Lesa Summerville

A population-based study of the clinical expression of the hemochromatosis gene

BACKGROUND AND METHODS Hereditary hemochromatosis is associated with homozygosity for the C282Y mutation in the hemochromatosis (HFE) gene on chromosome 6, elevated serum transferrin saturation, and excess iron deposits throughout the body. To assess the prevalence and clinical expression of the HFE gene, we conducted a population-based study in Busselton, Australia. In 1994, we obtained blood samples for the determination of serum transferrin saturation and ferritin levels and the presence or absence of the C282Y mutation and the H63D mutation (which may contribute to increased hepatic iron levels) in 3011 unrelated white adults. We evaluated all subjects who had persistently elevated transferrin-saturation values (45 percent or higher) or were homozygous for the C282Y mutation. We recommended liver biopsy for subjects with serum ferritin levels of 300 ng per milliliter or higher. The subjects were followed for up to four years. RESULTS Sixteen of the subjects (0.5 percent) were homozygous for the C282Y mutation, and 424 (14.1 percent) were heterozygous. The serum transferrin saturation was 45 percent or higher in 15 of the 16 who were homozygous; in 1 subject it was 43 percent. Four of the homozygous subjects had previously been given a diagnosis of hemochromatosis, and 12 had not. Seven of these 12 patients had elevated serum ferritin levels in 1994; 6 of the 7 had further increases in 1998, and 1 had a decrease, although the value remained elevated. The serum ferritin levels in the four other homozygous patients remained in the normal range. Eleven of the 16 homozygous subjects underwent liver biopsy; 3 had hepatic fibrosis, and 1, who had a history of excessive alcohol consumption, had cirrhosis and mild microvesicular steatosis. Eight of the 16 homozygous subjects had clinical findings that were consistent with the presence of hereditary hemochromatosis, such as hepatomegaly, skin pigmentation, and arthritis. CONCLUSIONS In a population of white adults of northern European ancestry, 0.5 percent were homozygous for the C282Y mutation in the HFE gene. However, only half of those who were homozygous had clinical features of hemochromatosis, and one quarter had serum ferritin levels that remained normal over a four-year period.

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.

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.

Lymphotoxin‐β receptor signaling regulates hepatic stellate cell function and wound healing in a murine model of chronic liver injury

Lymphotoxin‐beta (LTβ) is a proinflammatory cytokine and a member of the tumor necrosis factor (TNF) superfamily known for its role in mediating lymph node development and homeostasis. Our recent studies suggest a role for LTβ in mediating the pathogenesis of human chronic liver disease. We hypothesize that LTβ co‐ordinates the wound healing response in liver injury via direct effects on hepatic stellate cells. This study used the choline‐deficient, ethionine‐supplemented (CDE) dietary model of chronic liver injury, which induces inflammation, liver progenitor cell proliferation, and portal fibrosis, to assess (1) the cellular expression of LTβ, and (2) the role of LTβ receptor (LTβR) in mediating wound healing, in LTβR−/− versus wild‐type mice. In addition, primary isolates of hepatic stellate cells were treated with LTβR‐ligands LTβ and LTβ‐related inducible ligand competing for glycoprotein D binding to herpesvirus entry mediator on T cells (LIGHT), and mediators of hepatic stellate cell function and fibrogenesis were assessed. LTβ was localized to progenitor cells immediately adjacent to activated hepatic stellate cells in the periportal region of the liver in wild‐type mice fed the CDE diet. LTβR−/− mice fed the CDE diet showed significantly reduced fibrosis and a dysregulated immune response. LTβR was demonstrated on isolated hepatic stellate cells, which when stimulated by LTβ and LIGHT, activated the nuclear factor kappa B (NF‐κB) signaling pathway. Neither LTβ nor LIGHT had any effect on alpha‐smooth muscle actin, tissue inhibitor of metalloproteinase 1, transforming growth factor beta, or procollagen α1(I) expression; however, leukocyte recruitment‐associated factors intercellular adhesion molecule 1 and regulated upon activation T cells expressed and secreted (RANTES) were markedly up‐regulated. RANTES caused the chemotaxis of a liver progenitor cell line expressing CCR5. Conclusion: This study suggests that LTβR on hepatic stellate cells may be involved in paracrine signaling with nearby LTβ‐expressing liver progenitor cells mediating recruitment of progenitor cells, hepatic stellate cells, and leukocytes required for wound healing and regeneration during chronic liver injury. (HEPATOLOGY 2009;49:227–239.)

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.

Molecular and cellular characterization of transferrin receptor 2

Iron is an essential component of many biological processes. However, an excess of iron in the body is also toxic; thus, the levels of this element are tightly regulated. Our knowledge of the mechanism by which iron levels are maintained has been bolstered by the dramatic increase in the discovery of novel molecules implicated in iron homeostasis. The transferrin receptor-transferrin pathway is the main mechanism by which cells take up iron. The recently identified homolog of transferrin receptor, its characterization and its role in iron metabolism is the subject of this review.

[119] TARGETED DISRUPTION OF THE HEPATIC TRANSFERRIN RECEPTOR 2 GENE IN MICE LEADS TO IRON OVERLOAD

BACKGROUND & AIMS Transferrin receptor 2 (TfR2) plays a key role in the regulation of iron metabolism. Mutations of TfR2 in humans cause type 3 hereditary hemochromatosis. Although highly expressed in liver, several studies have reported TfR2 expression in other tissues. To determine the contribution of liver expressed TfR2 in iron homeostasis, we have generated and characterized a liver-specific TfR2-knockout (KO) mouse. METHODS Liver-specific TfR2-KO mice were generated by crossing TfR2-floxed mice with transgenic albumin-Cre mice. Tissue and serum from homozygous TfR2-floxed mice with and without albumin-Cre were analyzed. Serum transferrin saturation, hepatic, and splenic iron concentrations were determined. The expression of iron-related mRNA transcripts was analyzed by real-time PCR. Levels of the iron-related proteins TfR1, TfR2, ferritin, and prohepcidin were analyzed by immunoblotting. RESULTS Liver-specific TfR2-KO mice develop significant iron overload comparable to complete TfR2-KO mice. At all ages studied, transferrin saturation, hepatic iron concentration, and hepatic ferritin were significantly elevated. Hepatic TfR2 mRNA and protein were absent in the livers of liver-specific TfR2-KO mice, and TfR1 expression was reduced consistent with liver iron loading. At 5 weeks of age, hepcidin1 mRNA, and prohepcidin protein were decreased in liver-specific TfR2-KO compared to control mice. CONCLUSIONS The significant iron loading and modulation of expression of iron-related genes in liver-specific TfR2-KO mice demonstrates that the liver is the primary site for TfR2 expression and activity and that liver-expressed TfR2 is required for the regulation of hepcidin1.