DHPLC Scan of Iron Genes in Consecutive Patients with Suspected Iron Overload.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3206-3206
Author(s):  
Domenico Girelli ◽  
Giorgio Biasiotto ◽  
Barbara Foglieni ◽  
Claudia Bozzini ◽  
Anna Polotti ◽  
...  
Keyword(s):  
Iron Overload ◽  
Functional Role ◽  
Iron Homeostasis ◽  
Clinical Phenotype ◽  
Tertiary Care ◽  
Hereditary Hemochromatosis ◽  
Rapid Identification ◽  
Hfe Gene ◽  
Heterozygous Male ◽  
Juvenile Hemochromatosis

Abstract Background: Hereditary Hemochromatosis, once considered a monogenic disorder, is now seen as a polygenic disease, with clinical phenotype also influenced by several environmental factors. Besides the classic HFE gene, other genes involved in modulation of iron homeostasis and clinical phenotype include those coding for hemojuvelin and hepcidin (both responsible for Juvenile Hemochromatosis), ferroportin, and, possibly, H-ferritin. Methods: we used DHPLC to scan mutations in the above mentioned genes in 55 consecutive patients recently referred to our tertiary care unit for iron overload disorders. Many of them had at least biochemical signs of iron overload not explained, or not completely explained, by classic or rare HFE mutations. Main results: the −72 C→T variation in the promoter of hepcidin gene, near the putative TATA box, was found in a H63D heterozygous male with unexplained biochemical signs (serum ferritin 660 μg/L, TS 45%), who, rather, should have been protected by several previous blood donations. We recently found this new hepcidin mutation in a family from another cohort (Biasiotto et al., in press): genotype/phenotype correlation data were also consisting with a functional role of this mutation. Two other hepcidin variations were found in this series, the 108 G→A (new), and the 212 G→A. While the first appeared silent, familial study suggests that the second may be functional in association with C282Y. No new or functional variations were found in hemojuvelin or H-ferritin genes. Several polymorphisms were detected in the ferroportin gene, including two new sequence variations (−116 T→C in the promoter; 691+20C C→T in intron 4), that occurred in two unrelated subjects with wild-type HFE genotype. Their functional role is currently under investigation by extensive family studies and/or quantitative evaluation of hepatic iron by MRI/liver biopsy. Conclusions: DHPLC scan of iron genes appears as a helpful tool for integrating clinical data in selected patients referring for suspected iron overload, as well as for rapid identification of new mutations.

scholarly journals Hepcidin in iron overload disorders

Blood ◽  
2005 ◽  
Vol 105 (10) ◽  
pp. 4103-4105 ◽  
Author(s):  
George Papanikolaou ◽  
Michalis Tzilianos ◽  
John I. Christakis ◽  
Dionisios Bogdanos ◽  
Konstantina Tsimirika ◽  
...  
Keyword(s):  
Iron Overload ◽  
Transferrin Receptor ◽  
Iron Homeostasis ◽  
Iron Absorption ◽  
Hereditary Hemochromatosis ◽  
Congenital Dyserythropoietic Anemia ◽  
Cellular Iron ◽  
Juvenile Hemochromatosis ◽  
Hepcidin Gene

Abstract Hepcidin is the principal regulator of iron absorption in humans. The peptide inhibits cellular iron efflux by binding to the iron export channel ferroportin and inducing its internalization and degradation. Either hepcidin deficiency or alterations in its target, ferroportin, would be expected to result in dysregulated iron absorption, tissue maldistribution of iron, and iron overload. Indeed, hepcidin deficiency has been reported in hereditary hemochromatosis and attributed to mutations in HFE, transferrin receptor 2, hemojuvelin, and the hepcidin gene itself. We measured urinary hepcidin in patients with other genetic causes of iron overload. Hepcidin was found to be suppressed in patients with thalassemia syndromes and congenital dyserythropoietic anemia type 1 and was undetectable in patients with juvenile hemochromatosis with HAMP mutations. Of interest, urine hepcidin levels were significantly elevated in 2 patients with hemochromatosis type 4. These findings extend the spectrum of iron disorders with hepcidin deficiency and underscore the critical importance of the hepcidin–ferroportin interaction in iron homeostasis.

scholarly journals Correlation of genotype and phenotype in 32 patients with hereditary hemochromatosis in China

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Liyan Wu ◽  
Wei Zhang ◽  
Yanmeng Li ◽  
Donghu Zhou ◽  
Bei Zhang ◽  
...  
Keyword(s):  
Iron Overload ◽  
Iron Homeostasis ◽  
Clinical Phenotype ◽  
Hereditary Hemochromatosis ◽  
Clinical Manifestations ◽  
Pairwise Comparison ◽  
Asian Population ◽  
Chinese Patients ◽  
Pathogenic Variants ◽  
Significant Difference

Abstract Background Hereditary hemochromatosis (HH) is widely recognized and clinical manifestations of hemochromatosis-related (HFE-related) HH is well studied in European populations. Less is known about the clinical and laboratory characteristics of non-HFE related HH in Asian population. We aimed to explore the relationship between genotype and clinical phenotype in Chinese patients with non-HFE related hereditary hemochromatosis. Methods Peripheral blood samples and clinical data of patients with primary iron overload were collected from the China Registry of Genetic/Metabolic Liver Diseases. Sanger sequencing was performed in cases with primary iron overload, for 5 known HH related genes (HFE, HJV, HAMP, TFR2 and SLC40A1) and 2 novel iron homeostasis-related genes (DENND3 and SUGP2). The correlation of genotype and clinical phenotype in these patients was analyzed. Results Of the 32 patients with primary iron overload (23 were males and 9 were females), non-HFE variants were detected in 31 (31/32, 97%), including 8 pathogenic variants in HJV, 7 pathogenic variants in SLC40A1, 8 likely pathogenic variants in SUGP2 and 5 likely pathogenic variants in DENND3 cases. Among these 31 cases, 4 cases harbored homozygous variants, 2 cases harbored homozygous + heterozygous variants, 19 cases harbored heterozygous or combined heterozygous variants, and 6 cases harbored no any damaging variants. None of investigated cases carried damaging HAMP and TFR2 variants were found. 8 cases were classified as type 2A HH and 6 cases as type 4 HH, 10 cases as non-classical genotype, and 6 cases had no pathogenic variants from 31 cases. During the statistical analysis, we excluded one case (SLC40A1 IVS3 + 10delGTT + SUGP2 p. R639Q(homo)) with difficulty in grouping due to combined damaging variants. Cases with type 2A HH have an earlier age at diagnosis (p = 0.007). The iron index of cases in type 2A HH and type 4 HH was higher than that in other groups (p = 0.01). Arthropathy was relatively rare in all groups. None of cases with type 2A HH developed cirrhosis. Cirrhosis and diabetes are more prevalent in type 4 HH. The incidence of cirrhosis (p = 0.011), cardiac involvement (p = 0.042), diabetes (p = 0.035) and hypogonadism (p = 0.020) was statistically significant in the four groups. However, due to the limited sample size, the pairwise comparison showed no significant difference. Conclusions This is the first comprehensive analysis about the gene variant spectrum and phenotypic aspects of non-HFE HH in China. The results will be useful to the identification, diagnosis and management of HH in China.

scholarly journals Correlation of Genotype and Phenotype in 32 Patients with Hereditary Hemochromatosis in China

2021 ◽  
Author(s):  
Liyan Wu ◽  
Wei Zhang ◽  
Yanmeng Li ◽  
Donghu Zhou ◽  
Bei Zhang ◽  
...  
Keyword(s):  
Iron Overload ◽  
Iron Homeostasis ◽  
Clinical Phenotype ◽  
Hereditary Hemochromatosis ◽  
Clinical Manifestations ◽  
Asian Population ◽  
Chinese Patients ◽  
Pathogenic Variants ◽  
The Relationship ◽  
Metabolic Liver Diseases

Abstract Background: Hereditary hemochromatosis (HH) is widely recognized and clinical manifestations of HFE-related HH is well studied in European populations. Less is known about the clinical and laboratory characteristics of non-HFE related HH in Asian population. We aimed to explore the relationship between genotype and clinical phenotype in Chinese patients with non-HFE related hereditary hemochromatosis.Methods: Peripheral blood samples and clinical data of patients with primary iron overload were collected from the China Registry of Genetic/Metabolic Liver Diseases. Sanger sequencing was performed in cases with primary iron overload, for 5 known HH related genes (HFE, HJV, HAMP, TFR2 and SLC40A1) and 2 novel iron homeostasis-related genes (DENND3 and SUGP2). The correlation of genotype and clinical phenotype in these patients was analyzed.Results: Of the 32 patients with primary iron overload (23 were males and 9 were females), non-HFE variants were detected in 31 (31/32, 97%), including 8 pathogenic variants in HJV, 7 pathogenic variants in SLC40A1, 8 likely pathogenic variants in SUGP2 and 5 likely pathogenic variants in DENND3 cases. Among these 31 cases, 4 cases harbored homozygous variants, 2 cases harbored homozygous+ heterozygous variants, 19 cases harbored heterozygous or combined heterozygous variants, and 6 cases harbored no any damaging variants. None of investigated cases carried damaging HAMP and TFR2 variants were found. 8 cases were classified as type 2A HH and 6 cases as type 4 HH, 10 cases as non-classical genotype, and 6 cases had no pathogenic variants from 31 cases. During the statistical analysis, we excluded one case (SLC40A1 IVS3+10delGTT+SUGP2 p. R639Q(homo)) with difficulty in grouping due to combined damaging variants. Cases with type 2A HH have an earlier age at diagnosis. The iron index of cases in type 2A HH and type 4 HH was higher than that in other groups. Arthropathy was relatively rare in all groups. None of cases with type 2A HH developed cirrhosis. Cirrhosis and diabetes are more prevalent in type 4 HH.Conclusions: This is the first comprehensive analysis about the gene variant spectrum and phenotypic aspects of non-HFE HH in China. The results will be useful to the identification, diagnosis and management of HH in China.

scholarly journals Iron overload in the Asian community

Blood ◽  
2009 ◽  
Vol 114 (1) ◽  
pp. 20-25 ◽  
Author(s):  
Chun Yu Lok ◽  
Alison T. Merryweather-Clarke ◽  
Vip Viprakasit ◽  
Yingyong Chinthammitr ◽  
Somdet Srichairatanakool ◽  
...  
Keyword(s):  
Iron Overload ◽  
Hereditary Hemochromatosis ◽  
Severe Form ◽  
Hfe Gene ◽  
Dominant Form ◽  
Multiple Organs ◽  
Juvenile Hemochromatosis ◽  
Increased Risk ◽  
Autosomal Dominant Form ◽  
Secondary Iron Overload

Abstract Hereditary hemochromatosis is an iron overload disorder that can lead to the impairment of multiple organs and is caused by mutations in one or more different genes. Type 1 hemochromatosis is the most common form of the disease and results from mutations in the HFE gene. Juvenile hemochromatosis (JH) is the most severe form, usually caused by mutations in hemojuvelin (HJV) or hepcidin (HAMP). The autosomal dominant form of the disease, type 4, is due to mutations in the SLC40A1 gene, which encodes for ferroportin (FPN). Hereditary hemochromatosis is commonly found in populations of European origin. By contrast, hemochromatosis in Asia is rare and less well understood and can be masked by the presence of iron deficiency and secondary iron overload from thalassemia. Here, we provide a comprehensive report of hemochromatosis in a group of patients of Asian origin. We have identified novel mutations in HJV, HAMP, and SLC40A1 in countries not normally associated with hereditary hemochromatosis (Pakistan, Bangladesh, Sri Lanka, and Thailand). Our family studies show a high degree of consanguinity, highlighting the increased risk of iron overload in many countries of the developing world and in countries in which there are large immigrant populations from these regions.

scholarly journals Expression of Human Hemojuvelin (HJV) Is Tightly Regulated by Two Upstream Open Reading Frames in HJV mRNA That Respond to Iron Overload in Hepatic Cells

2015 ◽  
Vol 35 (8) ◽  
pp. 1376-1389 ◽  
Author(s):  
Cláudia Onofre ◽  
Filipa Tomé ◽  
Cristina Barbosa ◽  
Ana Luísa Silva ◽  
Luísa Romão
Keyword(s):  
Iron Overload ◽  
Iron Homeostasis ◽  
Open Reading Frames ◽  
Initiation Factor ◽  
Translational Repression ◽  
Translational Efficiency ◽  
Hepatic Cells ◽  
Juvenile Hemochromatosis ◽  
Upstream Open Reading Frames ◽  
Reading Frames

The gene encoding human hemojuvelin (HJV) is one of the genes that, when mutated, can cause juvenile hemochromatosis, an early-onset inherited disorder associated with iron overload. The 5′ untranslated region of the human HJV mRNA has two upstream open reading frames (uORFs), with 28 and 19 codons formed by two upstream AUGs (uAUGs) sharing the same in-frame stop codon. Here we show that these uORFs decrease the translational efficiency of the downstream main ORF in HeLa and HepG2 cells. Indeed, ribosomal access to the main AUG is conditioned by the strong uAUG context, which results in the first uORF being translated most frequently. The reach of the main ORF is then achieved by ribosomes that resume scanning after uORF translation. Furthermore, the amino acid sequences of the uORF-encoded peptides also reinforce the translational repression of the main ORF. Interestingly, when iron levels increase, translational repression is relieved specifically in hepatic cells. The upregulation of protein levels occurs along with phosphorylation of the eukaryotic initiation factor 2α. Nevertheless, our results support a model in which the increasing recognition of the main AUG is mediated by a tissue-specific factor that promotes uORF bypass. These results support a tight HJV translational regulation involved in iron homeostasis.

Physiologic systemic iron metabolism in mice deficient for duodenal Hfe

Blood ◽  
2007 ◽  
Vol 109 (10) ◽  
pp. 4511-4517 ◽  
Author(s):  
Maja Vujic Spasic ◽  
Judit Kiss ◽  
Thomas Herrmann ◽  
Regina Kessler ◽  
Jens Stolte ◽  
...  
Keyword(s):  
Iron Metabolism ◽  
Iron Homeostasis ◽  
Iron Absorption ◽  
Binding Capacity ◽  
Hereditary Hemochromatosis ◽  
Direct Interaction ◽  
Hfe Gene ◽  
Hepatic Iron ◽  
Primary Role ◽  
Genes Encoding

Abstract Mutations in the Hfe gene result in hereditary hemochromatosis (HH), a disorder characterized by increased duodenal iron absorption and tissue iron overload. Identification of a direct interaction between Hfe and transferrin receptor 1 in duodenal cells led to the hypothesis that the lack of functional Hfe in the duodenum affects TfR1-mediated serosal uptake of iron and misprogramming of the iron absorptive cells. Contrasting this view, Hfe deficiency causes inappropriately low expression of the hepatic iron hormone hepcidin, which causes increased duodenal iron absorption. We specifically ablated Hfe expression in mouse enterocytes using Cre/LoxP technology. Mice with efficient deletion of Hfe in crypt- and villi-enterocytes maintain physiologic iron metabolism with wild-type unsaturated iron binding capacity, hepatic iron levels, and hepcidin mRNA expression. Furthermore, the expression of genes encoding the major intestinal iron transporters is unchanged in duodenal Hfe-deficient mice. Our data demonstrate that intestinal Hfe is dispensable for the physiologic control of systemic iron homeostasis under steady state conditions. These findings exclude a primary role for duodenal Hfe in the pathogenesis of HH and support the model according to which Hfe is required for appropriate expression of the “iron hormone” hepcidin which then controls intestinal iron absorption.

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>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.

scholarly journals Discrepancy between Serum Ferritin and Liver Iron Concentration in a Patient with Hereditary Hemochromatosis – The Value of T2* MRI

2020 ◽  
Vol 13 (2) ◽  
pp. 712-715
Author(s):  
Mustafa A. Al-Tikrity ◽  
Mohamed A. Yassin
Keyword(s):  
Iron Overload ◽  
Serum Ferritin ◽  
Ferritin Level ◽  
Hereditary Hemochromatosis ◽  
Iron Concentration ◽  
Hfe Gene ◽  
Liver Iron Concentration ◽  
C282y Mutation ◽  
Liver Iron ◽  
T2 Mri

Primary hemochromatosis is an inherited disorder, and the homeostatic iron regulator (HFE) gene C282Y mutation is a common cause of hemochromatosis in Europe. We are reporting a case of a 56-year-old female known to have hemochromatosis with the HFE gene C282Y mutation with a serum ferritin level of 482 μg/L who underwent heart and liver T2* MRI which showed no evidence of iron overload – neither in the heart nor in the liver. This indicates that there is a discrepancy between serum ferritin and liver iron concentration by MRI and the superiority of T2* MRI in diagnosis and follow-up of iron overload in patients with hereditary hemochromatosis.

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 ◽  
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.

An Essential Role For Transferrin Receptor 2 In Erythropoiesis During Iron Restriction

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 429-429
Author(s):  
Daniel F Wallace ◽  
Cameron J McDonald ◽  
Eriza S Secondes ◽  
Lesa Ostini ◽  
Gautam Rishi ◽  
...  
Keyword(s):  
Iron Deficiency ◽  
Iron Overload ◽  
Iron Deficiency Anemia ◽  
Transferrin Receptor ◽  
Iron Homeostasis ◽  
Hereditary Hemochromatosis ◽  
Deficiency Anemia ◽  
Erythroid Cells ◽  
Iron Restriction ◽  
Extramedullary Erythropoiesis

Abstract Iron deficiency and iron overload are common clinical conditions that impact on the health and wellbeing of up to 30% of the world’s population. Understanding mechanisms regulating iron homeostasis will provide improved strategies for treating these disorders. The liver-expressed proteins matriptase-2 (encoded by TMPRSS6), HFE and transferrin receptor 2 (TFR2) play important and opposing roles in systemic iron homeostasis by regulating expression of the iron regulatory hormone hepcidin. Mutations in TMPRSS6 lead to iron refractory iron deficiency anemia, whereas mutations in HFE and TFR2 lead to the iron overload disorder hereditary hemochromatosis. To elucidate the competing roles of these hepcidin regulators, we created mice lacking matriptase-2, Hfe and Tfr2. Tmprss6 -/-/Hfe-/-/Tfr2-/- mice had iron deficiency anemia resulting from hepatic hepcidin over-expression and activation of Smad1/5/8, indicating that matriptase-2 predominates over Hfe and Tfr2 in hepcidin regulation. Surprisingly, this anemia was more severe than in the Tmprss6-/- mice, demonstrated by more extensive alopecia, lower hematocrit and significant extramedullary erythropoiesis in the spleen. There was increased expression of erythroid-specific genes in the spleens of Tmprss6-/-/Hfe-/-/Tfr2-/- mice, consistent with the extramedullary erythropoiesis. Expression of Tfr2 but not Hfe in the spleen was increased in the Tmprss6-/- mice compared to wild type and correlated with the expression of erythroid genes, suggesting that Tfr2 is expressed in erythroid cells. Further analysis of gene expression in the bone marrow suggests that the loss of Tfr2 in the erythroid cells of Tmprss6-/-/Hfe-/-/Tfr2-/- mice causes a delay in the differentiation process leading to a more severe phenotype. In conclusion, our results indicate that Hfe and Tfr2 act upstream of matriptase-2 in hepcidin regulation or in a way that is overridden when matriptase-2 is deleted. These results indicate that inhibition of matriptase-2 would be useful in the treatment of iron overload conditions such as hereditary hemochromatosis. We have also identified a novel role for Tfr2 in erythroid differentiation that is separate from its canonical role as a regulator of iron homeostasis in the liver. This important role of Tfr2 in erythropoiesis only becomes apparent during conditions of iron restriction. Our results provide novel insights into mechanisms regulating and linking iron homeostasis and erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

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