Examination of ALAS2, ABC7, Rag1, and IL6 Genes as Candidate Modifiers of Iron Overload in HFE C282Y Homozygotes with Severe Iron Overload.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3724-3724
Author(s):  
Pauline L. Lee ◽  
James C. Barton ◽  
Carol West ◽  
Karen Crain ◽  
Sreenivas V. Rao ◽  
...  
Keyword(s):  
Iron Overload ◽  
Transferrin Saturation ◽  
Allele Frequencies ◽  
Iron Loading ◽  
Promoter Polymorphisms ◽  
Sideroblastic Anemia ◽  
Iron Storage ◽  
Exon 2 ◽  
Hepcidin Expression ◽  
Control Subjects

Abstract The variable penetrance of HFE hemochromatosis led us to examine candidate modifier genes in 24 adult probands homozygous for the HFE C282Y mutation with severe iron overload. Mutations of the X-linked genes ALAS2 and ABC7 are associated with sideroblastic anemia and are such candidate genes. One C282Y homozygote had a P520L mutation in ALAS2 and another had an an ABC7 ivs2+1 G->A mutation that would result in the loss of a splice site and predict skipping of exon 2 with an in-frame deletion of 27 amino acids from the mature protein. Examination of respective family members demonstrated that inheritance of the ALAS2 P520L mutation or the ABC7 ivs2+1 mutation alone was not associated with excessive iron storage or sideroblastic anemia. Only subjects homozygous for HFE C282Y and hemizygous or heterozygous for these ALAS2 or ABC7 mutations had iron overload. Because the families were small, it was not possible to establish conclusively that these mutations affected the severity of iron overload. Nevertheless, these data suggest that if ALAS2 and ABC7 mutations are modifers of HFE hemochromatosis, they occur at a low frequency and do not account for the majority of cases of severe iron overload in C282Y homozygotes. Mice expressing double knockouts of rag1 and hfe or beta 2 microglobulin exhibit more severe iron overload than mice with knockouts of either gene alone. Thus, we sought to determine if mutations in RAG1 were associated with severe iron overload in 20 HFE C282Y homozygotes with severe iron overload. Four non-synonymous SNPs were identified in the HFE C282Y homozygotes: R249H, R449K, K820R, and M1006V. These mutations occurred at allele frequencies of 0.3500, 0.0238, 0.1190, and 0.0227, respectively. In 364 control subjects, the allele frequencies of R249H (0.3750) and K820R (0.1133) did not differ significantly from those in HFE C282Y homozygotes. A promoter polymorphism in IL6 at nt −174 has been shown to be associated with IL-6 expression: the G allele with high expression and the C allele with low expression. Lower levels of IL-6 expression might result in lower levels of hepcidin expression, further decreasing the already lower levels of hepcidin resulting from HFE C282Y homozygosity. Analysis of 340 control whites disclosed 62 IL6 -174C/C subjects had a mean transferrin saturation of 28% and 107 IL6 -174G/G subjects had a mean transferrin saturation of 25% (p <0.05). We found that the C allele frequency was 0.4800 in C282Y homozygotes with severe iron overload; this did not differ significantly from the frequency of 0.4338 in white control subjects. These data suggest that the IL6 -174 C allele is not significantly associated with severe iron overload in HFE C282Y homozygotes. We conclude that mutations of ALAS2 and ABC7 may be rare causes of severe iron loading in HFE C282Y homozygotes, but it is unlikely that RAG1 and the IL6 promoter polymorphisms contribute significantly to the iron loading of such subjects.

Target TMPRSS6 for the Treatment of Hereditary Hemochromatosis

Blood ◽  
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.

X-Linked Sideroblastic Anemia Is a Rare Cause of Microcytosis and Iron Overload in Adults: About 3 Cases with 2 New ALAS2 Gene Mutations Improved after Iron Chelation by Phlebotomy.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4619-4619
Author(s):  
Mohamed Touati ◽  
Franck Trimoreau ◽  
Marie-Pierre Gourin-Chaury ◽  
Caroline Kannengiesser ◽  
Pascal Turlure ◽  
...  
Keyword(s):  
Serum Level ◽  
Iron Overload ◽  
Gene Mutation ◽  
Molecular Analysis ◽  
Aminolevulinic Acid ◽  
Transferrin Saturation ◽  
Gene Mutations ◽  
Variable Response ◽  
Sideroblastic Anemia ◽  
Ring Sideroblasts

Abstract Introduction: X-linked sideroblastic anemia (XLSA), a rare disease characterized by an inherited microcytic and hypochromic anemia with high ferritin serum level and dyserythropoiesis with ring sideroblasts in bone marrow (BM), caused by mutations in the erythroid-specific 5-aminolevulinic acid synthase (ALAS2) gene located in the X chromosome is usually diagnosed in the early age. Anemia is often mild and well tolerated with variable response to pyridoxine treatment. The evolution can be dominated by iron overload due to hyperabsorption of iron and transfusional uptake. We report 3 adult cases, diagnosed after 30 years old, of XLSA transfusion free with iron overload. Case 1: A 34 y-old man, was seen in 2005 for a microcytic anemia and high ferritin serum, hemoglobin (Hb) 10.4 g/dl, MCV 70 fl and MCH 20.9 pg, dyserythropoiesis with 36% of ring sideroblasts (RS) on BM, ferritin serum level 2284 ng/ml (N: 30–300), transferrin sat 93% (N: 17–40). The hepatic MRI revealed a major iron overload at 350 μmol/g (N < 36) confirmed by biopsy showing a slight liver fibrosis. Molecular analysis of ALAS2 gene demonstrates a p.Arg452Gly mutation. Pyridoxine treatment and phlebotomy allowed a correction of anemia and reduction of the S-ferritin (371 ng/ml). Case 2: The family investigation of case 1 detect an affected first cousin, a 38 y-old man with Hb 12.9 g/dl, MCV 77 fl and MCH 25 pg, S-ferritin 559 ng/ml and transferrin saturation 91%. BM aspirate showed a dyserythropoiesis with 20% of ring sideroblasts. The molecular analysis of ALAS2 gene found the same mutation. The MRI indicates a marked liver iron overload (150 μmol/g) and elastography measurement (Fibroscan®) no fibrosis. Treatment by pyridoxine and phlebotomy every 2 weeks allowed a favourable outcome. Case 3: a 46 y-old man presented in 1994 a microcytosis without anemia Hb 13,2 g/dl, MCV 68 fl and MCH 22,5 pg, S-ferritin 1000 ng/ml transferrin saturation 63% and dyserythropoiesis with 66% of ring sideroblasts on BM. Treatment by pyridoxine was not efficient and iterative phlebotomies because of asthenia with arthralgies attributed to iron overload, with benefit for the patient. The molecular analysis of ALAS2 gene revealed a p.Arg572His mutation. Comments: Hereditary etiology due to ALAS2 gene mutations is a diagnostic rarely performed in adults, because of his rarity far behind primary acquired myelodysplastic syndromes (RARS) and secondary causes induced by drugs or toxics. The XLSA is the main cause of hereditary SA. More than 30 mutations have been identified. The 3 cases reported are XLSA due to 2 new ALAS2 gene mutations, never reported in the Human Gene Mutation Database. Conclusion: In XLSA with ALAS2 gene mutation, anemia often moderate, well tolerate and often unrecognized. Iron overload appears in this disease without any transfusion. Early diagnosis allows preventing the complications of the iron overload by iterative phlebotomies or by chelators. Pyridoxine treatment is indicated with variable response.

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

Blood ◽  
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&gt;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&gt;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.

RNAi-Mediated Inhibition of Tmprss6 Elevates Hamp1 Expression and Reduces Serum Iron Levels in Mice

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1045-1045 ◽  
Author(s):  
Ivanka Toudjarska ◽  
Zuhua Cai ◽  
Tim Racie ◽  
Stuart Milstein ◽  
Brian R Bettencourt ◽  
...  
Keyword(s):  
Iron Overload ◽  
Serum Iron ◽  
Iron Concentration ◽  
Transferrin Saturation ◽  
Elevated Serum ◽  
High Serum ◽  
Therapeutic Modality ◽  
Deficiency Anemia ◽  
Loss Of Function ◽  
Hepcidin Expression

Abstract Abstract 1045 The liver hormone Hepcidin (encoded by Hamp1) regulates serum iron levels by controlling the efflux of iron from intestinal enterocytes and macrophages. Maintaining sufficient iron levels to support erythropoiesis while preventing iron overload requires tight control of Hepcidin expression. Transcription of Hamp1 in hepatocytes is stimulated by high serum iron levels, via Transferrin Receptor signaling, as well as by activation of the BMP/SMAD pathway. The membrane serine protease Matriptase-2 (encoded by Tmprss6) inhibits BMP induced Hamp1 induction through the regulation of the BMP co-receptor, Hemojuvelin. In humans, loss of function mutations in TMPRSS6 lead to elevated Hepcidin levels resulting in iron-resistant iron-deficiency anemia (IRIDA). In diseases associated with iron overload, such as Thalassemia intermedia (TI) and Familial Hemochromatosis (FH), Hepcidin levels are low despite elevated serum iron concentrations. Studies in murine models of TI and FH have shown that elevating Hepcidin levels by genetic inactivation of Tmprss6 can prevent iron overload and correct aspects of the disease phenotype. Therefore, therapeutic strategies aimed at specifically inhibiting Tmprss6 expression could prove efficacious in these, and other, iron overloading diseases. Here we show that systemic administration of a potent lipid nanoparticle (LNP) formulated siRNA directed against Tmprss6 leads to durable inhibition of Tmprss6 mRNA in the mouse liver, with concomitant elevation of Hamp1 expression. This leads to significant decreases in serum iron concentration and Transferrin saturation, along with changes in hematologic parameters consistent with iron restriction. Further testing in mouse genetic models of TI and FH will support the rationale for developing LNP formulated Tmprss6 siRNA as a novel therapeutic modality. Disclosures: Toudjarska: Alnylam Pharmaceuticals, Inc.: Employment. Cai:Alnylam Pharmaceuticals, Inc.: Employment. Racie:Alnylam Pharmaceuticals, Inc.: Employment. Milstein:Alnylam Pharmaceuticals, Inc.: Employment. Bettencourt:Alnylam Pharmaceuticals, Inc.: Employment. Hettinger:Alnylam Pharmaceuticals, Inc.: Employment. Sah:Alnylam Pharmaceuticals, Inc.: Employment. Vaishnaw:Alnylam Pharmaceuticals, Inc.: Employment. Bumcrot:Alnylam Pharmaceuticals, Inc.: Employment.

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

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 481-481 ◽  
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.

Asymptomatic hemochromatosis subjects: genotypic and phenotypic profiles

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3707-3711 ◽  
Author(s):  
Ronald L. Sham ◽  
Richard F. Raubertas ◽  
Caroline Braggins ◽  
Joseph Cappuccio ◽  
Margaret Gallagher ◽  
...  
Keyword(s):  
Iron Overload ◽  
Hereditary Hemochromatosis ◽  
Transferrin Saturation ◽  
Entire Group ◽  
Iron Loading ◽  
Asymptomatic Patients ◽  
Liver Biopsies ◽  
The Relationship ◽  
Genotypic Profile ◽  
Compound Heterozygotes

Screening for hereditary hemochromatosis (HHC) by means of transferrin saturation (TS) levels has been advocated and will identify many patients who are asymptomatic. The purposes of this study were (1) to determine HFE genotypes among asymptomatic HHC patients and correlate this profile with the degree of iron overload and (2) to evaluate the relationship between mobilized iron (mob Fe), age, serum ferritin (SF), and quantitative hepatic iron (QHI) in this population. One hundred twenty-three asymptomatic HHC patients were evaluated; all had quantitative phlebotomy to determine mob Fe and genotyping for C282Y and H63D mutations. Liver biopsies with QHI determinations were performed on 72 of the 123 patients. Of the entire group, 60% were homozygous for C282Y, and 13% were compound heterozygotes (C282Y/H63D). Among asymptomatic patients, the prevalence of homozygous C282Y is lower compared with previous studies that include clinically affected patients. Of those patients with more than 4 g mob Fe, 77% were homozygous C282Y. Asymptomatic patients with lower iron burdens frequently had genotypes other than homozygous C282Y. There was no correlation between age and mob Fe in these patients; however, there was a correlation between mob Fe and both SF (r = 0.68) and QHI (r = 0.75). In conclusion, asymptomatic patients with moderate iron overload had a different genotypic profile than was seen in advanced iron overload. The significance of identifying patients with modest degrees of iron loading, who may not be homozygous for C282Y, must be addressed if routine TS screening is to be implemented.

Down Regulation of Hepcidin and Haemojuvelin Expression in the Hepatocyte Cell-Line HepG2 Induced by Thalassaemic Sera.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1556-1556
Author(s):  
Orly Weizer-Stern ◽  
Konstantin Adamsky ◽  
Ninette Amariglio ◽  
Carina Levin ◽  
Ariel Koren ◽  
...  
Keyword(s):  
Iron Overload ◽  
Iron Absorption ◽  
Transferrin Saturation ◽  
Reticuloendothelial System ◽  
Potential Effect ◽  
Thalassaemia Major ◽  
Healthy Individuals ◽  
Serum Factors ◽  
Hepcidin Expression ◽  
Plasma Factor

Abstract β-thalassaemia represents a group of diseases, in which ineffective erythropoiesis is accompanied by iron overload. In a mouse model of β-thalassaemia we observed that the liver expresses relatively low levels of hepcidin, which is a key factor in the regulation of iron absorption by the gut and of iron recycling by the reticuloendothelial system. We hypothesized that despite the overt iron overload, a putative plasma factor found in β-thalassaemia might suppress liver hepcidin expression. We therefore compared sera from β-thalassaemia and haemochromatosis (C282Y mutation) patients with those of healthy individuals in terms of their capacity to evoke changes in expression of key genes of iron metabolism in human HepG2 hepatoma cells. Sera from β-thalassaemia major patients evoked a major decrease in hepcidin (HAMP) and lipocalin2 (oncogene 24p3) (LCN2) expression, as well as a moderate decrease in haemojuvelin (HFE2) expression, compared to sera from healthy individuals. Significant correlation was found between the degree of downregulation of HAMP and HFE2 evoked by b-thalassaemia major sera (r=0.852, p<0.0009). Decreased HAMP expression was also found in HepG2 cells treated with sera collected from β-thalassaemia intermedia patients. In contrast, the majority of sera from hereditary haemochromatosis patients evoked an increase in HAMP expression, which correlated with their transferrin saturation (r=0.765, p<0.0099). Our results suggest that in β-thalassaemia, serum factors might override the potential effect of iron overload on HAMP expression, thereby providing an explanation for the failure to arrest excessive intestinal iron absorption.

Hfe Modulates the Response to Erythopoietic Stress In Wt Mice by Two Distinct Mechanisms

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4251-4251
Author(s):  
Pedro Ramos ◽  
Ella Guy ◽  
Robert W Grady ◽  
Maria de Sousa ◽  
Stefano Rivella
Keyword(s):  
Iron Overload ◽  
Iron Uptake ◽  
Serum Iron ◽  
Iron Absorption ◽  
Transferrin Saturation ◽  
Standard Diet ◽  
Iron Availability ◽  
Erythroid Cells ◽  
Hepcidin Expression

Abstract Abstract 4251 A deficient hepcidin response to iron is the principal mechanism responsible for increased iron uptake from the diet leading to iron overload. In hereditary hemochromatosis (HH), mutations in the HFE gene lead to iron overload through abnormally low levels of hepcidin. Interestingly, hepcidin has been shown to respond to a variety of stimuli, including iron, hypoxia, erythropoiesis and inflammation, requiring integration of the respective signals for its regulation. Further studies showed that HFE/Hfe could also modulate cellular iron uptake by associating with the transferrin receptor-1 (Tfrc), a crucial protein for iron uptake by erythroid cells. In addition, some studies have reported altered erythropoietic values in HH patients. Despite these findings, the role of Hfe in erythropoiesis was never explored. We hypothesized that Hfe influences erythropoiesis by two distinct mechanisms: 1) limiting hepcidin expression, thereby increasing iron availability, under conditions of simultaneous iron overload and stress erythropoiesis; 2) participating directly in the control of transferrin-bound iron uptake by erythroid cells. To test this hypothesis we investigated the role of Hfe in erythropoiesis, aiming to uncover the relative contribution of each of the aforementioned mechanisms. When erythropoiesis was challenged by phlebotomy, Hfe-KO animals were able to recover faster from anemia (p≤0.05) than either normal or iron overloaded wt mice. In Hfe-KO mice, despite their increased iron load, downregulation of hepcidin in response to phlebotomy or erythropoietin administration was comparable to that seen in wt mice. In contrast, iron overloaded wt mice showed increased hepcidin expression both at steady state and after erythropoietic stimulation compared to wt or Hfe-KO mice. In phlebotomized mice fed a standard diet, analysis of serum iron and transferrin saturation indicated that wt mice on the standard diet were able to increase their serum iron very rapidly. After 24 hours, both wt and Hfe-KO mice had similar serum iron and transferrin saturation levels. On the other hand, wt mice kept on an iron deficient diet over the course of phlebotomy, were unable to overcome the phlebotomy-induced anemia. In contrast, Hfe-KO mice fed the low iron diet were able to recover from anemia, although at a slower pace than either Hfe-KO or wt mice on a standard diet. These data indicate that gastrointestinal iron absorption in both wt and Hfe-KO mice is a major factor leading to recovery from anemia, although the excess iron in the liver of Hfe-KO mice contributes to restoration of the red blood cell reservoir. Phlebotomy is the main tool utilized to treat iron overload in HH patients. However, our data suggests that this treatment leads to both mobilization of iron from stores and increased gastrointestinal iron absorption. These observations suggest that patients might benefit from a controlled iron diet or from supplementation with hepcidin or an hepcidin agonist to limit iron absorption. Next, we determined that Hfe is expressed in erythroid cells and that it interacts with Tfrc in murine erythroleukemia cells. Moreover, we discovered that the level of Tfrc expression in Hfe-KO cells is 80% of that seen in wt cells, as measured by flow cytometry. This observation, together with measurement of iron uptake using 59Fe-saturated transferrin, indicated that Hfe-KO erythroid cells take up significantly more iron than wt cells. To confirm that Hfe plays a role in erythropoiesis independent from that in the liver, we transplanted Hfe-KO or wt bone marrow cells into lethally irradiated wt recipients and analyzed their recovery from phlebotomy. We observed that recovery from anemia was faster in Hfe→wt than in wt→wt and was associated with increased mean corpuscular hemoglobin levels, suggesting that lack of Hfe in the hematopoietic compartment can lead to increased hemoglobin production. In summary, our results indicate that lack of Hfe enhances iron availability for erythropoiesis by two distinct mechanisms. On the one hand, Hfe plays an important role in maintaining erythroid iron homeostasis by limiting the response of hepcidin to iron, particularly under conditions of erythropoietic stimulation. On the other hand, lack of Hfe contributes directly to increased iron intake by erythroid progenitors, even in the absence of iron overload. Disclosures: No relevant conflicts of interest to declare.

The Red Cell: How Erythropoiesis Modulates Hepcidin Expression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-24-SCI-24
Author(s):  
Yelena Ginzburg
Keyword(s):  
Bone Marrow ◽  
Iron Overload ◽  
Iron Metabolism ◽  
Conflicts Of Interest ◽  
Peptide Hormone ◽  
Well Being ◽  
Iron Loading ◽  
Ineffective Erythropoiesis ◽  
Hepcidin Expression ◽  
Erythroid Precursors

Abstract Abstract SCI-24 Erythroid precursors in the bone marrow require transferrin-bound iron for hemoglobin synthesis. Therefore, it is not surprising that the regulation of erythropoiesis and iron metabolism is interlinked. Iron demand for erythropoiesis is communicated to the iron-regulatory machinery through incompletely understood mechanisms. At the core of systemic iron homeostasis is the peptide hormone hepcidin, restricting cellular iron export to plasma by inducing the endocytosis and proteolysis of ferroportin. Hepcidin, a liver-synthesized peptide hormone, is released in response to increased iron load, and there is early evidence that circulating hepcidin concentrations affect the distribution of iron between the macrophage storage compartment (favored by higher hepcidin concentrations) and parenchymal cells, including cardiac myocytes and hepatocytes (favored by low hepcidin). Furthermore, ferroportin has recently been identified on erythroid precursors. Its purpose in this cell type and its function in the interface between erythropoiesis and iron metabolism are unclear. Additionally, in response to bleeding or the administration of erythropoietin, expansion of erythroid precursors suppresses hepcidin, most likely through one or more mediators released by the bone marrow and acting on hepatocytes. Iron-loading anemias with ineffective erythropoiesis, in particular β-thalassemia, demonstrate the effects of pathological “erythroid regulators” of hepcidin. Although erythrocyte transfusions are the main cause of iron loading in patients who receive them (β-thalassemia major), lethal iron overload is seen also in patients who are rarely or never transfused (β-thalassemia intermedia). Here, iron hyperabsorption is the cause of iron overload and, as in hereditary hemochromatosis, is caused by low hepcidin. Decreased hepcidin expression in β-thalassemia, with concurrent ineffective erythropoiesis and iron overload, indicates that the “erythroid regulator” may play an even more substantial role in iron metabolism than the “stores regulator.” Two members of the bone morphogenetic protein (BMP) family, growth differentiation factor (GDF) 15 and Twisted Gastrulation (TWSG1), have been implicated as candidate bone marrow-derived hepcidin suppressors in β-thalassemia. Neither factor is responsible for the physiologic hepcidin suppression in response to hemorrhage-induced stress erythropoiesis, and the physiologic suppressor is not known. We focus here on the current state of knowledge regarding the regulation of iron metabolism and attempt to elucidate the interface between iron regulation and erythropoiesis using evidence in part derived from animal models of β-thalassemia. A more complete understanding of the coregulation of erythropoiesis and iron metabolism may lay the foundation for improving diagnosis, increasing treatment options, and ultimately impacting the well-being of patients afflicted with different anemias and/or iron overload related-disorders. Disclosures: No relevant conflicts of interest to declare.

An RNAi-Therapeutic Targeting Tmprss6, in Conjunction With Oral Chelator Therapy, Ameliorates Anemia and Additively Diminishes Secondary Iron Overload In a Mouse Model Of β-Thalassemia Intermedia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1019-1019
Author(s):  
Paul J Schmidt ◽  
Tim Racie ◽  
Jim S Butler ◽  
Kevin Fitzgerald ◽  
Mark D Fleming
Keyword(s):  
Mouse Model ◽  
Iron Overload ◽  
Iron Deposition ◽  
Iron Loading ◽  
Thalassemia Intermedia ◽  
Ineffective Erythropoiesis ◽  
Liver Iron ◽  
Therapeutic Targeting ◽  
Hepcidin Expression ◽  
Secondary Iron Overload

Abstract β-Thalassemias are a group of inherited blood disorders caused by loss of β-globin synthesis and are characterized by anemia, extramedullary hematopoiesis and ineffective erythropoiesis leading to secondary iron overload. Increased iron absorption is due to inappropriately low levels of the liver hormone, hepcidin (HAMP). The membrane serine protease Matriptase-2 (TMPRSS6) attenuates BMP-mediated HAMP induction by cleaving the BMP co-receptor, hemojuvelin (HJV). Previously, we demonstrated that an RNAi-therapeutic targeting Tmprss6 elevates hepcidin expression and reduces disease severity in the Hbbth3/+ mouse model of β-Thalassemia intermedia (Blood. 2013; 14;121(7):1200-8). To further interrogate the efficacy of this therapeutic approach, Hbbth3/+ animals were treated with a siRNA directed against Tmprss6 on a replete 50ppm iron diet, a low iron diet (3-5ppm iron) or a 50ppm iron diet containing deferiprone. Systemic administration of an siRNA directed against Tmprss6 in the three diet conditions leads to significant inhibition of Tmprss6 mRNA in the livers of Hbbth3/+ mice with concomitant elevation in hepcidin expression. In correspondence with earlier studies, we demonstrate here that Tmprss6 silencing in animals under each of the three diet regimens leads to a significant improvement in the anemia of Hbbth3/+ mice as evidenced by increased total hemoglobin. Furthermore, hallmarks of ineffective erythropoiesis, including splenomegaly and reticulocytosis, were decreased in all Tmprss6 silenced Hbbth3/+ animals. If untreated, excessive iron loading in humans with β-Thalassemia leads to tissue iron deposition and eventual organ damage and failure. Importantly, here we demonstrate that the total body iron burden of Hbbth3/+ mice, as assessed by non-heme liver iron, is decreased by almost 30% in animals chelated with oral deferiprone and treated with Tmprss6 siRNA. A similar diminution of iron deposition is not evident in animals on a low iron diet or in mice fed deferiprone alone. Taken together, this data suggest that siRNA suppression of Tmprss6, in conjunction with chelation therapy, may provide an improved modality for treatment of the anemia and secondary iron loading seen in hemoglobinopathies such as β-Thalassemia. Disclosures: Racie: Alnylam Pharmaceutical, Inc: Employment. Butler:Alnylam Pharmaceutical, Inc: Employment. Fitzgerald:Alnylam: Employment. Fleming:Alnylam Pharmaceutical, Inc: Research Funding.

Sign in / Sign up

Export Citation Format

Share Document