Massive Iron Overload Research Articles

A novel hepcidin mutation.

The bioactive peptide hormone hepcidin-25 regulates iron levels by inhibiting iron transport to plasma via ferroportin. Hepcidin-25 is synthesized in the liver where the 84 amino acids pro-hepcidin is cleaved into the bioactive hepcidin-25. A patient admitted to the hospital presented with infertility and fatigue. Genomic DNA was purified from whole blood using the Maxwell 16 system (Promega). MLPA analysis was performed to detect large genomic rearrangements using the SALSA MLPA kit # P347, Hemochromatosis (MRC Holland, Holland). Plasma hepcidin measurements were performed using liquid chromatography/tandem mass spectrometry (LC-MS/MS). A novel HAMP mutation (homozygous one base deletion in c.215delG, p.Cys72Serfs*?) was detected. The deletion in nucleotide 215 causes a frameshift altering the predicted protein sequence from cysteine13 in mature peptide. Whether this leads to nonsense mediated decay of the mRNA or synthesis of an aberrant peptide in unknown, but bioactive hepcidin-25 was undetectable in plasma. The patient had massive iron overload with ferritin up to 8360µg/L. He was anaemic with a Hb at 7.0mmol/L (11.3g/dL) and suffered from hypogonadotropic hypogonadism with a total testosterone of 1.2nmol/l. Continued treatment with venesection and gonadotropins led to reduced fatigue, reduction in iron overload, a normalized Hb and improvement of semen quality. A novel hepcidin mutation was detected in a patient with massive iron overload, fatigue and hypogonadotropic hypogonadism.

High Serum Ferritin Levels among Blood Transfused Thalassemic Patients Admitted to the Department of Paediatrics in a Tertiary Care Centre: A Descriptive Cross-Sectional Study.

Raised serum ferritin levels often indicate iron overload, but they are not specific as the levels are elevated in inflammatory disorders, liver diseases, alcohol excess, or malignancy. If regular transfusions are required for the patient with thalassemia, this doubles the rate of iron accumulation leading to earlier massive iron overload and iron-related damage. The aim of this study aimed to find out the prevalence of high serum ferritin levels among blood-transfused thalassemic patients admitted to the Department of Paediatrics in a tertiary care centre. A descriptive cross-sectional study was conducted at a tertiary care centre from 1 March 2022 to 31 December 2022. Ethical approval was taken from the Institutional Review Committee (Reference number: 078/79-017/HG). Children who were confirmed by haemoglobin electrophoresis on regular blood transfusion were included in the study. Those who did not gave consent were excluded from the study. Convenience sampling method was used. Point estimate and 90% Confidence Interval were calculated. Out of 53 cases, the prevalence of high serum ferritin level was seen in 46 (88.79%) (80.30-97.28, 95% Confidence Interval). Among 46, 34 (73.91%) had serum ferritin levels of more than 1000 to 2500 ng/ml whereas 12 (26.09%) had more than 25000 ng/ml. The prevalence of high serum ferritin levels among blood transfused thalassemic patients admitted to the Department of Paediatrics in a tertiary care centre was found to be higher than in other studies done in similar settings. blood transfusion; ferritin; thalassemia.

Erythropoietin-driven dynamic proteome adaptations during erythropoiesis prevent iron overload in the developing embryo.

Erythropoietin (Epo) ensures survival and proliferation of colony-forming unit erythroid (CFU-E) progenitor cells and their differentiation to hemoglobin-containing mature erythrocytes. A lack of Epo-induced responses causes embryonic lethality, but mechanisms regulating the dynamic communication of cellular alterations to the organismal level remain unresolved. By time-resolved transcriptomics and proteomics, we show that Epo induces in CFU-E cells a gradual transition from proliferation signature proteins to proteins indicative for differentiation, including heme-synthesis enzymes. In the absence of the Epo receptor (EpoR) in embryos, we observe a lack of hemoglobin in CFU-E cells and massive iron overload of the fetal liver pointing to a miscommunication between liver and placenta. A reduction of iron-sulfur cluster-containing proteins involved in oxidative phosphorylation in these embryos leads to a metabolic shift toward glycolysis. This link connecting erythropoiesis with the regulation of iron homeostasis and metabolic reprogramming suggests that balancing these interactions is crucial for protection from iron intoxication and for survival.

Ferroptosis in Hepatocellular Carcinoma: Mechanisms, Drug Targets and Approaches to Clinical Translation.

Simple SummaryIn recent decades, scientific discoveries brought up several new treatments and improvements for patients suffering from hepatocellular carcinoma (HCC). However, increasing resistance to current therapies, such as sorafenib, worsen the outcome of HCC patients, leading to a search for alternative therapeutic strategies. The term ferroptosis describes a novel form of regulated cell death, which is different from apoptosis and necroptosis in a mechanistical and morphological manner. The main mechanism, which leads to cell death, is lipid peroxidation, caused by iron overload and the accumulation of polyunsaturated fatty acids. Recent studies demonstrate that ferroptosis can hamper the carcinogenesis in several tumor entities and in HCC. Therefore, a better understanding and a deeper insight in the processes of ferroptosis in HCC and the possible application of it in the clinical practice are of extreme importance.Ferroptosis, an iron and reactive oxygen species (ROS)-dependent non-apoptotic type of regulated cell death, is characterized by a massive iron overload and peroxidation of polyunsaturated fatty acids (PUFAs), which finally results in cell death. Recent studies suggest that ferroptosis can influence carcinogenesis negatively and therefore may be used as a novel anti-cancer strategy. Hepatocellular carcinoma (HCC) is a deadly malignancy with poor chances of survival and is the second leading cause of cancer deaths worldwide. Diagnosis at an already late stage and general resistance to current therapies may be responsible for the dismal outcome. As the liver acts as a key factor in iron metabolism, ferroptosis is shown to play an important role in HCC carcinogenesis and, more importantly, may hold the potential to eradicate HCC. In this review, we summarize the current knowledge we have of the role of ferroptosis in HCC and the application of ferroptosis as a therapy option and provide an overview of the potential translation of ferroptosis in the clinical practice of HCC.

Increased Bone Marrow Iron at Diagnosis Is Associated with Inferior Prognosis in Patients with Myelodysplastic Syndromes

Introduction: Iron storage in patients (pts) with myelodysplastic syndromes at the time of diagnosis may vary from normal to iron overload. Even before the first blood transfusion, storage iron can be increased due to down-regulation of hepcidin and subsequent increase in duodenal iron uptake. Iron overload is known to worsen the prognosis of MDS patients, partly due to iron-related organ damage after long-term transfusion therapy, and partly due to an increased risk of infections. However, it is unclear whether increased storage iron at the time of diagnosis already has a prognostic influence. We assessed bone marrow iron stores at the time of MDS diagnosis and correlated them with clinical outcome.Methods: In a retrospective analysis of 3762 adult MDS patients from the Düsseldorf MDS Registry, Prussian blue staining of marrow smears was performed in our cytology lab to assess iron stores according to the following categories: normal or decreased iron stores versus increased iron stores versus iron overload. Patients were followed up for survival and AML evolution until June 2021. Median time of follow-up was 20 months. 67.4% of the patients died during the course of the disease.Results: The study included 3.762 adult patients who received their initial diagnosis of MDS between 1970 and 2021. 58% were diagnosed as non-blastic MDS ( MDS SLD (RS) (n=240), MDS MLD (RS) (n=350), MDSdel(5q) (n=107), and MDS-U (n=25). Iron stores were decreased in 8% of the patients, normal in 44%, increased in 41%, and strongly increased in 7% (massive iron overload).In 282 cases, histologic assessment of storage iron was available. When comparing cytologic and histologic assessment, we found a strong correlation (p<0.0005), since 87% of the patients with increased iron on cytomorphology also showed increased iron as assessed by histopathology. However, 37% of the patients who cytologically showed normal iron stores, were reported to have slightly increased iron as assessed by histopathology. Median and mean serum ferritin values of patients with normal or decreased iron stores were 295 and 629 µg/l, respectively, as compared to 548 and 902 µg/l, respectively, in patients with increased iron stores.The cumulative risk of AML evolution was not associated with the results of iron staining. Regarding survival, we found that patients with decreased or normal storage iron had a median survival of 31 months, whereas those with increased iron had a median survival of 28 months (p=0.007). Focusing on patients with non-blastic MDS, the difference was not significant (46 vs 44 ms). However, patients who presented as EB I (n=435), EBII (n=510), AML MRC (n=264), CMML I (n=254), or CMML II (n=77), showed a prognostic impact of storage iron; patients with increased iron had a median survival of 11 months, as compared to 16 months in patients with normal or decreased iron (p<0.0005).Conclusion: Increased tissue iron in the bone marrow at the time of diagnosis is associated with inferior survival in patients with MDS, primarily in patients with higher risk MDS. At diagnosis, patients are not yet transfusion-dependent. This suggests that increased iron reflects a prolonged period of increased duodenal iron uptake as a consequence of ineffective erythropoiesis. Therefore, increased marrow iron at the time of MDS diagnosis seems to be a surrogate parameter of hematopoietic insufficiency, which is the real cause of inferior prognosis. DisclosuresNachtkamp: Jazz: Honoraria; Bsh medical: Honoraria; Celgene: Other: Travel Support. Gattermann: Novartis: Honoraria; Takeda: Research Funding; Celgene: Honoraria. Germing: Jazz Pharmaceuticals: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Other: advisory activity, Research Funding; Janssen: Honoraria; Novartis: Honoraria, Research Funding.

Simultaneous hepatic iron and fat quantification with dual-energy CT in a rabbit model of coexisting iron and fat.

Liver iron and fat are often co-deposited, synergistically aggravating the progression of chronic liver disease. Accurate determination of liver iron and fat content is helpful for patient management. To assess the accuracy of hepatic iron/fat decomposition using dual-energy computed tomography (DECT) for simultaneously quantifying hepatic iron and fat when both are present. Sixty-eight New Zealand rabbits on a high-fat/cholesterol diet plus iron injections were used to establish a model of coexisting hepatic iron/fat. Abdominal imaging was performed using dual-source DECT. The iron and fat fractions (Iron-CT and Fat-CT, respectively) were calculated using a 3-material decomposition algorithm. The spectroscopic liver iron concentration (LIC) grading (normal, mild, moderate, severe, and massive iron overload) and the histopathological fat fraction (Fat-ref) grading (normal, mild, moderate, severe steatosis) were used as references. Correlations between the DECT parameters and the references were analyzed. Hepatic iron/fat quantification equations were established and validated. Analysis of covariance was used to assess the influence of fat on iron measurements and vice versa. Iron-CT highly correlated with LIC (r=0.94, P<0.001), and Fat-CT highly correlated with Fat-ref (r=0.88, P<0.001). Both the Iron-CT- and Fat-CT-derived LIC and fat fraction showed good agreement with spectroscopy/histology. The linear relationship between Iron-CT and spectroscopic LIC was not affected by the grade of hepatic fat (F=1.93, P=0.16). The linear relationship between Fat-CT and Fat-ref was unaffected by hepatic iron grades from normal to severe (F=0.18, P=0.91). However, with massive iron overload [>15.0 mg Fe/g (270 µmol/g)] the regression began to deviate, causing fat underestimation (F=5.50, P=0.04). Our DECT-based iron/fat decomposition algorithm accurately measured hepatic iron and fat when both were present in a rabbit model. Hepatic fat may be underestimated when there is massive iron overload.

Defective palmitoylation of transferrin receptor triggers iron overload in Friedreich ataxia fibroblasts

Friedreich ataxia (FRDA) is a frequent autosomal recessive disease caused by a GAA repeat expansion in the FXN gene encoding frataxin, a mitochondrial protein involved in iron-sulfur cluster (ISC) biogenesis. Resulting frataxin deficiency affects ISC-containing proteins and causes iron to accumulate in the brain and heart of FRDA patients. Here we report on abnormal cellular iron homeostasis in FRDA fibroblasts inducing a massive iron overload in cytosol and mitochondria. We observe membrane transferrin receptor 1 (TfR1) accumulation, increased TfR1 endocytosis, and delayed Tf recycling, ascribing this to impaired TfR1 palmitoylation. Frataxin deficiency is shown to reduce coenzyme A (CoA) availability for TfR1 palmitoylation. Finally, we demonstrate that artesunate, CoA, and dichloroacetate improve TfR1 palmitoylation and decrease iron overload, paving the road for evidence-based therapeutic strategies at the actionable level of TfR1 palmitoylation in FRDA.

Massive iron overload and acute-on-chronic liver failure in a patient with Diamond\u2013Blackfan anaemia: a case report

BackgroundGenetic anaemias lead us to reflect on the classic ‘trolley dilemma’, when there are two choices but neither one is satisfactory. Either we do not treat anaemia and the patient suffers from chronic tiredness and fatigue, or we do treat it through blood transfusions, leading to iron overload, which is a quite harmful consequence.Case presentationWe present the case of a 34-year-old woman with Diamond–Blackfan anaemia (DBA). Bone marrow stem cell transplantation had not been accessible during her childhood, so she had been submitted to monthly blood transfusions throughout her life, leading to a hepatitis C virus infection (which was treated, achieving a sustained virological response when she was 18 years old), and secondary haemochromatosis. Despite chelation therapy, diffuse iron deposition was occurring in multiple organs, markedly in the heart and liver. Her serum ferritin was higher than 21,000 ng/mL and transferrin saturation reached 102%. When she faced heart decompensation, this congestive condition led to an acute liver injury overlapping pre-existing hepatic fibrosis. She progressed to haemodynamic and hepatic failure, with clinical features of acute-on-chronic liver failure (ACLF). Despite therapeutic optimisation, she died of respiratory insufficiency. An autopsy was performed and revealed the macroscopic and microscopic findings of a massive iron deposition in the liver, heart, lungs, spleen, bone marrow, thyroid and adrenal glands. We found marked advance of liver fibrosis (chronic damage), as well as necrosis of hepatocytes in zone 3 of the Rappaport acinus (acute damage), supporting the hypothesis of ACLF. The main feature responsible for acute liver decompensation seemed to be heart insufficiency.ConclusionThis is the first case reporting the sequence: DBA, multiple blood transfusions, secondary haemochromatosis, advanced liver fibrosis, heart failure, ACLF and death. A multidisciplinary team is essential to care for DBA patients, since there is a significant emotional burden related to the disease, which might impair an effective chelation therapy and lead to severe consequences due to iron deposition.

NEONATAL HEMOCHROMATOSIS: A RARE CASE REPORT WITH CONSANGUINITY

Background: Neonatal hemochromatosis (NH) is a rare and severe liver disease of mainly intra-uterine onset, characterized by neonatal liver failure, hepatic and extrahepatic iron acquisition.NH is also called as Gestational alloimmune liver disease (GALD). This leads to an altered iron metabolism with resulting siderosis ,multi-organ failure and infants may be stillborn or present with advance, overwhelming liver disease. The disease represents the most common cause of liver failure in neonates and is also the most common indication for neonatal liver transplantation. We present an neonate who died at 15 days of age,and who was found to have massive iron overload in the liver. Initial treatment consisted of chelation therapy and antioxidants, but lack of laboratory and clinical improvement led to an exchange transfusion followed by intravenous immunoglobulin (IVIG). Irrespective of all above treatment no improvement of general condition of the patient. The unfavourable course of the disease is described in this case report.

Extensive iron overload in bone marrow: A cause of pancytopenia in a thalassemia major patient – A case report

Iron overload-associated organ damage in transfusion-dependent anemias is a well-known phenomenon. Here, we discuss a case of 28-year-old, poorly chelated thalassemia major patient, whose blood workup revealed pancytopenia and moderately raised serum ferritin levels. His bone marrow examination was performed which revealed massive iron overload. Aggressive iron chelation led to successful recovery of peripheral blood counts in his patient. This case focuses on the importance of early detection and timely management of reversible iron overload toxicities. Serum ferritin although is convenient marker to asses iron overload, but it should not be relied upon to assess the severity of iron overload. Hence, organ-specific diagnostic modalities must be used along with serum ferritin to assess the severity of iron overload to prevent long-term complications in patients with regular blood transfusions.

Impact of HFE gene Variants on Iron Overload, Overall Survival and Leukemia-Free Survival in Myelodysplastic Syndromes

Myelodysplastic syndromes (MDS) are myeloid neoplasms defined by peripheral cytopenia, dysplasia of myeloid cells and an increased risk to transform to secondary acute myeloid leukemia (AML). Although several laboratory and molecular risk factors and scoring systems have been established, little is known about genetic features contributing to disease evolution, AML development, and survival. Accumulating evidence suggests that iron metabolism and transfusion frequency are important prognostic determinants in MDS. It has also been described that patients with MDS frequently display HFE gene variants. However, little is known about the clinical impact of HFE variants. We examined the HFE status by restriction fragment length polymorphism (RFLP) analysis covering 2 common variants (H63D and C282Y) in 167 patients with MDS (observation-period: 1992-2019) and in 494 healthy Austrian controls. The median age of our MDS patients at diagnosis was 69 years (range 27-85 years) with a f:m ratio of 1:1.13. In the MDS cohort the international prognostic scoring systems, IPSS and IPSS-R, were applied, and in 94 patients a ´myeloid mutation´ screen was performed by next-generation sequencing (NGS). In all patients, serum ferritin levels were recorded at diagnosis and during follow up. Variant HFE species (H63D and/or C282Y) were detected in 63/167 patients with MDS (38.4%) and in 170/494 controls (34.4%). Median ferritin levels at diagnosis were slightly higher in HFE-mutated patients (409 mg/dL; range: 23-7415 mg/dL) compared to non-mutated patients (346.5 mg/dL; range: 10-5450 mg/dL) (p=0.62). During the first 2 years of transfusion therapy, HFE-mutated patients showed a slightly faster increase in serum ferritin levels compared to non-mutated patients. However, again, the difference was not significant, and patients with massive iron overload were found in both cohorts. When examining French-American-British (FAB) subgroups of MDS, the percentage of patients withHFE variants was higher in the group with refractory anemia, RA (22/53=41.5%) or RA with ring sideroblasts, RARS (17/39=43.9%) than in the group of RA with excess of blasts, RAEB (16/46=34.8%) or RAEB in transformation (5/17=29.4%). In CMML, 5/12 patients (41.7%) were found to carry a HFE variant. Clear differences were also observed between subgroups of MDS patients classified by World Health Organization (WHO) criteria. The prevalence of HFE mutations was 50.0% (13/26) in patients with ring sideroblasts (RS) and multilineage dysplasia (MDS-RS-MLD), 50.0% in MDS-MLD (6/12), 43% (7/18) in MDS with RS and single-lineage dysplasia (MDS-RS-SLD), 42.9% (12/28) in patients with excess of blasts-1 (MDS-EB1), 38.9% (7/18) in MDS-SLD, 21.4% (6/28) in MDS-EB2, and 20% (2/10) in MDS with isolated 5q-. When splitting patients according to the IPSS and IPSS-R, patients at highest risk were found to have a lower prevalence of HFE variants (IPSS high: 20%; IPSS-R very high: 15.4%) compared to other risk groups (IPSS low: 34.9%, int-1: 42.0%, int-2: 31.0%; and IPSS-R very low: 33.3%, low: 38.5%, int: 36.8%, high: 42.9%). Next, we examined correlations between HFE variants and somatic myeloid mutations in our MDS patients (n=94). No significant correlations were found when comparing expression of HFE variants with somatic mutations in ASXL1, ASXL1, SF3B1, SRSF2, TET2, DNMT3A, RUNX1, NRAS, TP53, IDH2 or EZH2 detected by NGS. Finally, we examined the HFE status in the context of clinical end points. In these studies, we found a clear difference in AML-free survival between HFE-mutated and non-mutated patients, although the difference did not reach statistical significance (p=0.089) (Figure 1A). No differences in overall survival (OS) were found when comparing patients with or without HFE variants (Figure 1B). In conclusion, the HFE variants H63D and C282Y are frequently detected in Austrian patients with MDS. These variants are more commonly detectable in low risk FAB patients and correlate roughly with higher ferritin levels at diagnosis and during transfusion therapy. In addition, HFE-mutated MDS patients have a slightly better AML-free survival. However, the correlation is weak. Therefore, we currently do not recommend screening for HFE gene variants at diagnosis in patients with MDS unless ferritin levels are excessively high or increase very rapidly during transfusion suggesting the possibility of a concomitant primary hemochromatosis. Disclosures Valent: Blueprint: Research Funding; Celgene: Honoraria; Deciphera: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Sperr:Novartis: Honoraria; Celgene: Consultancy, Honoraria.

Hepcidin and Erythroferrone in the Anemia of Low-Risk Myelodysplastic Syndromes

BACKGROUND: Anemia is the most common manifestation of low-risk myelodysplastic syndromes (MDS). Iron-overload in MDS can occur before transfusion dependence in the context of ineffective erythropoiesis. Significantly lower hepcidin levels have been described in patients with sideroblastic refractory anemia compared to higher risk MDS, promoting inadequate iron absorption that leads to higher iron overload. Erythroferrone (ERFE) is a hormone that stimulates erythropoiesis and regulates iron homeostasis; in physiological conditions, is stimulated by erythropoietin and increases iron absorption inhibiting hepcidin. There are no studies describing the activity of ERFE in MDS. The objective of this study was to describe the relationship among hepcidin, ERFE and iron overload in 31 patients with low-risk MDS.METHODS: 50 samples were analized, 31 from patients (16 males, 17 females) with low-risk MDS: 10 low IPSS-R and 21 very-low IPSS-R; and 19 from healthy controls. 13 patients showed severe anemia with transfusion dependence, 4 patients received only erythropoiesis stimulating agents (ESA) and 14 patients did not receive any treatment for the anemia. Patient characteristics are summarized in table 1. Hepcidin levels were measured using the DRG Hepcidin 25 (bioactive) HS ELISA Kit (DRG Diagnostics GmbH), and ERFE was measured with the FAM132B (Human) OKEH02395 ELISA kit (Aviva Systems). For the analysis, two groups of patients were considered: 13 with severe and transfusion dependent anemia and 18 with mild/moderate anemia.RESULTS: Patients with severe anemia showed higher serum ferritin levels (median 2143ng/mL vs 204ng/mL, p<0.001) compared to patients without transfusion dependence. Hepcidin levels were significantly higher in patients with transfusion dependent anemia (mean 59.85 vs 16.11ng/mL, p=0.001) compared to patients with transfusion independence, and were also higher in these last patients compared to healthy controls (mean 16.11 vs 10.6ng/mL, p<0.001). Regarding ERFE, patients with transfusion dependent anemia showed significantly higher ERFE levels (mean 281.92 vs. 62.83pg/mL, p=0.016) than patients with mild/moderate anemia. However, there were no significant differences between patients with mild/moderate anemia and healthy controls. There was no correlation between hepcidin and ERFE levels (p=0.46).CONCLUSIONS: To the best of our knowledge, this is the first simultaneous analysis of hepcidine and ERFE in MDS. Patients with severe anemia showed significantly higher ERFE levels compared to those with moderate anemia, suggesting a higher erythropoietic stimulus. Patients with severe anemia showed significantly superior hepcidin levels, hindering iron absorption in situations of massive iron overload. Accordingly, ERFE did not show negative correlation with hepcidin in either cohort, supporting the abnormal iron metabolism in MDS. Larger studies are required to define the relationship between hepcidin and ERFE in low-risk MDS. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Variable expressivity of HJV related hemochromatosis: “Juvenile” hemochromatosis?

Juvenile hemochromatosis is a rare autosomal recessive disease due to variants in the Hemojuvelin (HJV) gene. Although biological features mimic HFE hemochromatosis, clinical presentation is worst with massive iron overload diagnosed during childhood.Our study describes clinical features and results of genetic testing for a group of patients initially referred for a hepcidino-deficiency syndrome and for whom HJV hemochromatosis was finally diagnosed. 662 patients with iron overload and high serum transferrin saturation were tested, and five genes (HFE, HJV, HAMP, TFR2, SLC40A1) were sequenced.Among our cohort, ten unrelated patients were diagnosed with HJV hemochromatosis. Genetic testing revealed five previously published and five undescribed variants: p.Arg41Pro, p.His180Arg, p.Lys299Glu, p.Cys361Arg and p.Ala384Val.Surprisingly, this study revealed a late age of onset in some patients, contrasting with the commonly accepted definition of “juvenile” hemochromatosis. Five of our patients were 30 years old or older, including two very late discoveries. Biological features and severity of iron overload were similar in younger and older patients.Our study brings new insight on HJV hemochromatosis showing that mild phenotype and late onset are possible. Genetic testing for HJV variants should thus be performed for all patients displaying a non-p.Cys282Tyr homozygous HFE hemochromatosis with hepcidin deficiency phenotype.

Smad1/5 is required for erythropoietin-mediated suppression of hepcidin in mice

AbstractAnemia suppresses liver hepcidin expression to supply adequate iron for erythropoiesis. Erythroferrone mediates hepcidin suppression by anemia, but its mechanism of action remains uncertain. The bone morphogenetic protein (BMP)–SMAD signaling pathway has a central role in hepcidin transcriptional regulation. Here, we explored the contribution of individual receptor-activated SMADs in hepcidin regulation and their involvement in erythroferrone suppression of hepcidin. In Hep3B cells, SMAD5 or SMAD1 but not SMAD8, knockdown inhibited hepcidin (HAMP) messenger RNA (mRNA) expression. Hepatocyte-specific double-knockout Smad1fl/fl;Smad5fl/fl;Cre+ mice exhibited ∼90% transferrin saturation and massive liver iron overload, whereas Smad1fl/fl;Smad5fl/wt;Cre+ mice or Smad1fl/wt;Smad5fl/fl;Cre+ female mice with 1 functional Smad5 or Smad1 allele had modestly increased serum and liver iron, and single-knockout Smad5fl/fl;Cre+ or Smad1fl/fl;Cre+ mice had minimal to no iron loading, suggesting a gene dosage effect. Hamp mRNA was reduced in all Cre+ mouse livers at 12 days and in all Cre+ primary hepatocytes. However, only double-knockout mice continued to exhibit low liver Hamp at 8 weeks and failed to induce Hamp in response to Bmp6 in primary hepatocyte cultures. Epoetin alfa (EPO) robustly induced bone marrow erythroferrone (Fam132b) mRNA in control and Smad1fl/fl;Smad5fl/fl;Cre+ mice but suppressed hepcidin only in control mice. Likewise, erythroferrone failed to decrease Hamp mRNA in Smad1fl/fl;Smad5fl/fl;Cre+ primary hepatocytes and SMAD1/SMAD5 knockdown Hep3B cells. EPO and erythroferrone reduced liver Smad1/5 phosphorylation in parallel with Hamp mRNA in control mice and Hep3B cells. Thus, Smad1 and Smad5 have overlapping functions to govern hepcidin transcription. Moreover, erythropoietin and erythroferrone target Smad1/5 signaling and require Smad1/5 to suppress hepcidin expression.

Angiocrine Bmp2 signaling in murine liver controls normal iron homeostasis

AbstractMicrovascular endothelial cells (ECs) display a high degree of phenotypic and functional heterogeneity among different organs. Organ-specific ECs control their tissue microenvironment by angiocrine factors in health and disease. Liver sinusoidal endothelial cells (LSECs) are uniquely differentiated to fulfill important organ-specific functions in development, under homeostatic conditions, and in regeneration and liver pathology. Recently, Bmp2 has been identified by us as an organ-specific angiokine derived from LSECs. To study angiocrine Bmp2 signaling in the liver, we conditionally deleted Bmp2 in LSECs using EC subtype-specific Stab2-Cre mice. Genetic inactivation of hepatic angiocrine Bmp2 signaling in Stab2-Cre;Bmp2fl/fl (Bmp2LSECKO) mice caused massive iron overload in the liver and increased serum iron levels and iron deposition in several organs similar to classic hereditary hemochromatosis. Iron overload was mediated by decreased hepatic expression of hepcidin, a key regulator of iron homeostasis. Thus, angiocrine Bmp2 signaling within the hepatic vascular niche represents a constitutive pathway indispensable for iron homeostasis in vivo that is nonredundant with Bmp6. Notably, we demonstrate that organ-specific angiocrine signaling is essential not only for the homeostasis of the respective organ but also for the homeostasis of the whole organism.

Quantitative ultrashort echo time imaging for assessment of massive iron overload at 1.5 and 3 Tesla.

Hepatic iron content (HIC) quantification via transverse relaxation rate (R2*)-MRI using multi-gradient echo (mGRE) imaging is compromised toward high HIC or at higher fields due to the rapid signal decay. Our study aims at presenting an optimized 2D ultrashort echo time (UTE) sequence for R2* quantification to overcome these limitations. Two-dimensional UTE imaging was realized via half-pulse excitation and radial center-out sampling. The sequence includes chemically selective saturation pulses to reduce streaking artifacts from subcutaneous fat, and spatial saturation (sSAT) bands to suppress out-of-slice signals. The sequence employs interleaved multi-echo readout trains to achieve dense temporal sampling of rapid signal decays. Evaluation was done at 1.5 Tesla (T) and 3T in phantoms, and clinical applicability was demonstrated in five patients with biopsy-confirmed massively high HIC levels (>25 mg Fe/g dry weight liver tissue). In phantoms, the sSAT pulses were found to remove out-of-slice contamination, and R2* results were in excellent agreement to reference mGRE R2* results (slope of linear regression: 1.02/1.00 for 1.5/3T). UTE-based R2* quantification in patients with massive iron overload proved successful at both field strengths and was consistent with biopsy HIC values. The UTE sequence provides a means to measure R2* in patients with massive iron overload, both at 1.5T and 3T. Magn Reson Med 78:1839-1851, 2017. © 2017 Wiley Periodicals, Inc.

Biallelic Mutations in Hamp in Type 2b Hemochromatosis

▪IntroductionHereditary hemochromatosis (HH) is a group of genetic iron overload disorders caused by mutations in genes encoding HFE (HFE1), transferrin receptor 2 (TFR2), hepcidin (HAMP), hemojuvelin (HFE2) or ferroportin (SLC40A1), respectively. HH is classified into 4 different categories according to the gene causing the disease. Type1 is the most common form arising from homozygosity for HFE C282Y mutation at the homozygous state. Type2 HH includes type 2A caused by mutations in HJV and type 2B , caused by mutations in HAMP. Hepcidin is synthesized as a pro-peptide of 84amino acids, which is cleaved into peptides of 20, 22, and 25 amino acids, the last being the active form of hepcidin. It is established that hepcidin negatively regulates iron export by direct interaction with ferroportin. Mutations or deficiency in Hepcidin result in excessive iron absorption that can lead to massive iron overload. We report a patient with a severe iron overload related to an unusual genotype of two previously unreported HAMP variants in combination with the H63D HFE mutation.Case report:A 69 year old male patient was diagnosed to have a severe iron overload with serum ferritin (SF) 5076 µg/l , transferrin saturation (TS) 48% and liver iron concentration (LIC) 290 µmolFe/g. Factors contributing to the hyperferritinemia included a chronic daily consumption of alcohol (100 g /days with gGT : 150 UI/l). Genetic analysis revealed heterozygosity for the common HFE1 H63D mutation and two new mutations in HAMP: one located in the promoter ( -274 G>C ) and the other in the stop codon (c. 255 G>C , p. Ter85TyrextTer21) . After one year of phlebotomy therapy, 5.5 g iron was removed and serum ferritin dropped to 1700 µg/L. Phlebotomy therapy was interrupted when patient was lost to follow-up and one year later SF was 2730 µg/L. Hepcidin was measured using mass spectrophotometry before resuming the phlebotomy (27.6 ng/ml, nle : 1 to 20 ng/ml).Methods :In order to analyze the involvement of the two HAMP mutations in the iron overload, further DNA studies were performed including MLPA (MRC Holland , P347 Hemochromatosis probemix , version A2) and Next generation sequencing (NGS, MiseqTM, Illumina ). The iron regulatory gene panel included the following gene sets : HFE-1; HFE-2 ; TFR2; HAMP; SLC40A1; FTH1; TF; FTL; SLC11A2; TMPRSS6; HEPH; CP; ALAS2; BMP6;BMP4; SMAD4. The Hepcidin functional tests were performed by using HEK293T cells expressing FPN1. Subsequently, cells were grown with preconditioned supernatants obtained from cells transfected with hepcidinwt or hepcidin255C. Promoter mutation was tested using HepG2 cells lines. Linkage between the two mutations was tested by means of DDPCR (Bio-Rad, QX200™ Droplet Digital™). We used simultaneously two of hydrolysis probes with different labeling and results were analyzed by a Poisson statistical comparison of double labeled drops count versus simple labeled drop.Results of these studies showed that :1- No other significant mutation could be retrieved with the NGS analysis and MLPA analysis of HFE, HFE2, HAMP, TFR2 and SLC40A1 did not show any pathogenic copy number variant.2- The elongated hepcidin was unable to induce internalization of ferroportin.3- The elongated hepcidin did not display any dominant effect (ie: unability to suppress the effect of wt hepcidin)4- The functional study of the -274 G>C promoter mutation fails to show a reproducible and significant decreased function compared to wt5- The two mutations were located in transDiscussion and conclusion.The reason why in some HAMP-P individuals iron overload was comparable to that of wild-type patients could be due to the fact that they are heterozygous for the SNP and, therefore, show an incomplete penetrance or compensatory mechanisms. We report a patient in which the only iron metabolism related mutations are the Hamp:c.255 G>C (p. Ter85TyrextTer21 mutation) and an SNP in the promoter: Hamp:c.-274 G>C mutation. We show that the elongated hepcidin is a loss of function mutant. Unfortunately, we could not have access to other family members for the segregation study of the HAMP mutations. Serum Hepcidin level was determined when ferritin was 2730 µg/l and appears to be low according to iron parameters. Our observations suggest a hypothetical role of the HAMP-P -274 G>C SNP on promoter reactivity. From this set of data, we propose that the patient is affected by a new Hamp linked HH (HFE 2B) probably worsened by drinking habits. DisclosuresSaunier:Bio-Rad: Employment.

Hepatic Fibrosis as a Consequence of Iron Overload in Pyruvate Kinase Deficiency

Pyruvate kinase deficiency, which results in a non-spherocytic hemolytic anemia, can be associated with secondary hepatic iron overload and fibrosis (1). We describe a patient with pyruvate kinase deficiency heterozygous for the H63D mutation in HFE who developed hepatic iron overload with advanced fibrosis. A 21-year-old female presented with abdominal pain, scleral icterus, and jaundice. She was diagnosed with pyruvate kinase deficiency at birth, received multiple blood transfusions, and had a splenectomy at age 5. Current laboratory studies revealed a hemoglobin of 6.8 g/dL, a ferritin of 5101 ng/mL, and a transferrin saturation of 100%. Liver enzymes, bilirubin, and LDH were also elevated (Table 1). Tests for viral hepatitis, alpha 1 antitrypsin, ceruloplasmin, AMA, and ANA were negative. A CT of the abdomen showed a homogeneous liver. Liver biopsy revealed massive iron overload and bridging fibrosis (Figure 1). Chelation therapy with deferoxamine was initiated to reduce hepatic iron stores, hoping to prevent progression to cirrhosis. Chelation resulted in improvement in ferritin, liver enzymes, bilirubin, and LDH levels at 24 months (Table 1).Figure 1Table 1: Hemoglobin, Ferritin, Transferrin Saturation, Liver Enzymes, Total Bilirubin, and LDH at Baseline and 24 Months Post-Deferoxamine TherapyIron overload is commonly described in thalassemia, but less frequently in pyruvate kinase deficiency. Salem et al. described a patient with iron overload and cirrhosis in pyruvate kinase deficiency (2). Liver iron concentrations above a critical threshold of 350-400 mumoles/g can cause hepatocellular damage (3). Chelation has been shown to reduce iron overload in pyruvate kinase deficiency (4). Alternative therapies are important for non-responders to chelation who have not progressed to cirrhosis. One group reported successful bone marrow transplantation in a child with pyruvate kinase deficiency (5). Gene targeting and liver transplant may be options in secondary iron overload, and warrant further investigation.

Comparison of Bleomycin-Detectable Iron and Labile Plasma Iron Assays

To the Editor: Non–transferrin-bound iron (NTBI)1 is detected in the plasma of patients with diseases in which the transferrin-binding capacity for iron is exceeded by a massive iron overload, such as in certain forms of thalassemia and hemochromatosis, repeated blood transfusions, or bone marrow failure. NTBI may occur in plasma as insoluble polynuclear ferrihydrate species, as Fe(III) citrate or acetate complexes, or bound to certain flavonoids, amino acids, albumin, or modified albumin. Excess iron is harmful because it causes oxidative stress, accumulates in and damages tissues, and enhances the growth of pathogens. Therefore, the measurement of NTBI is of clinical importance, because NTBI may be used as an indicator of systemic iron overload and iron toxicity (1, 2). We compared 2 NTBI assays: a labile plasma iron (LPI) assay that uses desferrioxamine as the iron chelator (3) and a microwell modification of a bleomycin-detectable iron (BDI) assay (2). The BDI assay is an indirect method based on the formation redox-active complexes between NTBI in the sample and the bleomycin reagent. …

New variant of unclassified congenital dyserythropoietic anaemia: the concept of the erythroid regulator?

New variant of unclassified congenital dyserythropoietic anaemia: the concept of the erythroid regulator?