Atypical haemochromatosis: phenotypic spectrum and beta2-microglobulin candidate gene analysis.

Beta2-microglobulin was investigated in atypical haemochromatosis patients not homozygous for the C282Y mutation of HFE (OMIM *235200), because the HFE protein binds beta2-microglobulin, and in mice beta2-microglobulin gene knockout causes hepatic iron overload. Six unrelated patients with atypical haemochromatosis were studied. Five patients had normal HFE coding sequence and the sixth was heterozygous for C282Y. We show that the spectrum of atypical haemochromatosis includes two distinct familial forms: juvenile haemochromatosis (OMIM *602390) and a novel form of familial iron overload, with apparently autosomal dominant inheritance, predominant Kupffer cell siderosis, and possible minimal dyserythropoiesis on bone marrow examination. Serial serum beta2-microglobulin estimation showed normal levels in all patients. Southern blot analysis showed normal beta2-microglobulin gene structure, excluding major gene rearrangement. Several corrections to the published beta2-microglobulin sequence were identified, but all six patients had normal beta2-microglobulin sequence. Western blot analysis of serum showed beta2-microglobulin protein of normal size. In conclusion, we found no evidence to implicate beta2-microglobulin mutation in atypical haemochromatosis. Two forms of familial iron overload appear unrelated to either HFE or beta2-microglobulin. Linkage studies are required to identify the genes involved, which may encode novel proteins crucial to the regulation of iron metabolism. Identification of these loci will aid the diagnosis, counselling, and treatment of iron overload disorders.

p53 mutations in british patients with hepatocellular carcinoma: clustering in genetic hemochromatosis.

BACKGROUND & AIMS: Environmental factors are important in the etiology of hepatocellular carcinoma (HCC). Aflatoxin B1 causes a specific point mutation in the p53 tumor-suppressor gene in exposed individuals. In Western populations, mutations of this gene seem to be less frequent. We have investigated the role of p53 mutations in tumorigenesis in British patients with HCC. The aim of this study was to determine the frequency and mutational spectrum of the p53 gene in HCCs from British patients. METHODS: DNA from 170 HCCs, of well-defined etiology, in British patients was analyzed by single-stranded conformational polymorphism using the polymerase chain reaction technique. Mutations were then characterized by direct sequencing. RESULTS: Twenty-nine percent of tumors had p53 mutations. Ten of 14 (71%) hemochromatotic cancers had mutations within the p53 gene, and clustering of these mutations at codon 220 (A-G) was found in 5 cases; 3 others had T-A mutations. No clustering was found in HCCs with other etiologies. CONCLUSIONS: p53 mutations are more common than was thought in Northern European HCCs. This is the first demonstration of p53 mutational clustering in HCCs from hemochromatotic subjects.

A 6p22 reference map of leukocyte DNA: exclusion of rearrangement in four cases of atypical haemochromatosis.

We describe a 4 Mb reference map of the haemochromatosis gene region in leukocyte DNA from seven controls and four atypical haemochromatosis patients. Three patients had normal coding sequence for HFE, the candidate gene for genetic haemochromatosis (GH). The fourth patient had classical GH but was heterozygous for Cys282Tyr with otherwise normal coding sequence. The genomic DNA was mapped by pulsed-field gel electrophoresis (PFGE) using five rare-cutting enzymes. Seventeen probes including HFE were positioned on the map. Despite proximity to the highly polymorphic major histocompatibility complex (MHC), no polymorphism was observed in the control group with these telomeric probes. Furthermore, major rearrangement of the HFE region was excluded as a mutation contributing to iron overload in these atypical patients. Maps of cloned DNA are linked through genes and other probes to this reference map of the HFE region in uncloned genomic DNA.

Isolation, characterization, and functional studies of rat liver iron regulatory protein 1.

Ferritin mRNAs are translationally regulated by the binding of either of two cytosolic proteins, iron regulatory protein 1 (IRP1) or IRP2, to the iron responsive element (IRE) located in their 5' untranslated region (UTR). Rat liver IRP1 was purified by anion exchange, gel filtration, and affinity chromatography using a concatemerized version of the IRE. Two bands with M(r) of 95,000 and 100,000 were observed by reducing SDS-PAGE. A single protein was responsible for both bands since: (1) [32P]IRE RNA specifically cross-linked to both components; (2) alkylation with iodoacetamide resulted in formation of a single species with M(r) of 95,000; and (3) they possessed identical peptide patterns after digestion with cyanogen bromide. The N-terminal sequence of rat liver IRP1 was MKNPFAHLAEPLDPAQPGKKFNLNKLEDSRYGRLPFXIRVLLEAAV which is identical to the sequence deduced from the cDNA. Rat liver IRP1 has an amino acid composition similar to that of bovine liver caconitase. Several species of IRP1 were observed by two-dimensional gel electrophoresis with pIs ranging from 7.5 to 8.0. Rat liver IRP1 bound the IRE with high affinity (K(D) = 0.04 nM) and repressed translation of ferritin mRNA in vitro. IRP1 bound 100-fold less well to an IRE variant and failed to significantly repress translation of a ferritin mRNA containing the mutated IRE. We conclude that decreases in the affinity of interaction between IRP1 and the IRE, of a magnitude similar to that observed when the binding protein in converted to c-aconitase, are sufficient to significantly enhance translation of ferritin mRNA in vitro.

Hepatic and extrahepatic hepatitis C virus replication in relation to response to interferon therapy.

Response to a 1-yr course of interferon-alpha 2b was assessed in 18 patients with chronic hepatitis C virus infection in relation to clinical, biochemical and histological parameters and to the presence or absence of hepatitis C virus RNA and the presumed replicative form of the virus (negative-strand hepatitis C virus RNA) in serum, liver and peripheral blood mononuclear cells. The findings were compared with those in seven untreated patients studied over the same period. At the start of the study, positive-strand hepatitis C virus RNA was found in sera of all 25 patients, in livers of 24 and in peripheral-blood mononuclear cells of 19 of 22 tested; negative strand was found in livers of 11 and in peripheral-blood mononuclear cells of 15 of 22. Negative-strand hepatitis C virus RNA was not found in the serum of any patient at any stage. All of the five treated patients considered to show complete response during the study period cleared hepatic hepatitis C virus RNA, and four also became seronegative, but three had evidence suggestive of viral replication in their peripheral-blood mononuclear cells; two of these last patients subsequently relapsed. Loss of hepatic hepatitis C virus RNA was the only significant difference between these five and the seven partial and six nonresponders, but it is uncertain whether the observed changes were due specifically to interferon-induced modulation of virus expression because similar (apparently spontaneous) changes were seen in four of the untreated patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Ferritin gene expression in health and malignancy.

Intracellular iron can be stored in the protein shell of ferritin to protect the cell against the toxic action of the iron. In response to increased cell iron, more ferritin subunits are synthesized using translational and transcriptional mechanisms. Translational control involves a unique stem-loop structure in the 5' untranslated region of the subunit messengers. When iron level is low, a protein binds to this stem-loop structure and prevents translation. When intracellular iron level rises, the repressor protein is discharged and the large population of messengers begins to translate subunits. Similar stem-loop motifs occur in the 3' untranslated region of the transferrin receptor messenger where they regulate breakdown of the receptor mRNA. Finally, the presence of excess iron preferentially stimulates transcription of more ferritin message of one type (L-mRNA) which produces ferritin shells favoring iron storage. In this way, protection of the cell against iron excess is enhanced by coordinate changes in rate of synthesis of ferritin mRNA of the L-type, by release of ferritin mRNA stored in the cytoplasm, and by a reduction in the number of receptors for accepting iron into the cell. The application of these principles with reference to malignant cells is discussed.

Interferon-alpha receptor expression and regulation in chronic hepatitis B virus infection.

Interferon-alpha elicits antiviral and immunoregulatory activities by binding to specific receptors on the cell surface. In this study, binding characteristics of interferon-alpha to peripheral blood mononuclear cells in patients with chronic hepatitis B virus infection were studied using radioiodinated recombinant interferon-alpha 2b to determine interferon-alpha receptor numbers and dissociation constants. A single class of interferon-alpha receptor was demonstrated on peripheral blood mononuclear cells and mononuclear subsets. Peripheral blood mononuclear cells from patients with chronic hepatitis B virus infection (n = 20) and controls (n = 16) expressed a similar number of interferon-alpha receptors (484 +/- 175 vs. 511 +/- 168 sites/cell respectively, p = NS) with a similar dissociation constant (dissociation constant approximately 0.2 to 0.7 nmol/L). Expression of interferon-alpha receptors was similar in monocyte-enriched and lymphocyte-enriched fractions in both groups. Similar changes were observed in patients receiving alpha-interferon therapy. There was no correlation between interferon-alpha receptors expression and serum transaminase, serum HBsAg, serum HBV DNA, liver histological findings or the response to interferon-alpha therapy. After incubation of lymphocytes in vitro with interferon-alpha 2b (10 to 1,000 U/ml), interferon-alpha receptors number dropped by 42% to 80%, but this was associated with an increase in binding affinity (dissociation constant approximately 0.05 to 0.15 nmol/L) in both patients and controls. There was significant delay in the initial phase of receptor recovery in the patients with chronic hepatitis B virus infection compared with normal controls (days 1 and 2, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Differential expression of transferrin receptor in duodenal mucosa in iron overload. Evidence for a site-specific defect in genetic hemochromatosis.

In genetic hemochromatosis, metabolic studies have demonstrated inappropriately increased iron absorption by cells of the duodenal mucosa. It is not clear whether this reflects an intrinsic abnormality of iron homeostasis at this site or is a consequence of a more generalized defect in cellular iron metabolism particularly involving the liver. We have previously used the expression of iron-related proteins as markers of iron homeostasis and have demonstrated normal regulation of the transferrin receptor and ferritin in the liver in this condition. In the present study we used immunohistochemical techniques to study transferrin-receptor expression in the gastrointestinal epithelium in normal subjects and patients with iron overload. In untreated genetic hemochromatosis and normal subjects, villus epithelial cells expressed receptor in the basolateral, subnuclear region. In contrast, in patients with secondary iron overload, receptor staining was absent in villus epithelial cells. The cells in the duodenal crypts showed intense staining for the transferrin receptor in all subjects investigated, a finding consistent with the known behavior of this receptor in proliferating cells. Given that body iron stores in both types of iron overload were comparable, these findings indicating a failure of down-regulation of the villus enterocyte transferrin receptor in genetic hemochromatosis may reflect the presence of a regulatory defect associated with the inability to control iron absorption in this condition.

Transferrin and its receptor: their roles in cell function.

Transferrin, its receptor and the entry of iron into the cell have sprung into prominence because of recent evidence that proliferation of various cell types involves regulation of this sequence of events, as evidenced especially by changes in receptor number. A third component functionally linked to transferrin and its receptor is the intracellular iron-storage protein, ferritin, which ensures against toxic levels of free ferrous iron, which might otherwise cause peroxidative damage to cell membranes and other cell structures (1). In this article, we shall focus on interactions between these three proteins of iron exchange, their roles in homeostasis and especially their role in relation to the liver which is a major organ of iron storage.

The natural history of arthritis in idiopathic haemochromatosis: progression of the clinical and radiological features over ten years.

The natural history of arthritis associated with idiopathic haemochromatosis was studied in 18 male patients over a 10 year period. Chondracalcinosis was present radiologically in at least on joint in seven patients initially and developed later in 13, with the articular cartilages of the wrists and knees affected most frequently. During the period of observation seven patients developed chondrocalcinosis in the wrists, seven in the knees, three in the hips, one in the symphysis pubis and two in the spine. In no patient did the chondrocalcinosis decrease or disappear. The development and progression of chondrocalcinosis was not affected by treatment of iron overload by venesection. The presence of chondrocalcinosis in at least one joint was not related to the age of the patient at the initial or follow-up examination or to the amount of iron removed by venesection (p greater than 0.05, Wilcoxon rank sum test). Arthritis (loss of joint space, cysts, destruction of articular surfaces) usually with associated symptoms and signs affected the hands in eight patients initially and developed later in 13. Arthritis of large joints was uncommon and none of the patients in the present series developed destructive arthritis of hips or knees. This contrast with the finding in a retrospective survey of 93 patients with idiopathic haemochromatosis seen since 1967 at King's College Hospital in which seven patients had destructive arthritis affecting the hips.