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Clinical and experimental studies on HFE and other genes involved in iron homeostasis

thesis
posted on 2024-09-03, 01:38 authored by Petra Holmström

In this thesis, the clinical significance of HFE mutations was further investigated, and the molecular mechanisms underlying the disturbed iron homeostasis during hemochromatosis, inflammation and hepatocellular carcinoma were studied in experimental models.

Hereditary hemochromatosis (HH) is a prevalent autosomal recessive iron overload disorder caused by increased intestinal absorption and deposition of iron in vital organs, leading to organ damage. Most HH patients are homozygous for the C282Y mutation in the HFE gene. The initial study served to further explore genetics of HH with respect to the S65C mutation for which the clinical significance was disputed. However, we found that half of the S65C-positive patients had evidence of mild or moderate hepatic iron overload, but without signs of extensive fibrosis in the liver biopsies. In conclusion, our data suggest that the HFE S65C mutation may lead to mild-to-moderate hepatic overload but possibly with a negligible risk for iron-associated liver cirrhosis. The S65C mutation is a rare variant in Sweden (allele frequency 1.6%). HFE mutation frequencies in Sweden were similar to other populations with a Northern European ancestry.

Porphyria cutanea tarda (PCT) is a disorder of heme biosynthesis presenting with cutaneous symptoms. PCT is known to involve iron, and an association with HH was suggested. The second study was undertaken to retrospectively investigate the association of overt PCT with the presence of HFE mutations. HFE mutations were clearly enriched among Swedish PCT patients with (PCT), and occurred almost to the same degree as in individuals with clinical suspicion of iron overload. Our finding indicates that the inheritance of HFE mutations plays a role for the susceptibility to PCT, by contributing to the hepatic iron overload often seen in patients with symptomatic PCT.

The rat HFE gene was found to consist of six exons and five introns, and its exon-intron boundaries were well conserved when comparisons were made with the murine HFE gene. In the rat, we detected HFE transcripts at low levels in multiple tissues. To our knowledge, we present the first evidence of HFE expression in red bone marrow. The strongest expression of rat HFE mRNA was observed in the liver. We showed for the first time, independently of species, that HFE expression occurs in the hepatocytes. In addition, hepatocytes exhibited the highest expression of HFE mRNA in the liver, being twice as high as in Kupffer cells. These findings initiate discussions about the function of HFE in the iron metabolism of hepatocytes.

We demonstrate that genes involved in the maintenance of iron homeostasis are regulated by both inflammation, as induced by exposure to the proinflammatory TNF-?, and iron perturbations in primary rat hepatocytes. In the rat, induction of HAMP mRNA in response to iron overload is not directly mediated by iron itself, but rather depending upon factors not present in hepatocyte culture, such as other liver cells. Interestingly, we observed a differential regulation between TfR1 and its close homologue TfR2 with respect to both iron and inflammation. In iron overloaded hepatocytes, expression of TfR1, DMT1 and IREG1, involved in cellular iron uptake and release, were orchestrated in order to prevent further accumulation. In addition, we observed a decrease in IREG1 expression in hepatocytes exposed to TNF-?, suggesting that iron will be bound intracellularly during inflammation.

Hepatocellular carcinomas (HCCs) display alterations of their iron metabolism, which may be of importance for tumor growth. The iron-deficient phenotype of HCCs was studied in a rat model and illustrated by histochemical absence of iron and low liver iron content. The altered iron metabolism in HCC involved increased TfR1 and DMT1-IRE mRNA, active in cellular iron uptake, reflecting the high iron demand. We propose that HCCs maintain their iron-deficient phenotype by a transcriptional upregulation of the iron exporter IREG1, whereas the regulation of HAMP expression is unaltered.

List of scientific papers

I. Holmstrom P, Marmur J, Eggertsen G, Gafvels M, Stal P (2002). Mild iron overload in patients carrying the HFE S65C gene mutation: a retrospective study in patients with suspected iron overload and healthy controls. Gut. 51(5): 723-30.
https://pubmed.ncbi.nlm.nih.gov/12377814

II. Harper P, Floderus Y, Holmstrom P, Eggertsen G, Gafvels M (2004). Enrichment of HFE mutations in Swedish patients with familial and sporadic form of porphyria cutanea tarda. J Intern Med. 255(6): 684-8.
https://pubmed.ncbi.nlm.nih.gov/15147533

III. Holmstrom P, Dzikaite V, Hultcrantz R, Melefors O, Eckes K, Stal P, Kinnman N, Smedsrod B, Gafvels M, Eggertsen G (2003). Structure and liver cell expression pattern of the HFE gene in the rat. J Hepatol. 39(3): 308-14.
https://pubmed.ncbi.nlm.nih.gov/12927914

IV. Dzikaite V, Holmstrom P, Eckes K, Hagen K, Stal P, Eggertsen G, Gafvels M, Melefors O, Hultcrantz R (2004). Effects of the TNF-alpha and iron overload on the expression of genes involved in iron metabolism in rat hepatocytes.

V. Holmstrom P, Gafvels M, Eriksson L, Dzikaite V, Hultcrantz R, Eggertsen G, Stal P (2004). Expression of iron regulatory genes in hepatocellular carcinoma in the rat. [Manuscript]

History

Defence date

2004-10-08

Department

  • Department of Laboratory Medicine

Publication year

2004

Thesis type

  • Doctoral thesis

ISBN-10

91-7140-073-7

Number of supporting papers

5

Language

  • eng

Original publication date

2004-09-17

Author name in thesis

Holmström, Petra

Original department name

Department of Laboratory Medicine

Place of publication

Stockholm

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