepatic iron concentration normalized after 3 and 5 months of iron chelation therapy with deferasirox, which was also associated with reduced insulin demands. During short term treatment there was no clinical or imaging evidence for significant effects on brain iron overload.ConclusionsAceruloplasminemia can show an incomplete clinical penetrance but is invariably associated with iron accumulation in the liver and in the brain. Iron accumulation in aceruloplasminemia is a result of defective cellular iron export, where hepcidin regulation is appropriate for the degree of iron overload. Iron chelation with deferasirox was effective in mobilizing hepatic iron but has no effect on brain iron.

Musculoskeletal Complications of Hereditary Hemochromatosis: A Case-Control Study

Arthropathy that mimics osteoarthritis (OA) and osteoporosis (OP) is considered a classic complication of hereditary hemochromatosis (HH) but has never been investigated in a cross-sectional study. We investigated whether HH is associated with increased prevalence of OA and OP and related factors. A self-administered questionnaire was completed by 306 patients with HH and 304 age and sex-matched unaffected controls. The mean age of patients was 60.1 ± 11.3 years, and 47.4% were women. More patients with HH than controls had been given a diagnosis of OA: 50.5% vs 28.9% [adjusted OR (aOR) 2.5, 95% CI 1.8-3.6]. Compared with controls, patients with HH had a higher risk of knee (aOR 5.3, 95% CI 1.1-25.6) and hip replacement prosthesis (aOR 5.2, 95% CI 2.2-11.9). More patients than controls had been diagnosed with OP: 23.3% vs 4.6% (aOR 7.3, 95% CI 3.2-17.0). Patients with HH showed an increased prevalence, although not significant, of wrist (aOR 1.7, 95% CI 0.9-3.0) or vertebral fractures (aOR 1.7, 95% CI 0.8-3.8). The severity of iron overload, as defined by a ferritin level > 1000 μg/l at diagnosis, was associated with OA (p = 0.0005), OP (p = 0.01), presence of hip prosthesis (p = 0.04), wrist fractures (p = 0.002), and vertebral fractures (p = 0.02). This case-control study strongly suggests a significant association among HH, OA, and OP. Joint involvement can be severe, especially in patients with substantially elevated ferritin levels.

Combined iron chelation therapy

Patients with thalassemia major accumulate body iron over time as a consequence of continuous red blood cell transfusions which cause hepatic, endocrine, and cardiac complications. Despite the availability of three iron chelators, some patients fail to respond adequately to monotherapy with any of them. Combination therapy, consisting in the use of two chelators on the same day, has been introduced to increase the efficacy and to induce negative iron balance in patients with severe iron overload. Extensive long-term experience has shown that combined chelation with deferiprone and deferoxamine (DFO) rapidly reduces liver iron, serum ferritin, and myocardial siderosis, improves cardiac function, reverses and prevents endocrine complications, reduces cardiac mortality, and improves survival. Side effects, though significant, are manageable if properly monitored. Preliminary promising results have been obtained using combined chelation with deferasirox and DFO. As more drug combination regimes are evaluated, it should be possible to better tailor iron chelation to the needs of the patients, minimizing toxicity and maximizing efficacy throughout life.