Petra Holmström

Mild iron overload in patients carrying the HFE S65C gene mutation: a retrospective study in patients with suspected iron overload and healthy controls

Background and aims: The role of the HFE S65C mutation in the development of hepatic iron overload is unknown. The aim of the present study was: (A) to determine the HFE S65C frequency in a Northern European population; and (B) to evaluate whether the presence of the HFE S65C mutation would result in a significant hepatic iron overload. Patients and methods: Biochemical iron parameters and HFE mutation analysis (for the C282Y, H63D, and S65C mutations) were analysed in 250 healthy control subjects and collected retrospectively in 296 patients with suspected iron overload (elevated serum ferritin and/or transferrin saturation). The frequency of patients having at least mild iron overload, and mean serum ferritin and transferrin saturation values were calculated for each HFE genotype. For patients carrying the S65C mutation, clinical data, liver biopsy results, and amount of blood removed at phlebotomy were determined. Results: The HFE S65C mutation was found in 14 patients and eight controls. In controls, the S65C allele frequency was 1.6%. The S65C allele frequency was enriched in non-C282Y non-H63D chromosomes from patients (4.9%) compared with controls (1.9%) (p<0.05). Serum ferritin was significantly increased in controls carrying the S65C mutation compared with those without HFE mutations. Fifty per cent of controls and relatives having the S65C mutation had elevated serum ferritin levels or transferrin saturation. The number of iron overloaded patients was significantly higher among those having HFE S65C compared with those without any HFE mutation. Half of patients carrying the S65C mutation (7/14) had evidence of mild or moderate hepatic iron overload but no signs of extensive fibrosis in liver biopsies. Screening of relatives revealed one S65C homozygote who had no signs of iron overload. Compound heterozygosity with S65C and C282Y or H63D did not significantly increase the risk of iron overload compared with S65C heterozygosity alone. Conclusions: The HFE S65C mutation may lead to mild to moderate hepatic iron overload but neither clinically manifest haemochromatosis nor iron associated extensive liver fibrosis was encountered in any of the patients carrying this mutation.

Structure and liver cell expression pattern of the HFE gene in the rat

BACKGROUND/AIMS Very little is known about the HFE gene in the rat. The aim of the present study was to determine: (1) the structure of the rat HFE gene; and (2) the tissue expression of the HFE mRNA in the rat, with special emphasis on the liver. METHODS Cloning of the rat HFE gene was performed using library screening and PCR. Exon-intron borders were assigned by DNA sequencing. Parenchymal and non-parenchymal liver cells were isolated by fractionation of normal rat liver. HFE mRNA levels were determined by Northern blot (tissues) and real-time PCR (isolated liver cells). RESULTS The rat HFE gene contained six exons and five introns. The HFE gene is expressed in multiple tissues in the rat, including bone marrow, with the highest expression in the liver. We observed HFE transcripts in several categories of isolated rat liver cells. Unexpectedly, expression also occurred in rat hepatocytes. CONCLUSIONS The exon-intron pattern of the HFE gene is strongly conserved between rat and mouse. The pattern of tissue expression of the HFE gene is rather similar in humans and rodents. The finding of HFE gene expression in rat hepatocytes raises interesting questions regarding its role in the hepatocyte iron metabolism.

Expression of iron regulatory genes in a rat model of hepatocellular carcinoma.

Abstract: Background/Aims: The altered iron metabolism in hepatocellular carcinomas (HCCs), characterized by the iron‐deficient phenotype, is suggested to be of importance for tumour growth. However, the underlying molecular mechanisms remain poorly understood. We asked whether these iron perturbations would involve altered expression of genes controlling iron homeostasis.

A novel mutation in the biliverdin reductase-A gene combined with liver cirrhosis results in hyperbiliverdinaemia (green jaundice)

Background: Hyperbiliverdinaemia is a poorly defined clinical sign that has been infrequently reported in cases of liver cirrhosis or liver carcinoma, usually indicating a poor long‐term prognosis.

Enrichment of HFE mutations in Swedish patients with familial and sporadic form of porphyria cutanea tarda

Dear Sir, Porphyria cutanea tarda (PCT) is caused by a strongly reduced activity of the hepatic enzyme uroporphyrinogen decarboxylase (UROD; EC 4.1.1.37). The reduced activity leads to accumulation of phototoxic porphyrins, primarily uroporphyrinogen and heptacarboxylated porphyrinogens that cause the characteristic clinical pattern with skin fragility and blisters on skin areas exposed to the sun [1, 2]. Iron seems to play a central role in the pathogenesis of PCT, because mild hepatic siderosis is present in most of the patients with overt disease [3, 4]. Removal of iron by repeated phlebotomies always leads to clinical and biochemical remission, even in patients without increased liver or total body iron [2, 5]. The disease process is enhanced by several heterogeneous factors such as alcohol abuse, estrogens and hepatic viral infections [for review see 6]. An association between hereditary haemochromatosis and PCT has been suspected for decades [3, 4, 7]. After the identification of the haemochromatosis-associated HFE gene [8], a high percentage of HFE mutations in patients with manifest PCT have been reported in many countries [9–16]. The present study was undertaken to retrospectively investigate the frequency of three HFE mutations (C282Y, H63D and S65C) in 117 unrelated Swedish PCT patients, with clinically and biochemically confirmed diagnosis [6]. The HFE allele frequencies in PCT patients were compared with the frequencies found in a control group of 250 Swedish healthy subjects and also to a group consisting of 296 patients with suspected clinical iron overload (elevated serum ferritin and/or transferrin saturation). The latter group has been characterized and reported before [17]. Written informed consent was obtained from all the patients and the study was approved by the local Ethics Committee of the Karolinska Institute (Dnr 167/99), Stockholm 1 . Based on the UROD activity in erythrocyte lysates [18, 19], the PCT patients had been classified at the time of diagnosis as being either of familial (F-PCT, n 1⁄4 53) or sporadic (S-PCT, n 1⁄4 64) form of PCT [20]. Genomic DNA was extracted from peripheral blood, which had been kept frozen at)80 C since the time of PCT diagnosis. The HFE genotypes were determined by sequencing two of the HFE gene regions flanking Cys282 (exon 4) and His63/Ser65 (exon 2). Comparisons of the frequencies of the HFE C282Y, H63D and S65C mutations in patients with PCT, healthy controls and patients with suspected clinical iron overload were performed with chisquare test, and P < 0.05 was considered statistically significant (Table 1). Of the 53 patients with F-PCT, six were homozygous for the C282Y mutation, five were C282Y/ H63D compound heterozygous and one patient was homozygous for the H63D mutation. In the group of 64 S-PCT patients, 14 were homozygous for the C282Y mutation, six were compound heterozygous (C282Y/H63D) and three were homozygous for the H63D mutation. The presence of the third HFE mutation (S65C) was found in two S-PCT cases, one heterozygous and one compound heterozygous (S65C/H63D). Amongst PCT patients, there was a clear overrepresentation of C282Y homozygosity and C282Y/H63D compound heterozygosity compared with healthy controls (P < 0.001), which is in accordance with the results published by Bulaj et al. [16]. The HFE genotypes in PCT patients were not statistically different from those found in patients with suspected clinical iron overload (Table 1). The frequency of the carrier condition of the C282Y mutation in the S-PCT group was 44%, which is the same frequency as reported in British, Australian and North American S-PCT patients [9, 11, 13]. Journal of Internal Medicine 2004; 255: 684–687

Iron-regulatory gene expression during liver regeneration

Abstract Background. In rat, the first 18–24 h after partial hepatectomy (PH) are characterized by an acute-phase reaction, after which liver regeneration predominates. Interleukin-6 (IL-6) induces the iron hormone hepcidin, which blocks iron uptake and may compromise iron uptake in the growing liver. The expressions of hepcidin and the iron-regulatory pathway of hepcidin gene expression during the late phase of liver regeneration are unknown. Aim. To characterize the expression pattern of hepcidin and the iron-sensing pathway of hepcidin regulation during liver regeneration. Methods. Rats were subjected to PH or sham operation. Liver weights, number of S-phase nuclei, and serum levels of iron and IL-6 were determined. Messenger-RNA levels of hepcidin, ferritin, hemojuvelin, transferrin receptor 1 and 2, HFE, divalent metal transporter 1, ferroportin, and ceruloplasmin were determined with qPCR at different time points. Protein levels of STAT3 and SMAD4 were determined with western blot. Results. During the acute-phase response, IL-6 release induced STAT3 protein and hepcidin mRNA, whereas mRNA levels of proteins in the iron-sensing pathway (HFE, hemojuvelin, and transferrin receptor 2) decreased. The mRNA levels of proteins involved in cellular iron uptake were increased and cellular iron export unchanged. During liver regeneration >24 h after PH, gene expressions in the iron-sensing pathway were continuously suppressed and hepcidin mRNA levels declined 3–7 days after surgery. Conclusions. Hepcidin gene expression peaks during the acute-phase response, but a sustained down-regulation of the iron-sensing pathway of hepcidin regulation gradually reduces hepcidin gene expression until regeneration is complete, thereby promoting iron mobilization to the regenerating liver.