Increased Susceptibility to Mycobacterium avium in Hemochromatosis Protein HFE-Deficient Mice

ABSTRACT Mycobacterium avium is an opportunistic infectious agent in immunocompromised patients, living inside macrophage phagosomes. As for other mycobacterial species, iron availability is a critical factor for M. avium survival and multiplication. Indeed, an association between host secondary iron overload and increased susceptibility to these mycobacteria is generally acknowledged. However, studies on the impact of primary iron overload on M. avium infection have not been performed. In this work, we used animal models of primary iron overload that mimic the human disease hereditary hemochromatosis. This pathology is characterized by increased serum transferrin saturation with iron deposition in parenchymal cells, mainly in the liver, and is most often associated with mutations in the gene encoding the molecule HFE. In this paper, we demonstrate that mice of two genetically determined primary iron overload phenotypes, Hfe−/− and β2m−/−, show an increased susceptibility to experimental infection with M. avium and that during infection these animals accumulate iron inside granuloma macrophages. β2m−/− mice were found to be more susceptible than Hfe−/− mice, but depleting Hfe−/− mice of CD8+ cells had no effect on resistance to infection. Overall, our results suggest that serum iron, rather than total liver iron, levels have a considerable impact on susceptibility to M. avium infection.

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Target TMPRSS6 Using Antisense Technology for the Treatment of Hereditary Hemochromatosis and β-Thalassemia

Blood ◽

10.1182/blood.v120.21.481.481 ◽

2012 ◽

Vol 120 (21) ◽

pp. 481-481 ◽

Cited By ~ 1

Author(s):

Shuling Guo ◽

Carla Casu ◽

Sara Gardenghi ◽

Sheri Booten ◽

Andy Watt ◽

...

Keyword(s):

Treatment Group ◽

Iron Overload ◽

Hereditary Hemochromatosis ◽

Iron Concentration ◽

Transferrin Saturation ◽

Control Group ◽

Mrna Levels ◽

Liver Iron ◽

Hepcidin Expression ◽

Antisense Technology

Abstract Abstract 481 Hepcidin, the master regulator of iron homeostasis, is a peptide that is mainly expressed and secreted by the liver. Low levels of hepcidin are associated with increased iron absorption. In conditions in which hepcidin is chronically repressed, such as hereditary hemochromatosis and b-thalassemia, patients suffer from iron overload and very severe pathophysiological sequelae associated with this condition. Hepcidin expression is regulated predominantly at the transcriptional level by multiple factors. TMPRSS6, a transmembrane serine protease mutated in iron-refractory, iron-deficient anemia, is a major suppressor of hepcidin expression. It has been demonstrated that hepcidin expression is significantly elevated in Tmprss6−/− mice and reduction of Tmprss6 expression in hereditary hemochromatosis (Hfe−/−) mice ameliorates the iron overload phenotype (Finberg et al. Nature Genetics, 2008; Du et al. Science 2008; Folgueras et al. Blood 2008; Finberg et al., Blood, 2011). It has also been demonstrated that hepcidin up-regulation using either a hepcidin transgene or Tmprss6−/− significantly improves iron overload and anemia in a mouse model of β-thalassemia intermedia (th3/+ mice) (Gardenghi et al. JCI, 120:4466, 2010; Nai et al. Blood, 119: 5021, 2012). In this report, we have examined whether reduction of Tmprss6 expression using antisense technology is an effective approach for the treatment of hereditary hemochromatosis and β-thalassemia. Second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6 were identified. When normal male C57BL/6 mice were treated with 25, 50 and 100mg/kg/week ASO for four weeks, we achieved up to >90% reduction of liver Tmprss6 mRNA levels and up to 5-fold induction of hepcidin mRNA levels in a dose-dependent manner. Dose-dependent reductions of serum iron and transferrin saturation were also observed. ASOs were well tolerated in these animals. In Hfe−/− mice (both males and females), ASOs were administrated at 100 mg/kg for six weeks. This treatment normalized transferrin saturation (from 92% in control animals to 26% in treatment group) and significantly reduced serum iron (from >300ug/dl in control group to <150ug/dl in treatment group), as well as liver iron accumulation. Histopathological evaluation and Prussian's Perl Blue staining indicated that iron was sequestered by macrophages, which led to an increase in spleen iron concentration. The mouse model of thalassemia intermedia that we utilized mimics a condition defined as non-transfusion dependent thalassemia (NTDT) in humans. These patients exhibit increased iron absorption and iron overload due to ineffective erythropoiesis and suppression of hepcidin; iron overload is the most frequent cause of morbidity and mortality. Th3/+ animals exhibit ineffective erythropoiesis, characterized by increased proliferation and decreased differentiation of the erythroid progenitors, apoptosis of erythroblasts due to the presence of toxic hemichromes, reticulocytosis and shorter lifespan of red cells in circulation, leading to splenomegaly, extramedullary hematopoiesis and anemia (∼ 8 g/dL; Libani et al, Blood 112(3):875–85, 2008). Five month old th3/+ mice (both males and females) were treated with Tmprss6 ASO for six weeks. In th3/+ mice, ∼85% Tmprss6 reduction led to dramatic reductions of serum transferrin saturation (from 55–63% in control group down to 20–26% in treatment group). Liver iron concentration (LIC) was also greatly reduced (40–50%). Moreover, anemia endpoints were significantly improved with ASO treatment, including increases in red blood cells (∼30–40%), hemoglobin (∼2 g/dl), and hematocrit (∼20%); reduction of splenomegaly (∼50%); decrease of serum erythropoietin levels (∼50%); improved erythroid maturation as indicated by a strong reduction in reticulocyte number (50–70%) and in a normalized proportion between the pool of erythroblasts and enucleated erythroid cells. Hemichrome analysis showed a significant decrease in the formation of toxic alpha-globin/heme aggregates associated with the red cell membrane. This was consistent with a remarkable improvement of the red cell distribution width (RDW) as well as morphology of the erythrocytes. In conclusion, these data demonstrate that targeting TMPRSS6 using antisense technology is a promising novel therapy for the treatment of hereditary hemochromatosis and β-thalassemia. Disclosures: Guo: Isis Pharmaceuticals: Employment. Booten:Isis Pharmaceuticals: Employment. Watt:Isis Pharmaceuticals: Employment. Freier:Isis Pharmaceuticals: Employment. Rivella:Novartis Pharmaceuticals: Consultancy; Biomarin: Consultancy; Merganser Biotech: Consultancy, Equity Ownership, Research Funding; Isis Pharma: Consultancy, Research Funding. Monia:Isis Pharmaceuticals: Employment.

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A High Through-Put Screen Identifies MCP-1 (CCL2) As a Novel Regulator of Iron Homeostasis and a Modifier of Hereditary Hemochromatosis Disease Severity

Blood ◽

10.1182/blood.v118.21.685.685 ◽

2011 ◽

Vol 118 (21) ◽

pp. 685-685

Author(s):

Martina U. Muckenthaler ◽

Maja Vujic Spasic ◽

Katarzyna Mleczko-Sanecka ◽

Mingang Zhu ◽

Rainer Pepperkok ◽

...

Keyword(s):

Iron Overload ◽

Mrna Expression ◽

Disease Severity ◽

Iron Homeostasis ◽

Expression Profiles ◽

Hereditary Hemochromatosis ◽

Transferrin Saturation ◽

Human Cells ◽

Liver Iron ◽

Deficient Mice

Abstract Abstract 685 To identify genes that modify the severity of human iron disorders we pre-selected 74 genes from gene expression profiles of cells and tissues with altered iron levels and assessed whether siRNA-mediated knock-down of these genes affects uptake of transferrin, a key cellular process to acquire iron. This screen identifies the monocyte chemoattractant protein-1 (MCP-1), also known as CCL2, as a critical suppressor of transferrin receptor mRNA expression in human cells. We next analyzed CCL2-deficient mice and demonstrate profound alterations of parameters of systemic iron homeostasis. Specifically, CCL2 knock-out mice show decreased serum iron levels and transferrin saturation, strong iron-overload in the spleen and duodenum as well as mild iron accumulation in the liver. Iron imbalance in CCL2−/− mice is unlikely explained by an impairment of the major control system of systemic iron homeostasis, the hepcidin/ferroportin regulatory system: hepcidin mRNA expression is unaltered and splenic ferroportin protein expression is strongly increased in CCL2−/− mice, as would be expected as a consequence of splenic iron overload. We speculate that increased iron absorption from the plasma, possibly mediated by inappropriately high levels of TfR1 in the spleen, duodenum and liver, may be responsible for tissue iron overload. It is of note that CCL2 levels are strongly decreased in Hfe-deficient mice and patients with Hfe-associated Hereditary Hemochromatosis (HH). We therefore asked whether CCL2 levels could modify disease severity of HH. Analysis of 51 HH patients, all homozygous for the C282Y HFE mutation, confirms significantly lower MCP-1 levels in the serum compared to a group of 23 sex- and age-matched normal controls. Importantly, CCL2 levels in HH patients show a significant negative correlation with liver iron overload at the time point of diagnosis. Furthermore, low CCL2 concentrations are significantly associated with the HLA-A3 genotype and the CD8+ T lymphocyte phenotype, both traits previously shown to correlate with iron overload in HH patients. These patient data and the data from CCL2-deficient mice suggest that appropriate CCL2 expression is required to prevent iron overload. Taken together our data demonstrate the power of siRNA screens to identify novel regulators of iron metabolism in human cells that are critically involved in maintaining systemic iron homeostasis in the mouse and that play a role in modifying hepatic iron overload in the frequent iron overload disorder Hereditary Hemochromatosis. Disclosures: No relevant conflicts of interest to declare.

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Hepcidin Peptidomimetics - Oral Efficacy in Pre-Clinical Disease Model of Iron Overload

Blood ◽

10.1182/blood-2020-141720 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 47-48

Author(s):

Roopa Taranath ◽

Gregory Bourne ◽

Jie Zhang ◽

Brian Frederick ◽

Tran T Tran ◽

...

Keyword(s):

Mouse Model ◽

Iron Overload ◽

Polycythemia Vera ◽

Serum Iron ◽

Iron Homeostasis ◽

Hereditary Hemochromatosis ◽

Iron Concentration ◽

Transferrin Saturation ◽

Private Company ◽

Liver Iron

Hepcidin peptidomimetics that are orally stable and systemically active will mark a paradigm change in management of blood disorders that exhibit aberrant iron homeostasis (e.g. hereditary hemochromatosis) and in conditions that can be influenced by modulating stressed iron homeostasis (e.g. polycythemia vera). Hepcidin modulates the iron exporter membrane protein ferroportin and is the master regulator of iron homeostasis in the body. Orally bioavailable "Minihepcidins" have been previously shown to be efficacious in lowering serum iron in mice when dosed peroral (PO) (Preza GC et. al., Journal of Clinical Investigation 2011). Here we describe hepcidin mimetic peptides that are metabolically stable in the gastrointestinal tract, systemically absorbed when delivered orally, and pharmacodynamically active in reducing serum iron parameters in pre-clinical models. Further, we also demonstrate improvement in disease parameters in a mouse model for hereditary hemochromatosis. The oral peptides, PN20076 and PN20089, have EC50 of 16.5 nM and 1.39 nM respectively in cell based ferroportin internalization assay (Table 1). In comparison EC50 was 67.8 nM for Hepcidin and 6.12 nM for PTG-300. (PTG-300 is an injectable hepcidin mimetic currently in Phase 2 clinical studies for polycythemia vera and hereditary hemochromatosis.) Oral stability of the peptides was evaluated in a panel of assays, including in vitro matrices simulating the gastric and intestinal conditions, and ex vivo matrices of serum/plasma from different species. Table 1 shows data for peptides PN20018, PN20076 and PN20089. PN20076 demonstrated extended stability in gastric and intestinal conditions, and degradation half-life of >24 hr in mouse plasma and 14.8 hr in rat serum. Based on their stability and potency data from the above battery of screening assays, the peptides were selected for in vivo evaluation in healthy mice to characterize their pharmacodynamic (PD) and pharmacokinetic (PK) properties. PN20076 and PN20089 showed equivalent PD response of reduction in serum iron concentration in wild type mice. After two successive PO doses of PN20076 or PN20089 approximately 24 hr apart, serum iron concentration was reduced from ~30 µM to ~10 µM (group averages), i.e. ~66% reduction, at 4.5 hr post-second dose for both peptides (Fig. 1). At 4.5 hr post-dose, the serum concentration of PN20076 was ~262 nM. PN20076 was further evaluated for its effect in lowering iron overload in a mouse model for hemochromatosis (HFE2-/- with homozygous deletion of hemojuvelin, a positive regulator of hepcidin expression). This mouse model is marked by hyper-absorption of dietary iron, higher transferrin saturation and deposition of excessive iron in liver, all manifestations of aberrant iron homeostasis caused by the genetic disruptions of the hepcidin-iron pathway. Liver iron accumulation was significantly prevented in groups treated with PN20076 once daily (QD) by PO administration for over two weeks, as compared to vehicle treated controls (Fig. 2). The reduction in non-heme iron concentration in liver homogenates (measured using a colorimetric iron assay) was statistically significant in the female group treated with PN20076. We have described orally stable and systemically active hepcidin mimetic peptides and demonstrated oral activity in preventing liver iron overload in hemochromatosis mice. The effective reduction of iron absorption from the diet and the steady state lowering of transferrin-saturation can potentially prevent tissue iron toxicity in hereditary hemochromatosis. Similarly, the sustained reduction of systemic iron levels with an oral hepcidin mimetic to control stressed iron homeostasis should reduce excessive erythrocytosis, a hallmark of polycythemia vera and other congenital and acquired erythropoietic disorders. Disclosures Bourne: Protagonist Therapeutics: Current Employment, Other: shareholder. Zhang:Protagonist Therapeutics: Current Employment, Other: shareholder. Frederick:Protagonist Therapeutics: Current Employment, Other: shareholder. Tran:Protagonist Therapeutics: Current Employment, Other: shareholder. Vengalam:Protagonist Therapeutics: Current Employment, Current equity holder in private company. McMahon:Protagonist Therapeutics: Current Employment, Other: shareholder. Huie:Protagonist Therapeutics: Current Employment, Other: shareholder. Ledet:Protagonist Therapeutics: Current Employment, Other: shareholder. Zhao:Protagonist Therapeutics: Current Employment, Other: shareholder. Tovera:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Lee:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Yang:Protagonist Therapeutics: Current Employment, Other: shareholder. Dion:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Yuan:Protagonist Therapeutics: Current Employment, Other: shareholder. Zemede:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Nguyen:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Masjedizadeh:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Cheng:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Mattheakis:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Liu:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Smythe:Protagonist Therapeutics: Current Employment, Other: shareholder.

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Iron Overload in C282Y Heterozygotes: Identification of New Rare HFE Gene Mutants and a Step Strategy for Diagnosis.

Blood ◽

10.1182/blood.v112.11.1859.1859 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1859-1859

Author(s):

Patricia Aguilar-Martinez ◽

Severine Cunat ◽

Fabienne Becker ◽

Francois Blanc ◽

Marlene Nourrit ◽

...

Keyword(s):

Iron Overload ◽

Iron Status ◽

Hereditary Hemochromatosis ◽

Genetic Defect ◽

Transferrin Saturation ◽

Hfe Gene ◽

Compound Heterozygous ◽

Exon 2 ◽

High Iron ◽

Iron Parameters

Abstract Introduction: Homozygozity for the p.Cys282Tyr (C282Y) mutation of the HFE gene is the main genotype associated with the common form of adult hereditary hemochromatosis. C282Y carriers do not usually develop iron overload, unless they have additional risk factors such as liver diseases, a dysmetabolic syndrome or an associated genetic defect. The commonest is the compound heterozygous state for C282Y and the widespread p.His63Asp (H63D) variant allele. However, a few rare HFE mutations can be found on the 6th chromosome in trans, some of which are of clinical interest to fully understand the disorder. Patients and Methods: We recently investigated four C282Y carrier patients with unusually high iron parameters, including increased levels of serum ferritin (SF), high transferrin saturation (TS) and high iron liver content measured by MRI. They were males, aged 37, 40, 42, 47 at diagnosis. Two brothers (aged 40 and 42) were referred separately. The HFE genotype, including the determination of the C282Y, H63D and S65C mutations was performed using PCR-RFLP. HFE sequencing was undertaken using the previously described SCA method (1). Sequencing of other genes (namely, HAMP, HJV/HFE2, SLC40A1, TFR2) was possibly performed in a last step using the same method. Results: We identified three rare HFE mutant alleles, two of which are undescribed, in the four studied patients. One patient bore a 13 nucleotide-deletion in exon 6 (c.[1022_1034del13], p.His341_Ala345>LeufsX119), which is predicted to lead to an abnormal, elongated protein. The two brothers had a substitution of the last nucleotide of exon 2 (c.[340G>A], p.Glu114Lys) that may modify the splicing of the 2d intron. The third patient, who bore an insertion of a A in exon 4 (c.[794dupA],p.[trp267LeufsX80]), has already been reported (1). Discussion: A vast majority of C282Y carriers will not develop iron overload and can be reassured. However, a careful step by step strategy at the clinical and genetic levels may allow to correctly identify those patients deserving further investigation. First, clinical examination and the assessment of iron parameters (SF and TS) allow identifying C282Y heterozygotes with an abnormal iron status. Once extrinsic factors such as heavy alcohol intake, virus or a dysmetabolic syndrome have been excluded, MRI is very useful to authenticate a high liver iron content. Second, HFE genotype must first exclude the presence of the H63D mutation. Compound heterozygozity for C282Y and H63D, a very widespread condition in our area, is usually associated with mild iron overload. Third, HFE sequencing can be undertaken and may identify new HFE variants as described here. The two novel mutations, a frameshift modifying the composition and the length of the C terminal end of the HFE protein and a substitution located at the last base of an exon, are likely to lead to an impaired function of HFE in association with the C282Y mutant. However, it is noteworthy that three of the four patients were diagnosed relatively late, after the 4th decade, as it is the case for C282Y homozygotes. Three further unrelated patients are currently under investigation in our laboratory for a similar clinical presentation. Finally, it can be noted that in those patients who will not have a HFE gene mutant identified, analysis of other genes implicated in iron overload must be performed to search for digenism or multigenism. None of our investigated patients had an additional gene abnormality.

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Comparing the Value of Serum Ferritin, Transfusion History and Magnetic Resonance Imaging for the Prediction of Iron Overload In MDS and AML Patients Undergoing Allogeneic Stem Cell Transplantation.

Blood ◽

10.1182/blood.v116.21.3493.3493 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3493-3493

Author(s):

Martin Wermke ◽

Jan Moritz Middeke ◽

Nona Shayegi ◽

Verena Plodeck ◽

Michael Laniado ◽

...

Keyword(s):

Iron Overload ◽

Serum Ferritin ◽

Iron Content ◽

Iron Concentration ◽

Transferrin Saturation ◽

Resonance Imaging ◽

Liver Iron Concentration ◽

Liver Iron ◽

Liver Iron Content ◽

Transfusion History

Abstract Abstract 3493 An increased risk for GvHD, infections and liver toxicity after transplant has been attributed to iron overload (defined by serum ferritin) of MDS and AML patients prior to allogeneic hematopoietic stem cell transplantation (allo-HSCT). Nevertheless, the reason for this observation is not very well defined. Consequently, there is a debate whether to use iron chelators in these patients prior to allo-HSCT. In fact, serum ferritin levels and transfusion history are commonly used to guide iron depletion strategies. Both parameters may inadequately reflect body iron stores in MDS and AML patients prior to allo-HSCT. Recently, quantitative magnetic resonance imaging (MRI) was introduced as a tool for direct measurement of liver iron. We therefore aimed at evaluating the accurateness of different strategies for determining iron overload in MDS and AML patients prior to allo-HSCT. Serologic parameters of iron overload (ferritin, iron, transferrin, transferrin saturation, soluble transferrin receptor) and transfusion history were obtained prospectively in MDS or AML patients prior to allo-SCT. In parallel, liver iron content was measured by MRI according to the method described by Gandon (Lancet 2004) and Rose (Eur J Haematol 2006), respectively. A total of 20 AML and 9 MDS patients (median age 59 years, range: 23–74 years) undergoing allo-HSCT have been evaluated so far. The median ferritin concentration was 2237 μg/l (range 572–6594 μg/l) and patients had received a median of 20 transfusions (range 6–127) before transplantation. Serum ferritin was not significantly correlated with transfusion burden (t = 0.207, p = 0.119) but as expected with the concentration of C-reactive protein (t = 0.385, p = 0.003). Median liver iron concentration measured by MRI was 150 μmol/g (range 40–300 μmol/g, normal: < 36 μmol/g). A weak but significant correlation was found between liver iron concentration and ferritin (t = 0.354; p = 0.008). The strength of the correlation was diminished by the influence of 5 outliers with high ferritin concentrations but rather low liver iron content (Figure 1). The same applied to transfusion history which was also only weakly associated with liver iron content (t = 0.365; p = 0.007). Levels of transferrin, transferrin saturation, total iron and soluble transferrin receptor did not predict for liver iron concentration. Our data suggest that serum ferritin or transfusion history cannot be regarded as robust surrogates for the actual iron overload in MDS or AML patients. Therefore we advocate caution when using one of these parameters as the only trigger for chelation therapy or as a risk-factor to predict outcome after allo-HSCT. Figure 1. Correlation of Liver iron content with Ferritin. Figure 1. Correlation of Liver iron content with Ferritin. Disclosures: No relevant conflicts of interest to declare.

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Target TMPRSS6 for the Treatment of Hereditary Hemochromatosis

Blood ◽

10.1182/blood.v118.21.1047.1047 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1047-1047

Author(s):

Sheri Booten ◽

Daniel Knox ◽

Luis Alvarado ◽

Shuling Guo ◽

Brett P. Monia

Keyword(s):

Iron Overload ◽

Genetic Disorder ◽

Hereditary Hemochromatosis ◽

Genetic Defect ◽

Transferrin Saturation ◽

Common Mutation ◽

C282y Mutation ◽

Iron Storage ◽

Hepcidin Expression ◽

Iron Deficient

Abstract Abstract 1047 Hereditary hemochromatosis (HH) is a genetic disorder in which hyperabsorption of dietary iron leads to accumulation of iron in multiple tissues including liver and heart. A common clinical manifestation in HH patients is cirrhosis and hepatocellular carcinoma as a result of iron-mediated injury in liver. The most prevalent genetic defect for HH is the failure to up-regulate hepcidin, a peptide hormone that inhibits the absorption of iron in duodenum and the release of iron from intracellular iron storage such as macrophages. Mutations in a number of genes have been identified as the cause for HH, including hepcidin itself. However, the most common mutation is C282Y mutation in HFE, which is a positive regulator for hepcidin expression. C282Y mutation represents about 85% of the HH population. HFE C282Y HH is an autosomal recessive disease with a ∼50% penetrance. Currently, the only treatment available for iron overload is phlebotomy which will continue throughout the patient's life. Hepcidin is mainly expressed and secreted by the liver and its expression is regulated predominantly at the transcription level. TMPRSS6, a transmembrane serine protease mutated in iron-refractory, iron-deficient anemia, is a major suppressor for hepcidin expression. It's been demonstrated that hepcidin expression is significantly elevated in Tmprss6−/− mice and reduction of TMPRSS6 in Hfe−/− mice could ameliorate the iron overload phenotype (Du et al. Science 2008; Folgueras et al. Blood 2008; Finberg KE et al., Blood, 2011). Using second generation antisense technology, we identified antisense oligonucleotides (ASOs) targeting mouse TMPRSS6 for the treatment of HH. These compounds were first identified through in vitro screens in mouse primary hepatocytes. After 4 weeks of treatment in C57BL/6 mice on normal chow, we observed an 80% to 90% reduction of liver TMPRSS6 mRNA with a subsequent 2–3 fold induction of liver hepcidin mRNA. Serum iron and transferrin saturation levels were reduced by ∼50%. These ASOs are currently being evaluated in a diet-induced iron overload model and an Hfe−/− iron overload model. Our preliminary results demonstrate that targeting TMPRSS6 is a viable approach for the treatment of hereditary hemochromatosis and possibly other iron-loading diseases associated with suppressed hepcidin levels. Disclosures: Booten: Isis Pharmaceuticals: Employment. Knox:Isis Pharmaceuticals: Summer Intern. Alvarado:Isis Pharmaceuticals: Employment. Guo:Isis Pharmaceuticals: Employment. Monia:Isis Pharmaceuticals: Employment.

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Role of Magnetic Ressonance Imaging – T2* in Hereditary Hemochromatosis.

Blood ◽

10.1182/blood.v120.21.2096.2096 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2096-2096

Author(s):

Reijane Alves de Assis ◽

Fernando Uliana Kay ◽

Paulo Vidal Campregher ◽

Gilberto Szarf ◽

Fabiana Mendes Conti ◽

...

Keyword(s):

Iron Overload ◽

Serum Ferritin ◽

Conflicts Of Interest ◽

Hereditary Hemochromatosis ◽

Transferrin Saturation ◽

Laboratory Data ◽

Surrogate Marker ◽

Control Group ◽

Double Mutation ◽

H63d Mutation

Abstract Abstract 2096 Introduction: Hereditary hemochromatosis (HH) is an autossomic recessive disorder characterized by increased iron absorption. Magnetic resonance imaging – T2* (MRI-T2*) has become a reliable and robust methodology to directly assess the iron burden, with better results in transfusional hemosiderosis compared to indirect methods, such as serum ferritin and transferrin saturation (TS). However, little is known about its role in HH. Objectives: Describe the demographic profile of HH type 1 patients as to the type of the HFE mutation and correlate laboratory parameters to MRI-T2*results. Methods: We collected data from patients with a positive HFE gene mutation who performed abdominal and/or cardiac MRI-T2* in our institution from 2004 to 2011. Images retrieved from the digital archive were analyzed by two blinded independent radiologists using the Thalassemia-Tools software (Cardiovascular Imaging Solutions, London, UK). Laboratory data available within 6 months before or after the MRI study were analyzed using the t-Student test, Exact Fisher's test analysis and multivariate analyses. Results: We analyzed 81 patients, 76 (93%) males and 5 (6.2%) females, with a median age of 48 years (21–80). Liver, pancreatic and splenic MRI-T2*values and LIC calculation were performed in 80 patients, and cardiac T2* assessment in 57 patients. The inter-observer T2* variation coefficient was 5%. Serum ferritin was abnormal in 70 patients (90.9%), while TS was abnormal in 34% of the tests. In our study sample, the H63D mutation was present in 70 patients (86.4%): 11 (13.6%) were homozygous, 59 (72.8%) heterozygous and 7 (8.6%) double heterozygous for C282Y/H63D. Only three patients (3.7%) were homozygous and 6 (7.4%) were heterozygous only for the C282Y mutation. The S65C mutation was detected in heterozygous state in 2 (2.5%) of cases. Two out 57 cases had a positive T2* result and were classified as light cardiac overload (T2*:18.98 e 19.14 ms). Both had the H63D mutation (1 homozygous and 1 heterozygous). Thirty seven out of 80 patients (46.3%) had liver overload in abdominal MRI (T2*: 3.8–11.4ms), being 33 (41.3%) light overload and four (5%) moderate overload (T2*:1.8–3.8ms). We found that 77.8% of patients with liver overload were C282Y carriers, of which 57.2% had double mutation and 40.3% had H63D mutation in hetero or homozigosity. Pancreatic overload was found in 20 patients (25.1%), while 30 patients (37.5%) had splenic overload. There was a slight correlation (r: 0.365) between liver T2* and splenic T2* (p=0.001). The presence of C282Y and H63D mutations was statistically associated with a higher frequency of abnormal liver T2* (p=0.017 and p=0.042, respectively). The H63D mutation was associated with iron accumulation in the liver (p=0,037) and homozygous carriers showed higher levels of liver overload (p=0,038). Conclusion: In our study, serum ferritin was a better surrogate marker for iron overload than ST. In addition, up to 40.3% of patients with H63D mutation had evidence of hepatic iron overload by MRI. These findings differ from the currente literature. The higher RMI positivity might be due to a higher sensitivity to detect lower levels of organic iron. Despite the lack of a control group and laboratory tests or MRI in all the cases studied, our results suggest that RMI-T2* is a promising methodology to guide the therapeutic management of HH patients. The clinical impact of this finding must be investigated in further studies. Disclosures: No relevant conflicts of interest to declare.

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Association of Iron Overload with Survival and Complications in Allogeneic Hematopoietic Cell Transplant Recipients: Prospective Cohort Study Using R2-MRI Measured Liver Iron Content

Blood ◽

10.1182/blood.v120.21.1961.1961 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1961-1961

Author(s):

Bryan J Trottier ◽

Todd E. Defor ◽

Linda J Burns ◽

Sarah Cooley ◽

Navneet S. Majhail

Keyword(s):

Iron Overload ◽

Research Funding ◽

Immune Reconstitution ◽

Cell Transplant ◽

Hematopoietic Cell Transplant ◽

Liver Iron ◽

Allogeneic Hct ◽

Liver Iron Content ◽

Significant Difference ◽

The Impact

Abstract Abstract 1961 Elevated pre-transplant ferritin levels have been associated with increased mortality and transplant-related complications in hematopoietic cell transplant (HCT) recipients. However, attempts to define the impact of iron overload on transplant outcomes using ferritin are confounded by its lack of specificity. Using liver magnetic resonance imaging (R2-MRI) to quantify liver iron content (LIC), we designed a prospective cohort study to determine the impact of iron overload on outcomes following allogeneic HCT. Our primary study objective was to determine the impact of pre-transplant iron overload on overall survival (OS); secondary objectives included cumulative incidence of non-relapse mortality (NRM) and post-HCT complications. Adult patients with hematologic malignancies being considered for allogeneic HCT were recruited for this study. Enrolled patients underwent baseline, pre-transplant ferritin measurements; patients with ferritin levels > 500 ng/ml had LIC quantified using liver R2-MRI. Patients were defined as no-iron overload (ferritin ≤ 500 or LIC ≤ 1.8 mg/gdw) and iron overload (LIC > 1.8). Of the 112 patients recruited for the study, 24 were excluded (disease progression=12, unable to complete MRI=9, transplant delays due to pre-HCT complications=3) and 88 were included in the final analysis (no-iron overload=28, iron overload=60). Four patients had ferritin >500, but on MRI had LIC ≤ 1.8 and were included in the no-iron overload group. Median ferritin in the two groups was 290 (range, 52–2023) and 1732 (range, 510–7137), respectively. The median LIC in the iron overload group was 4.3 (range, 1.9–25.4). Baseline ferritin moderately correlated with LIC (Spearman's R=0.58). There was no significant difference in recipient age, conditioning intensity, graft source, or HCT comorbidity index scores between the two groups. Patients with iron overload were more likely to have acute leukemia (55% vs 15%) and less likely to have high risk disease (40 vs 75%). We observed no significant difference in OS, NRM, relapse, acute or chronic graft-versus-host disease, organ failure, bacterial infections, viral infections, or fungal infections among patients without and with iron overload (see Table). We also found no difference in the composite endpoint of NRM, any infection, organ failure or hepatic veno-occlusive disease (1 yr cumulative incidence 71% vs 80%, P=0.44). In multivariate analyses that adjusted for other important prognostic variables, iron overload status did not impact risks of overall mortality (relative risk 2.3 (0.9–5.9) for iron overload vs. no-iron overload). We also evaluated outcomes based on an LIC threshold of ≤ 5 vs > 5 and observed similar results (see Table). Immune reconstitution studies were done in 55 patients at 3, 6 and/or 12 months post-HCT (no-iron overload=19, iron overload=36). On generalized linear mixed modeling, presence of iron overload was not associated with delay in recovery of absolute lymphocyte count, total NK cells, total T cells, CD4 cells, CD8 cells, or regulatory T cells. In conclusion, we did not find an association between pre-transplant iron-overload defined by R2-MRI measured LIC and OS, NRM, complications or immune reconstitution after allogeneic HCT in adults. Pre-transplant ferritin levels only moderately correlated with LIC. Future studies of iron overload in HCT should consider LIC to define iron overload instead of ferritin. Table. Outcomes by Iron-Overload Status Prob (95% CI) Prob (95% CI) P-value Ferritin ≤ 500 or LIC ≤ 1.8 LIC > 1.8 N 28 60 2 yr OS 78% (57–90) 58% (44–70) 0.12 2 yr NRM 18% (4–33) 21% (10–32) 0.91 1 yr Bacterial Infection 11% (0–22) 13% (5–22) 0.72 1 yr Fungal Infection 7% (0–17) 12% (4–20) 0.49 LIC ≤ 5.0 LIC > 5.0 N 65 23 2 yr OS 62% (49–73) 73% (49–87) 0.42 2 yr NRM 20% (10–30) 19% (3–35) 0.82 1 yr Bacterial Infection 12% (4–20) 13% (0–27) 0.98 1 yr Fungal Infection 12% (4–20) 4% (0–12) 0.28 Disclosures: Burns: Novartis Pharmaceuticals: Research Funding. Majhail:Novartis Pharmaceuticals: Research Funding.

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Impact Of Liver Iron Overload On Myocardial T2* Response In Transfusion-Dependent Thalassemia Major Patients Treated With Deferasirox For Up To 3 Years

Blood ◽

10.1182/blood.v122.21.1016.1016 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1016-1016 ◽

Cited By ~ 2

Author(s):

John Porter ◽

Ali T Taher ◽

Yesim Aydinok ◽

Maria D Cappellini ◽

Antonis Kattamis ◽

...

Keyword(s):

Iron Overload ◽

Board Of Directors ◽

Research Funding ◽

Iron Removal ◽

Advisory Committees ◽

Liver Iron ◽

Cardiac Iron ◽

Significant Difference ◽

Liver Iron Overload ◽

The Impact

Abstract Background Patients with myocardial iron overload require effective cardiac iron removal to minimize the risk of cardiac complications. The 3 year EPIC cardiac sub-study showed that the oral iron chelator, deferasirox (DFX), effectively reduced cardiac iron overload. Previous reports demonstrate that cardiac iron removal is slow and suggest that liver iron concentration (LIC) may affect cardiac iron removal rate by chelators (Pennell et al., 2012; Blood). The objective of these analyses was to evaluate the impact of the severity of the liver iron overload on the change in myocardial T2* (mT2*) for patients receiving up to 3 years of DFX treatment in the EPIC sub-study. Methods Inclusion and exclusion criteria have been described previously (Pennell et al., 2012; Haematologica). Patients were categorized into LIC ≤15 and >15 mg Fe/g dry weight (hereafter mg/g) at baseline (BL) and by LIC <7, 7–≤15 and >15 mg/g at 12, 24, and 36 months to assess the impact of BL LIC and changes in LIC overtime on mT2*, respectively. During study, LIC and mT2* were measured every 6 months. Efficacy was assessed in per-protocol population that entered third year extension. Here, mT2* is presented as the geometric mean (Gmean) ± coefficient of variation (CV) unless otherwise specified. Statistical significance was established at α-level of 0.05 using a 2-sided paired t-test for within group comparisons and ANOVA for multiple group comparisons. All p-values were of exploratory nature for this post-hoc analysis. Results Of the 71 patients, who continued into study year 3, 68 patients considered evaluable were included in this analysis (per protocol population); 59 patients had LIC values available at end of study (EOS). Mean age was 20.5 ±7.35 years and 61.8 % of patients were female. Mean actual dose of DFX (mg/kg/day) was 32.1 ±5.5 and 35.1 ±4.9 in patients with BL LIC ≤15 and >15 mg/g, respectively. At EOS, mean actual doses were 32.9 ±5.4 (LIC <7 mg/g), 38.0 ±3.4 (LIC 7–≤15 mg/g), and 37.6 ±3.1 (LIC >15 mg/g). Overall, patients had high BL LIC (Mean, 29.0 ±10.0 mg/g); 61 patients had LIC >15 (30.8 ±8.8) mg/g, only 7 patients had LIC ≤15 (12.7 ±1.1) mg/g, and no patients had LIC <7 mg/g. After 36 months, a significant mean decrease from BL in LIC of -7.6 ±4.6 mg/g (p = 0.0049) and -16.8 ±14.0 mg/g (p <0.001) was observed in patients with LIC ≤15 and >15 mg/g, respectively. Notably, 51.9% of patients with BL LIC >15 mg/g achieved EOS LIC <7 mg/g. Overall, mean mT2* was 12.8 ±4.6 ms. The impact of BL LIC on mT2* and LIC response was as follows: in patients with LIC ≤15 mg/g (Mean BL mT2*, 14.2 ±3.6 ms) and >15 mg/g (BL mT2*, 12.7 ±4.7 ms), mT2* increased by 52% (Mean abs. change, 7.5 ±4.1 ms, p=0.0016) and 46% (7.3 ±7.3 ms, p<0.001), respectively. Patients with BL LIC ≤15 normalized mT2* in 24 months (Mean, 20.0 ±6.0 ms) versus 36 months for patients with BL LIC >15 mg/g, (20.1 ±10.6 ms) displaying a lag of nearly 12 months. The relation between post-BL LIC on mT2* response at 12, 24 and 36 months is shown in the figure. At 12 months, there was no significant difference in mT2* that had occurred in patients with LIC <7 mg/g (24% increase; mean abs. change, 3.5 ±2.3 ms), LIC 7–≤15 mg/g (19% increase; 3.4 ±5.2 ms) and those with LIC >15 mg/g (13% increase; 1.9 ±3.2 ms). However, at 24 months, there was a statistically significant difference amongst the 3 subgroups in percent increase in the mT2* that had occurred; patients with LIC <7, LIC 7-≤15 and LIC >15 mg/g had 54% (Mean abs. change, 8.3 ±7.3 ms), 33% (5.2 ±5.2 ms) and 10% (2.1 ±4.3 ms) increase (p <0.001), respectively. Similarly, at 36 months, the mT2* had increased by 71% (Mean abs. change, 10.3 ±6.6 ms) in the LIC <7 mg/g group; a 31% increase (5.3 ±5.0 ms) had occurred in the LIC 7– ≤15 mg/g group; and an 18% (3.3 ±6.0 ms) increase (p <0.001) had occurred in the LIC >15mg/g group. At all-time points, in patients who achieved an LIC <7 mg/g, a statistically significant increase in T2* from BL had occurred. Discussion Overall, DFX treatment resulted in a significant decrease in LIC and improved mT2*. A greater difference in mT2* improvement was shown to have occurred in patients who achieved lower end-of-year LIC after treated with DFX. This divergence was progressive with time, being maximal at 36 months. Thus, a therapeutic response in LIC with DFX is associated with a greater likelihood of improving mT2*. This may assist in monitoring liver and cardiac response to DFX. Prospective evaluation of this relationship is indicated. Disclosures: Porter: Novartis Pharma: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria; Celgene: Consultancy. Taher:Novartis Pharma: Honoraria, Research Funding. Aydinok:Novartis Oncology: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Shire: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Cappellini:Novartis Pharma: Honoraria, Speakers Bureau; Genzyme: Honoraria, Membership on an entity’s Board of Directors or advisory committees. Kattamis:Novartis: Research Funding, Speakers Bureau; ApoPharma: Speakers Bureau. El-Ali:Novartis Pharma: Employment. Martin:Novartis Pharma: Employment. Pennell:Novartis: Consultancy, Honoraria, Research Funding; ApoPharma: Consultancy, Honoraria, Research Funding; Shire: Consultancy, Honoraria.

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Hereditary Hemochromatosis in an Adult Due to H63D Mutation: The Value of Estimating Iron Deposition By MRI T2* and Dissociation Between Serum Ferritin Concentration and Hepatic Iron Overload

Blood ◽

10.1182/blood.v124.21.4891.4891 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4891-4891

Author(s):

Mohamed A. Yassin ◽

Ashraf T Soliman ◽

Vincenzo Desanctis ◽

Sandara Abusamaan ◽

Ahmed Elsotouhy ◽

...

Keyword(s):

Iron Overload ◽

Basal Ganglion ◽

Serum Ferritin ◽

Normal Values ◽

Hereditary Hemochromatosis ◽

Transferrin Saturation ◽

Iron Deposition ◽

Ferritin Concentration ◽

H63d Mutation ◽

Serum Ferritin Concentration

Abstract Hereditary hemochromatosis (HH) is an autosomal recessive disorder characterized by excessive intestinal absorption of dietary iron, causing iron overload in different organs, especially the liver. Hemochromatosis may not be recognized until later in life. Patients are usually asymptomatic but may present with a variety of signs and symptoms. These include: hyper-pigmented skin, hepatomegaly, arthralgia, diabetes mellitusand/or heart failure/arrhythmia. The risk of HH related morbidity in HFE compound homozygotes patients (H63D /H63D) is considered rare, we report a male patient with H63D mutation who developed impaired glucose tolerance, and high hepatic enzymes due to significant iron accumulation in the liver as well as Parkinsonian-like syndrome due to iron deposition in the basal ganglia. A 40 year old Qatari male was referred for evaluation of a rise in hemoglobin and hematocrit values with normal MCV, total leucocyte and platelet counts. The patient was asymptomatic with normal vital signs, no depigmentation or hepato-splenomegaly. Hematologic findings included a hemoglobin concentration of Hb 16.5 g/dL, hematocrit 53%, mean corpuscular volume (MCV) 93 fL/red cell, leucocyte count of 7200/ μL and a platelet count of 199000/μL. His serum ferritin was 359 μg/l ( normal values: < 336 μg/l), serum iron: 37 μmol/l ( normal values <28.6μmol/l), fasting transferrin saturation: 64% (normal < 50%). A random glucose 6.5 and 6.4 mmol/L (normal values 5.5mmol/L ), A1C of 5,4 %, normal creatinine and electrolytes, alanine aminotransferase (ALT) of 66 U/l (normal < 40U/l), mild elevation of bilirubin 39 umol/l (normal <24umol/l), normal U&E Hepatitis B and C antibodies were negative. OGTT revealed impaired glucose tolerance. Thyroid function, morning serum cortisol, LH and FSH and serum total testosterone concentrations were in the normal range. A diagnosis of polycythemia vera was excluded on the basis of WHO Criteria 2008. The polymerase chain restriction assay was negative for the common mutation (C282Y) but positive for H63 D mutation. Family screening confirmed HH in his brother (homozygous), whereas his mother, two brothers and the sister were carriers (heterozygous). His four offspring were carriers. This suggested an autosomal recessive mode of inheritance. Conventional MRI study showed a normal liver size with diffuse fatty changes and focal areas of fatty sparing with some evidence of iron deposition. Whereas, T2-star (T2*) sequences showed a diffuse and significant decrease in liver signal intensity. A LIC liver concentration of 27 mg Fe/g dry wt was found (normalvalues:< 2 mg Fe/g dry wt; severe iron overload: ≥15 mg Fe/g dry wt). No significant iron deposition in the spleen, heart or pancreas was observed. At the age of 41 years the patient complained of tremors in both hands and arms while sitting or standing still (resting tremor) that improved with hands movements. A brain MRI revealed iron deposition in the basal ganglion. It was concluded that basal ganglionicn iron deposition mediated the neurological decline. Currently, the transferrin saturation and serum ferritin levels are within normal. Discussion: This is the first case of HH secondary to H63 D among an Arab family and the first reported case of Parkinsonism tremors secondary to this mutation. The H63D HFE variant is less frequently associated with HH, but its role in the neurodegenerative diseases has received a great attention. An accurate evaluation of iron overload is necessary to establish the diagnosis of HH and to guide iron chelation in HH by determination of liver iron concentration (LIC) by means of T2* MRI. Although serum ferritin concentration was only mildly increased a significant siderosis in the liver was detected by MRI T2* technique occurred. Liver siderosis was associated with mild impairment of liver function (increased serum ALT and bilirubin ). Conclusion: Our data further confirm that serum ferritin levels are not an accurate measure of total body iron stores in HH. Iron deposition in the liver and basal ganglion occurred despite mild elevation of ferritin. changes in basal ganglion may present by parkinsonian like tremors in these patients Use,T2* MRI should be encouraged in patients with HH for better evaluation of Iron overload and avoidance of Complications since serum ferritin can be misleading in these conditions. Disclosures Yassin: Qatar National research fund: Patents & Royalties, Research Funding. Aldewik:Qatar Ntional Research Fund: Patents & Royalties, Research Funding.

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