Arthropathy in hereditary haemochromatosis segregates with elevated erythrocyte mean corpuscular volume.

Objective: To evaluate the relationship between erythrocyte parameters and the presence or absence of arthritis in HFE C282Y homozygous hereditary haemochromatosis (HH) subjects compared to control groups of non-HH subjects with arthritis.Method: Erythrocyte and arthritis parameters [mean corpuscular volume (MCV) and mean cell haemoglobin (MCH)] were obtained from consecutive HH subjects (n = 119) who were referred for initial evaluation and management. For comparison, MCV and MCH values were collected from randomly selected non-HH subjects with rheumatoid arthritis (n = 100) and osteoarthritis (n = 100), consisting of equal numbers of men and women. Two other comparison groups comprised 16 men and women who were heterozygous for C282Y with arthritis, and 38 non-HH subjects with type 2 polyarticular osteoarthritis (T2POA).Results: MCV values were significantly higher in HH subjects with arthritis (95 ± 0.56 fL) than in HH subjects without arthritis (92.75 ± 0.50 fL, p = 0.037). HH subjects with or without arthritis demonstrated a higher mean MCV than the control groups of non-HH osteoarthritis (90.12 ± 0.46 fL, p < 0.001) and non-HH rheumatoid arthritis (90.94 ± 0.57 fL, p < 0.001). HH subjects with arthritis also demonstrated a higher MCV than heterozygous C282Y subjects with arthritis (93.18 ± 1.55 fL, p = 0.025) and non-HH subjects with a similar pattern of arthritis, notably T2POA (91.13 ± 0.50 fL, p < 0.01). An MCV of ≥ 97.85 fL provided a likelihood ratio of 2.2 for development of arthritis in HH subjects.Conclusion: This study demonstrated a relationship between elevated MCV and arthritis in incident cases of HH.

Patient-focused outcomes following detection in a hospital-based screening programme for C282Y haemochromatosis.

BACKGROUND: Haemochromatosis is a common genetic disease in populations of a northern European origin. However, there is uncertainty as to whether it is a condition that should be screened for. AIMS: To determine the proportion of persons, in a public hospital setting, who were homozygous for the C282Y mutation for hereditary haemochromatosis and the proportion of these persons who would benefit from therapeutic phlebotomy. METHODS: All persons who had blood submitted for pathology testing, had total iron-binding capacity and iron measured and transferrin saturation calculated, and where this result exceeded 40%, genotyping for the C282Y mutation was carried out. RESULTS: Of 18,779 patients screened, 887 (5.4%) were found to have transferrin saturation greater than 40%. Thirty-five of these were homozygous for the C282Y mutation. Fourteen were previously known to be affected and six of these were non-compliant with venesection. Venesection was commenced in 5 of the 21 newly diagnosed subjects. CONCLUSIONS: The proportion of detected subjects who commenced venesection was significant. Results suggest that clinical penetrance is higher in Australia than other countries and that even in the environment of a large tertiary teaching hospital, phenotypic screening identifies cases of hereditary haemochromatosis, which are likely to benefit from treatment.

Use of community genetic screening to prevent HFE-associated hereditary haemochromatosis.

HFE-associated hereditary haemochromatosis is a recessive, iron-overload disorder that affects about one in 200 north Europeans and that can be easily prevented. However, genetic screening for this disease is controversial, and so we assessed whether such screening was suitable for communities. Cheek-brush screening for the Cys282Tyr HFE mutation was offered to individuals in the workplace. Outcomes were assessed by questionnaires before and after testing. 11,307 individuals were screened. We recorded no increase in anxiety in individuals who were homozygous for the Cys282Tyr mutation or non-homozygous. Self-reported tiredness before testing was significantly higher in homozygous participants than in non-homozygous participants (chi2 test, p=0.029). Of the 47 homozygous individuals identified, 46 have taken steps to treat or prevent iron accumulation. Population genetic screening for HFE-associated hereditary haemochromatosis can be practicable and acceptable.

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.

Duodenal expression of iron transport molecules in untreated haemochromatosis subjects.

BACKGROUND AND AIMS: In HFE associated hereditary haemochromatosis, the duodenal enterocyte behaves as if iron deficient and previous reports have shown increased duodenal expression of divalent metal transporter 1 (DMT1) and iron regulated gene 1 (Ireg1) in affected subjects. In those studies, many patients had undergone venesection, which is a potent stimulus of iron absorption. Our study investigated duodenal expression of DMT1 (IRE and non-IRE), Ireg1, hephaestin, and duodenal cytochrome-b (Dyctb) in untreated C282Y homozygous haemochromatosis patients, iron deficient patients, and iron replete subjects. METHODS: Total RNA was extracted from duodenal biopsies and expression of the iron transport genes was assessed by ribonuclease protection assay. RESULTS: Expression of DMT1 (IRE) and Ireg1 was increased 3-5-fold in iron deficient subjects compared with iron replete subjects. Duodenal expression of DMT1 (IRE) and Ireg1 was similar in haemochromatosis patients and iron replete subjects but in haemochromatosis patients with elevated serum ferritin concentrations, both DMT1 (IRE) and Ireg1 expression were inappropriately increased relative to serum ferritin concentration. Hephaestin and Dcytb levels were not upregulated in haemochromatosis. DMT1 (IRE) and Ireg1 levels showed significant inverse correlations with serum ferritin concentration in each group of patients. CONCLUSIONS: These findings are consistent with DMT1 (IRE) and Ireg1 playing primary roles in the adaptive response to iron deficiency. Untreated haemochromatosis patients showed inappropriate increases in DMT1 (IRE) and Ireg1 expression for a given level of serum ferritin concentration, although the actual level of expression of these iron transport genes was not significantly different from that of normal subjects.

Effects of alcohol consumption on indices of iron stores and of iron stores on alcohol intake markers.

BACKGROUND: Alcohol increases body iron stores. Alcohol and iron may increase oxidative stress and the risk of alcohol-related liver disease. The relationship between low or "safe" levels of alcohol use and indices of body iron stores, and the factors that affect the alcohol-iron relationship, have not been fully characterized. Other aspects of the biological response to alcohol use have been reported to depend on iron status. METHODS: We have measured serum iron, transferrin, and ferritin as indices of iron stores in 3375 adult twin subjects recruited through the Australian Twin Registry. Information on alcohol use and dependence and smoking was obtained from questionnaires and interviews. RESULTS: Serum iron and ferritin increased progressively across classes of alcohol intake. The effects of beer consumption were greater than those of wine or spirits. Ferritin concentration was significantly higher in subjects who had ever been alcohol dependent. There was no evidence of interactions between HFE genotype or body mass index and alcohol. Alcohol intake-adjusted carbohydrate-deficient transferrin was increased in women in the lowest quartile of ferritin results, whereas adjusted gamma-glutamyltransferase, aspartate aminotransferase, and alanine aminotransferase values were increased in subjects with high ferritin. CONCLUSIONS: Alcohol intake at low level increases ferritin and, by inference, body iron stores. This may be either beneficial or harmful, depending on circumstances. The response of biological markers of alcohol intake can be affected by body iron stores; this has implications for test sensitivity and specificity and for variation in biological responses to alcohol use.

Population screening for HFE-associated haemochromatosis: should we have to pay for our genes?

Haemochromatosis associated with mutations in the HFE gene is the most common inherited disorder in Caucasian populations. Early diagnosis and treatment allows for normal life expectancy, whereas there is considerable morbidity and early mortality in those patients diagnosed late or untreated. Unfortunately, the development of symptoms and signs in haemochromatosis is usually associated with significant iron overload. For this reason, many clinicians and geneticists have advocated population screening. The recent identification of the HFE gene and the availability of a simple DNA-based diagnostic test have led to international debate as to the most cost-effective means of population screening for HFE-associated haemochromatosis. The present paper summarizes the evidence in favour of population screening and analyses the relative advantages of genotypic (DNA test) versus phenotypic (transferrin saturation) testing.

Lipid peroxidation in hepatic steatosis in humans is associated with hepatic fibrosis and occurs predominately in acinar zone 3.

BACKGROUND AND AIMS: Hepatic steatosis has been shown to be associated with lipid peroxidation and hepatic fibrosis in a variety of liver diseases including non-alcoholic fatty liver disease. However, the lobular distribution of lipid peroxidation associated with hepatic steatosis, and the influence of hepatic iron stores on this are unknown. The aim of this study was to assess the distribution of lipid peroxidation in association with these factors, and the relationship of this to the fibrogenic cascade. METHODS: Liver biopsies from 39 patients with varying degrees of hepatic steatosis were assessed for evidence of lipid peroxidation (malondialdehyde adducts), hepatic iron, inflammation, fibrosis, hepatic stellate cell activation (alpha-smooth muscle actin and TGF-beta expression) and collagen type I synthesis (procollagen alpha1 (I) mRNA). RESULTS: Lipid peroxidation occurred in and adjacent to fat-laden hepatocytes and was maximal in acinar zone 3. Fibrosis was associated with steatosis (P < 0.04), lipid peroxidation (P < 0.05) and hepatic iron stores (P < 0.02). Multivariate logistic regression analysis confirmed the association between steatosis and lipid peroxidation within zone 3 hepatocytes (P < 0.05), while for hepatic iron, lipid peroxidation was seen within sinusoidal cells (P < 0.05), particularly in zone 1 (P < 0.02). Steatosis was also associated with acinar inflammation (P < 0.005). alpha-Smooth muscle actin expression was present in association with both lipid peroxidation and fibrosis. Although the effects of steatosis and iron on lipid peroxidation and fibrosis were additive, there was no evidence of a specific synergistic interaction between them. CONCLUSIONS: These observations support a model where steatosis exerts an effect on fibrosis through lipid peroxidation, particularly in zone 3 hepatocytes.

Role of myofibroblasts in tumour encapsulation of hepatocellular carcinoma in haemochromatosis.

BACKGROUND/AIMS: Hepatocellular carcinoma is a common malignancy and a major complication of untreated haemochromatosis. Encapsulation of liver tumours has been associated with a better prognosis and longer disease-free periods following resection. This study investigated the source of the tumour capsule in patients with haemochromatosis and coexisting hepatocellular carcinoma and examined potential factors influencing development. METHODS: Five haemochromatosis patients with encapsulated hepatocellular carcinoma were studied. Myofibroblasts were identified using combined immunohistochemistry and in situ hybridisation for alpha-smooth muscle actin and procollagen alpha1(I) mRNA, respectively. Immunohistochemistry was also performed for transforming growth factor (TGF)-beta1, platelet-derived growth factor (PDGF)-beta receptor and malondialdehyde. RESULTS: Procollagen alpha1(I) mRNA co-localised to alpha-smooth muscle actin positive myofibroblasts. The number of myofibroblasts was maximal within the capsule and decreased away from the tumour. TGF-beta1 protein was expressed in iron-loaded cells in non-tumour liver at the interface of tumour capsule. PDGF-beta receptor expression was observed in mesenchymal cells in the tumour capsule and in portal tracts. Malondialdehyde adducts were observed in the tumour, non-tumour tissue and in the capsule. CONCLUSIONS: This study provides evidence that myofibroblasts are the cell type responsible for collagen production within the tumour capsule surrounding hepatocellular carcinoma in haemochromatosis. The production of TGF-beta1 by iron-loaded hepatic cells at the tumour capsule interface may perpetuate the myofibroblastic phenotype, resulting in the formation of the tumour capsule.

Effect of hemochromatosis genotype and lifestyle factors on iron and red cell indices in a community population.

BACKGROUND: Heterozygotes for the C282Y mutation of the HFE gene may have altered hematology indices and higher iron stores than wild-type subjects. METHODS: We performed a cross-sectional analysis of 1488 females and 1522 males 20-79 years of age drawn from the Busselton (Australia) population study to assess the effects of HFE genotype, age, gender, and lifestyle on serum iron and hematology indices. RESULTS: Male C282Y heterozygotes had increased transferrin saturation compared with the wild-type genotype. Neither male nor female heterozygotes had significantly increased ferritin values compared with the wild-type genotype. Younger (20-29 years) wild-type males, but not heterozygous males, had significantly lower ferritin values than wild-type males in the older age groups. Compound heterozygous subjects had increased means for serum iron, transferrin saturation, corpuscular volume, and corpuscular hemoglobin compared with the wild-type genotype, and the males also had increased ferritin values (medians 323 vs 177 microg/L; P = 0.003). In both male and female wild-type subjects, an increased body mass index was associated with decreased serum iron and transferrin saturation and increased ferritin values. There was a significant increase in ferritin concentrations in both genders with increasing frequency of red meat consumption above a baseline of 1-2 times per week and alcohol intakes >10 g/day. CONCLUSIONS: Male C282Y heterozygotes had significantly increased transferrin saturation values. Compound heterozygous (C282Y/H63D) subjects formed a separate category of C282Y heterozygotes in whom both iron and red cell indices were significantly increased compared with the wild-type genotype.

What's new in hemochromatosis.

Hereditary hemochromatosis (HHC) is an inherited disorder of iron metabolism affecting approximately 1 in 200-300 individuals of Northern European descent. Over time, the continued deposition of iron in parenchymal cells of many organs can eventually lead to diabetes mellitus, cardiomyopathy, and hepatic cirrhosis, the last of which is frequently followed by hepatocellular carcinoma. Although the complications of HHC can be devastating, its clinical management is simple and effective if the disease is identified early in its progression. The recent elucidation of the HFE gene has provided insight into the pathogenesis of HHC and provided a means for the early identification of individuals in whom HHC may develop. Two mutations have been implicated in HHC: C282Y and H63D. The former occurs in a homozygous state seen in 75-100% of patients with HHC. The high correlation of HFE to HHC has caused it to be considered as a candidate gene for population-based genetic testing for diagnosis and detection of predisposition to HHC. In addition, mechanisms of iron transport and metabolism are unfolding and are providing clues to the enigma of iron homeostasis and the pathophysiology of iron overload.

Detection of male DNA in the liver of female patients with primary biliary cirrhosis.

BACKGROUND/AIMS: Primary biliary cirrhosis is a chronic cholestatic liver disease characterized by progressive inflammatory destruction of bile ducts, with eventual hepatic fibrosis and cirrhosis. Since primary biliary cirrhosis affects predominantly middle-aged women and has pathological similarities to hepatic graft-versus-host-disease, we investigated whether fetal cell microchimerism might be involved in the development of this disease. METHODS: The presence of Y-chromosome-specific sequences was analyzed by polymerase chain reaction using peripheral blood mononuclear cells from women with primary biliary cirrhosis (n=18) and healthy (control) women (n=18), and by in situ hybridization of liver biopsy sections from women with primary biliary cirrhosis (n=19) and women with chronic hepatitis C or alcoholic liver disease (n=20). RESULTS: Male cells were detected in liver biopsy specimens of 8 of 19 patients (42%) with primary biliary cirrhosis. Y-chromosome-containing cells were not seen in any of the liver biopsy specimens from women with chronic hepatitis C or alcoholic liver disease. Male cells were detected in peripheral blood mononuclear cells from one healthy control at a level of 1 male cell per 10(6) female cells, but were not detected in peripheral blood mononuclear cells of women with primary biliary cirrhosis. CONCLUSIONS: The presence of male cells in the liver of women with primary biliary cirrhosis raises the possibility that fetal cell microchimerism may be involved in the pathogenesis of this chronic liver disease.

Haemochromatosis in the new millennium.

Hereditary haemochromatosis (HHC) is a common inherited disorder of iron metabolism characterised by progressive iron loading of parenchymal cells of the liver, pancreas, heart and other organs ultimately leading to cirrhosis and organ failure. Despite HLA studies which localised the defective gene to the short arm of chromosome 6, the haemochromatosis gene remained elusive until 1996, when the gene was identified by a massive positional cloning effort. The haemochromatosis gene (HFE) encodes a novel nonclassical MHC class-1-like molecule. Two missense mutations have been identified in patients with HHC, a G to A at nucleotide 845, resulting in a substitution of tyrosine for cysteine at amino acid 282 (referred to as the C282Y mutation) and a C to G at nucleotide 187, resulting in a substitution of aspartate for histidine at amino acid 63 (H63D). An average of 85-90% of patients with typical clinical features of HHC are homozygous for the C282Y mutation. H63D is not associated with the same degree of iron loading as C282Y. Clinical expression is variable depending on environmental (dietary) iron, physiological and pathological blood loss and as yet unidentified modifying genetic factors. One recent Australian study indicates that only about 50% of homozygous subjects are fully expressing and symptomatic and that about 30% show no clinical or biochemical expression. Genetic tests for identifying mutations in the HFE gene provide precise means for diagnosis, family testing and population screening and have led to re-evaluation of the indications for liver biopsy in this disease. At the present time, however, the most practical and cost-effective method of screening is for phenotypic expression by transferrin saturation or unsaturated iron binding capacity measurement. In the future, population screening by genotype should be feasible once the relevant technical, legal and ethical issues are resolved.

Effects of HFE C282Y and H63D polymorphisms and polygenic background on iron stores in a large community sample of twins.

The aim of this study was to assess and to compare the role of HFE polymorphisms and other genetic factors in variation in iron stores. Blood samples were obtained from 3,375 adult male and female twins (age range 29-82 years) recruited from the Australian Twin Registry. There were 1,233 complete pairs (562 monozygotic and 571 dizygotic twins). Serum iron, transferrin, transferrin saturation with iron, and ferritin were measured, and the HFE C282Y and H63D genotypes were determined. The frequency of the C282Y allele was.072, and that of the H63D allele was.141. Significant sources of variation in the indices of iron status included age, sex, age-sex interaction, body-mass index, and both the C282Y and H63D genotypes. The iron, transferrin, and saturation values of CC and CY subjects differed significantly, but the ferritin values did not. After correction for age and body-mass index, 23% and 31% of the variance in iron, 66% and 49% of the variance in transferrin, 33% and 47% of the variance in transferrin saturation, and 47% and 47% of the variance in ferritin could be explained by additive genetic factors, for men and women, respectively. HFE C282Y and H63D variation accounted for <5% of the corrected phenotypic variance, except for saturation (12% in women and 5% in men). We conclude that HFE CY and HD heterozygotes differ in iron status from the CC and HH homozygotes and that serum transferrin saturation is more affected than is serum ferritin. There are highly significant effects of other as-yet-unidentified genes on iron stores, in addition to HFE genotype.

p73 is up-regulated in a subset of hepatocellular carcinomas.

Loss of heterozygosity (LOH) at 1p36 occurs in a number of solid tumors including hepatocellular carcinoma (HCC). Recently, a novel gene, p73, has been identified at 1p36.33. p73 is structurally and functionally related to p53 located at 17p13.1, which is a target for inactivation in HCCs. p73 produces at least two splicing variants, p73alpha and beta, and a polymorphism in exon 2 results in two alleles, GC or AT. Initially, only the AT allele and p73alpha transcripts were identified in malignant cell lines, suggesting a role for these in the malignant phenotype. The aims of this study were to determine the extent of LOH at 1p36 and 17p13.1 in HCCs from Australia and South Africa, and to identify patterns of p73 mRNA and p73 and p53 protein expression. LOH at 1p36 was found in 8 of 25 Australian and 6 of 10 South African cases. p73 mRNA expression occurred in 8 HCCs, but not in nonmalignant liver tissue. Two of these 8 HCCs had LOH of 1p36. Both alpha and beta transcripts were observed in GC/GC homozygotes and GC/AT heterozygotes. No p73 protein expression was observed by immunohistochemistry in nonmalignant liver tissue or in HCC. p53 inactivation appeared to be associated with up-regulation of p73 expression, suggesting a compensatory role for p73 in this situation. The LOH at 1p36 implies a liver-specific tumor suppressor gene is in this region. However, the up-regulation of p73 mRNA suggests p73 is not the target of this loss.

Automated measurement of unsaturated iron binding capacity is an effective screening strategy for C282Y homozygous haemochromatosis.

BACKGROUND: C282Y hereditary haemochromatosis is an appropriate condition for population screening. Transferrin saturation, the best screening test to date, is relatively expensive, labour intensive, and cannot be automated. Unsaturated iron binding capacity is a surrogate marker of transferrin saturation and its measurement can be automated. AIMS: To evaluate a screening strategy for C282Y hereditary haemochromatosis in a tertiary hospital environment based on unsaturated iron binding capacity as the initial screening test. METHODS: Measurement of unsaturated iron binding capacity was adapted to the main laboratory analyser. An unsaturated iron binding capacity of less than 30 micromol/l was identified as an appropriate decision point and 5182 consecutive subjects were screened over 28 consecutive days. RESULTS: Of those screened, 697 had an unsaturated iron binding capacity less than 30 micromol/l. Of these, transferrin saturation was greater than 40% in 294. A total of 227 were able to be genotyped for the C282Y mutation. Nine subjects homozygous for C282Y were identified. Based on full cost recovery, affected persons were identified at a cost of Aus$2268.77 per case (approximately US$1496). CONCLUSION: Automated measurement of unsaturated iron binding capacity enables a cost effective, large scale population screening programme for C282Y hereditary haemochromatosis to be developed.