Daniel F. Wallace

First phenotypic description of transferrin receptor 2 knockout mouse, and the role of hepcidin

Background: Transferrin receptor 2 (TfR2) is a key molecule involved in the regulation of iron homeostasis. Mutations in humans cause type 3 haemochromatosis and a targeted mutation in mice leads to iron overload with a similar phenotype. We have previously described the generation of a complete TfR2-knockout (KO) mouse. Aims: The aims of this study were to determine the phenotype and analyse expression of iron related molecules in the liver, duodenum, and spleen of homozygous TfR2-KO, heterozygous, and wild-type mice. Methods: Serum and tissue iron levels were determined in 10 week old male mice. Expression of iron related mRNA transcripts were analysed in the liver, duodenum, and spleen using real time polymerase chain reaction. Expression of iron related proteins in the liver were analysed by immunoblotting and immunohistochemistry. Results: Homozygous TfR2-KO mice had no TfR2 protein expression and developed significant iron overload typical of TfR2 associated haemochromatosis. In the liver of TfR2-KO mice there was no upregulation of hepcidin mRNA or prohepcidin protein in response to iron loading. Conclusions: Our results suggest that TfR2 is required for iron regulated expression of hepcidin and is involved in a pathway related to Hfe and hemojuvelin.

Uncommon Mutations and Polymorphisms in the Hemochromatosis Gene

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron metabolism. Iron absorption from the gut is inappropriately high, resulting in increasing iron overload. The hemochromatosis gene (HFE) was identified in 1996 by extensive positional cloning by many groups over a period of about 20 years. Two missense mutations were identified. Homozygosity for one of these, a substitution of a tyrosine for a conserved cysteine (C282Y), has now clearly been shown to be associated with HH in 60-100% of patients. The role of the second mutation, the substitution of an aspartic acid for a histidine (H63D), is not so clear but compound heterozygotes for both these mutations have a significant risk of developing HH. Here we review other putative mutations in the HFE gene and document a number of diallelic polymorphisms in HFE introns.

A novel mutation in ferroportin1 is associated with haemochromatosis in a Solomon Islands patient

Background: A severe form of iron overload with the clinicopathological features of haemochromatosis inherited in an autosomal dominant manner has been described in the Solomon Islands. The genetic basis of the disorder has not been identified. The disorder has similarities to type 4 haemochromatosis, which is caused by mutations in ferroportin1. Aims: The aims of this study were to identify the genetic basis of iron overload in a patient from the Solomon Islands. Patient and methods: Genomic DNA was isolated from peripheral blood leucocytes of a Solomon Islands man with severe iron overload. The entire coding region and splice sites of the ferroportin1 gene was sequenced. Results and conclusions: A novel missense mutation (431A>C; N144T) was identified in exon 5 of the ferroportin1 gene. A novel restriction endonuclease based assay which identifies both the N144T and N144H mutations was developed which will simplify the diagnosis and screening of patients for iron overload in the Solomon Islands and other populations. This is the first identified mutation associated with haemochromatosis in the Solomon Islands population.

Hepcidin: Regulation of the master iron regulator

Iron, an essential nutrient, is required for many diverse biological processes. The absence of a defined pathway to excrete excess iron makes it essential for the body to regulate the amount of iron absorbed; a deficiency could lead to iron deficiency and an excess to iron overload and associated disorders such as anaemia and haemochromatosis respectively. This regulation is mediated by the iron-regulatory hormone hepcidin. Hepcidin binds to the only known iron export protein, ferroportin (FPN), inducing its internalization and degradation, thus limiting the amount of iron released into the blood. The major factors that are implicated in hepcidin regulation include iron stores, hypoxia, inflammation and erythropoiesis. The present review summarizes our present knowledge about the molecular mechanisms and signalling pathways contributing to hepcidin regulation by these factors.

Co-factors in liver disease: The role of HFE-related hereditary hemochromatosis and iron

The severity of liver disease and its presentation is thought to be influenced by many host factors. Prominent among these factors is the level of iron in the body. The liver plays an important role in coordinating the regulation of iron homeostasis and is involved in regulating the level of iron absorption in the duodenum and iron recycling by the macrophages. Iron homeostasis is disturbed by several metabolic and genetic disorders, including various forms of hereditary hemochromatosis. This review will focus on liver disease and how it is affected by disordered iron homeostasis, as observed in hereditary hemochromatosis and due to HFE mutations. The types of liver disease covered herein are chronic hepatitis C virus (HCV) infection, alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), end-stage liver disease, hepatocellular carcinoma (HCC) and porphyria cutanea tarda (PCT).

In Situ Proximity Ligation Assays Indicate That Hemochromatosis Proteins Hfe and Transferrin Receptor 2 (Tfr2) Do Not Interact

The hemochromatosis associated proteins HFE and Transferrin Receptor 2 (TFR2) have been shown to be important for the proper regulation of hepcidin. A number of in vitro studies using transient overexpression systems have suggested that an interaction between HFE and TFR2 is required for the regulation of hepcidin. This model of iron sensing which centers upon the requirement for an interaction between HFE and TFR2 has recently been questioned with in vivo studies in mice from our laboratory and others which suggest that Hfe and Tfr2 can regulate hepcidin independently of each other. To re-examine the postulated interaction between Hfe and Tfr2 we developed a novel expression system in which both proteins are stably co-expressed and used the proximity ligation assay to examine the interactions between Hfe, Tfr1 and Tfr2 at a cellular level. We were able to detect the previously described interaction between Hfe and Tfr1, and heterodimers between Tfr1 and Tfr2; however no interaction between Hfe and Tfr2 was observed in our system. The results from this study indicate that Hfe and Tfr2 do not interact with each other when they are stably expressed at similar levels. Furthermore, these results support in vivo studies which suggest that Hfe and Tfr2 can independently regulate hepcidin.

Increased iron stores correlate with worse disease outcomes in a mouse model of schistosomiasis infection.

Schistosomiasis is a significant parasitic infection creating disease burden throughout many of the worlds developing nations. Iron deficiency anemia is also a significant health burden resulting from both nutritional deficit as well as parasitic infection in these countries. In this study we investigated the relationships between the disease outcomes of Schistosoma japonicum infection and iron homeostasis. We aimed to determine if host iron status has an effect on schistosome maturation or egg production, and to investigate the response of iron regulatory genes to chronic schistosomiasis infection. Wild-type C57BL/6 and Transferrin Receptor 2 null mice were infected with S. japonicum, and sacrificed at the onset of chronic disease. Transferrin Receptor 2 null mice are a model of type 3 hereditary hemochromatosis and develop significant iron overload providing increased iron stores at the onset of infection. The infectivity of schistosomes and egg production was assessed along with the subsequent development of granulomas and fibrosis. The response of the iron regulatory gene Hepcidin to infection and the changes in iron status were assessed by real-time PCR and Western blotting. Our results show that Hepcidin levels responded to the changing iron status of the animals, but were not significantly influenced by the inflammatory response. We also show that with increased iron availability at the time of infection there was greater development of fibrosis around granulomas. In conclusion, our studies indicate that chronic inflammation may not be the primary cause of the anemia seen in schistosomiasis, and suggest that increased availability of iron, such as through iron supplementation, may actually lead to increased disease severity.

Iron storage disease in Asia‐Pacific populations: The importance of non‐HFE mutations

Hereditary hemochromatosis (HH) is a widely recognized and well‐studied condition in European populations. This is largely due to the high prevalence of the C282Y mutation of HFE. Although less common than in Europe, HH cases have been reported in the Asia‐Pacific region because of mutations in both HFE and non‐HFE genes. Mutations in all of the currently known genes implicated in non‐HFE HH (hemojuvelin, hepcidin, transferrin receptor 2, and ferroportin) have been reported in patients from the Asia‐Pacific region. This review discusses the molecular basis of HH and the genes and mutations known to cause non‐HFE HH with particular reference to the Asia‐Pacific region. Challenges in the genetic diagnosis of non‐HFE HH are also discussed and how new technologies such as next generation sequencing may be informative in the future.

Identification of ferroportin disease in the Indian subcontinent

Haemochromatosis is a common inherited disorder of iron metabolism, characterised by excessive iron absorption and deposition in tissues. The majority of cases are associated with mutations in the HFE gene and inherited in an autosomal recessive manner.1 Autosomal dominant forms of haemochromatosis have been reported, mainly associated with mutations in the ferroportin 1 gene.2 This syndrome, termed type 4 haemochromatosis or more recently ferroportin disease,3 is usually characterised by an early increase in serum ferritin with normal transferrin saturation. Iron accumulation is most prominent in Kupffer cells and other macrophages, in addition to hepatocytes. Some patients do not tolerate venesection therapy well and can develop anaemia. Hereditary iron overload disorders appear to be uncommon in Asia. Secondary iron overload due to beta thalassaemia is relatively common in the Indian subcontinent. However, primary iron overload disorders …

Ferroportin disease due to the A77D mutation in Australia

Ferroportin disease or type 4 haemochromatosis is an autosomal dominant iron overload disorder caused by mutations in the iron exporter ferroportin.1,2 Numerous mutations in ferroportin ( SLC40A1 ) have been identified (see review by Pietrangelo3). The A77D mutation of ferroportin has thus far only been reported in Italy.2 We report the first A77D mutation of ferroportin which resulted in hepatic iron overload in an Australian family. The study was approved by and performed in accordance with the ethical standards of the Queensland Institute of Medical Research Human Research Ethics Committee and the Helsinki Declaration of 1975, as revised in 1983. Informed and written consent was obtained from the patient and family members. The subject, a 45 year old Caucasian male, presented with complaints of lethargy and malaise. He had no risk factors for viral hepatitis, consumed minimal alcohol (20 g/week), and was married with two …