Porphyria cutanea tarda (PCT) is associated with cutaneous accumulation of porphyrins. This accumulation results from a deficiency of uroporphyrinogen decarboxylase occurring only in the liver. The classical presentation is blistering on sun-exposed areas.

A 59-year-old woman presented at the dermatology consultation for sclerotic lesions that had been present for one year. The remainder of the clinical examination and further investigations did not indicate systemic scleroderma. The sun-exposed nature of the lesions led us to perform an assay of urinary porphyrin, which was found to be elevated. Uroporphyrinogen decarboxylase levels were normal, confirming the diagnosis of type 1 PCT. Screening for a hepatic etiology revealed a heterozygous mutation H63D/C282Y of the hemochromatosis gene responsible for this clinical picture. The patient underwent regular bleeding, which led to complete disappearance of cutaneous sclerosis.

Sclerodermatous lesions are an unusual presentation of PCT and cause delays in diagnosis. The accumulation of uroporphyrins in the dermis stimulates fibroblasts, which then synthesize collagen, resulting in cutaneous sclerosis.

The hemochromatosis (HFE) gene encodes the HFE protein that regulates iron absorption. HFE mutations lead to the hemochromatosis disease of excessive iron absorption. HFE mutations may also influence the sustained virologic response (SVR, long-term virus suppression) in chronic hepatitis C patients treated with interferon-based antiviral therapy. We performed a meta-analysis of all English and Chinese language studies of HFE mutations and SVR in interferon-treated chronic hepatitis C patients indexed in the Medline, PubMed, Embase, and China National Knowledge Infrastructure databases to November 2011. Seven studies involving 605 patients with HFE mutations (homozygous or heterozygous mutation of C282Y, H63D or S65C) and 1279 with wild-type HFE (no mutation of C282Y, H63D or S65C for both alleles) were analyzed. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated with the fixed- or random-effect models. HFE mutations were associated with significantly higher SVR rate (vs. wild-type: OR = 1.56, 95% CI: 1.23-1.97, P < 0.001), indicating that mutation carriers were likely to achieve SVR in response to interferon-based antiviral therapy. Stratification analysis by HFE mutation type revealed that the H63D mutation was associated with a significantly higher SVR rate (OR = 1.60, 95% CI: 1.09-2.34, P = 0.020), while the C282Y mutation was not (OR = 1.19, 95% CI: 0.71-1.98, P = 0.510). Our meta-analysis results indicate that the H63D mutation in HFE is associated with a higher SVR rate in chronic hepatitis C patients treated with interferon-based antiviral therapy.

Contemporary sequencing studies often ignore the diploid nature of the human genome because they do not routinely separate or 'phase' maternally and paternally derived sequence information. However, many findings - both from recent studies and in the more established medical genetics literature - indicate that relationships between human DNA sequence and phenotype, including disease, can be more fully understood with phase information. Thus, the existing technological impediments to obtaining phase information must be overcome if human genomics is to reach its full potential.

Heterozygotes for the p.Cys282Tyr (C282Y) mutation of the HFE gene do not usually express a hemochromatosis phenotype. Apart from the compound heterozygous state for C282Y and the widespread p.His63Asp (H63D) variant allele, other rare HFE mutations can be found in trans on chromosome 6.

We performed molecular investigation of the genes implicated in hereditary hemochromatosis in six patients who presented with iron overload but were simple heterozygotes for the HFE C282Y mutation at first genetic testing. Functional impairment of new variants was deduced from computational methods including molecular modeling studies.

We identified four rare HFE mutant alleles, three of which have not been previously described. One mutation is a 13-nucleotide deletion in exon 6 (c.1022_1034del13, p.His341_Ala345 > LeufsX119), which is predicted to lead to an elongated and unstable protein. The second one is a substitution of the last nucleotide of exon 2 (c.340G > A, p.Glu114Lys) which modifies the relative solvent accessibility in a loop interface. The third mutation, p.Arg67Cys, also lies in exon 2 and introduces a destabilization of the secondary structure within a loop of the α1 domain. We also found the previously reported c.548T > C (p.Leu183Pro) missense mutation in exon 3. No other known iron genes were mutated. We present an algorithm at the clinical and genetic levels for identifying patients deserving further investigation. Conclusions Our results suggest that additional mutations in HFE may have a clinical impact in C282Y carriers. In conjunction with results from previously described cases we conclude that an elevated transferrin saturation level and elevated hepatic iron index should indicate the utility of searching for further HFE mutations in C282Y heterozygotes prior to other iron gene studies.

The incomplete phenotypic penetrance of high iron Fe genotypes in relation to hemochromatosis poses a practical problem in the interpretation of the genotyping results by clinicians. We carried out meta-analyses of the associations between hemochromatosis genotypes C282Y/C282Y, C282Y/H63D, C282Y/wild-type, H63D/H63D, H63D/wild-type, versus wild-type/wild-type and iron overload, both provisional (elevated serum iron markers) and documented (elevated serum iron markers associated with evidence of iron excess based on liver biopsy and/or quantitative phlebotomy).

After reviewing 3572 article titles and evaluating 92 articles in detail, odds ratios were pooled from 43 study populations (9986 cases and 25,492 controls) using a random-effects model.

Homozygosity for either variant or compound heterozygosity was associated with both provisional and documented iron overload. Single heterozygosity conferred no risk for elevated hepatic iron index and/or mobilizable iron by quantitative phlebotomy. In patients with clinical hereditary hemochromatosis, no evidence of provisional and documented iron overload with transferrin saturation (TS) values greater than 55% was evidenced for C282Y and H63D single heterozygotes whereas documented iron overload including TS of 45% to 50% was weakly associated with C282Y/wild-type genotype; H63D/H63D genotype was not associated with documented iron overload in patients with TS values of 45% to 50%.

The results, mainly from case-control studies, cannot necessarily be extrapolated to the general population.

Iron overload in humans is associated with a variety of genetic and acquired conditions. Of these, HFE hemochromatosis (HFE-HC) is by far the most frequent and most well-defined inherited cause when considering epidemiological aspects and risks for iron-related morbidity and mortality. The majority of patients with HFE-HC are homozygotes for the C282Y polymorphism [1]. Without therapeutic intervention, there is a risk that iron overload will occur, with the potential for tissue damage and disease. While a specific genetic test now allows for the diagnosis of HFE-HC, the uncertainty in defining cases and disease burden, as well as the low phenotypic penetrance of C282Y homozygosity poses a number of clinical problems in the management of patients with HC. This Clinical Practice Guideline will therefore, focus on HFE-HC, while rarer forms of genetic iron overload recently attributed to pathogenic mutations of transferrin receptor 2, (TFR2), hepcidin (HAMP), hemojuvelin (HJV), or to a sub-type of ferroportin (FPN) mutations, on which limited and sparse clinical and epidemiologic data are available, will not be discussed. We have developed recommendations for the screening, diagnosis, and management of HFE-HC.

Distinct from hereditary haemochromatosis, hepatic iron overload is a common finding in several chronic liver diseases. Many studies have investigated the prevalence, distribution and possible contributory role of excess hepatic iron in non-haemochromatotic chronic liver diseases. Indeed, some authors have proposed iron removal in liver diseases other than hereditary haemochromatosis. However, the pathogenesis of secondary iron overload remains unclear. The High Fe (HFE) gene has been implicated, but the reported data are controversial. In this article, we summarise current concepts regarding the cellular role of the HFE protein in iron homeostasis. We review the current status of the literature regarding the prevalence, hepatic distribution and possible therapeutic implications of iron overload in chronic hepatitis C, hepatitis B, alcoholic and non-alcoholic fatty liver diseases and porphyria cutanea tarda. We discuss the evidence regarding the role of HFE gene mutations in these liver diseases. Finally, we summarize the common and specific features of iron overload in liver diseases other than haemochromatosis.

Hereditary iron overload is mainly due to mutations of the HFE gene, implicated in most cases of hereditary hemochromatosis. Non-HFE-related hereditary iron overload is rare. It includes hereditary hemochromatosis related to mutations of other genes, ferroportin disease (also known as hemochromatosis type 4), and entities associated with specific clinical manifestations. Four genes have been implicated in hereditary hemochromatosis: HFE and TFR2 (which codes for the second transferrin receptor), both involved in adult forms of hereditary hemochromatosis, and HAMP and HJV, which code for hepcidin and hemojuvelin, respectively, and are responsible for juvenile hemochromatosis. All types of hereditary hemochromatosis share common clinical and biological characteristics, including an autosomal recessive inheritance pattern, elevation of transferrin saturation as the initial manifestation, hepatic parenchymal iron overload, and sensitivity to therapeutic phlebotomy. They are due to hyperabsorption of dietary iron and are linked to a deficit of hepcidin, the principal iron regulator in the body. Ferroportin disease is a special dominantly inherited clinical form of iron overload due to mutations of the SLC40A1 gene. Its expression differs significantly from that of hereditary hemochromatosis, and its mechanism is related to impairment of iron release from reticuloendothelial cells. Other causes of non-HFE-related hereditary iron overload are usually associated with recognizable clinical manifestations, such as anemia or neurological disorders.

The present study examines the frequency of the two main HFE mutations (C282Y and H63D) in a randomly selected population of 346 individuals including 201 DNA samples from women with cervical neoplasia (including high-grade squamous intraepithelial lesions and invasive squamous cell carcinoma) and a control population of 146 women from the same geographical area. We found a significantly lower risk of development of cervical neoplasia in H63D carriers (OR = 0.56; 95% CI 0.35-0.92; p = 0.01). Multivariate logistic regression analysis confirms this observation (OR = 0.55; 95% CI 0.35-0.88, p = 0.01). Regarding the C282Y mutation no association was found (OR = 1.32; 95% CI 0.53-3.33; p = 0.52). In addition, a significant difference between H63D carrier and non-carrier women on the time-to-onset of cervical lesions was observed (log-rank test: p = 0.0012). These results indicate that HFE could be considered a candidate modifier gene of viral-related neoplasia such as cervical carcinoma possibly by a dual role on iron metabolism and immunological system.

The frequencies of C282Y and H63D mutations of the HFE gene vary between different populations. A previous study showed an unexpectedly high H63D frequency in Chuetas (a population of Jewish descent). The present study addressed the question of the distribution of these mutations in Jewish populations from different origins and studied the possible causes of the high H63D frequency in Chuetas. Moreover, to improve the understanding of the controversial relationship between H63D homozygosity and iron overload, a group of patients with altered iron metabolism were studied. The high frequency of H63D mutation in Chuetas is not due to a high prevalence of this mutation in Sephardic Jews. Jewish populations have low C282Y and moderate H63D frequencies, suggesting slight gene flow from their surrounding populations. In accordance with historical and demographic data, genetic drift is the most probable cause for the singular H63D frequency in Chuetas. Clinically, this study of H63D homozygotes supports the conclusion that this genotype must be taken into account, because it confers an increased risk of iron overload and therefore genetic susceptibility to developing hereditary hemochromatosis or to aggravating other diseases.

The number of new genes implicated in iron metabolism has dramatically increased during the last few years. Alterations of these genes may cause hyperferritinemia associated or not with iron overload. Correct assignment of the specific disorder of iron metabolism requires the identification of the causative gene mutation. Here, we propose a rational strategy that allows targeting the gene(s) to be screened for a diagnostic purpose. This strategy relies on the age of onset of the disease, the type of clinical symptoms, the biochemical profile (elevated or normal serum transferrin saturation (TfSat)), the presence or not of visceral iron excess, and the mode of inheritance (autosomal recessive or dominant). Then, two main entities can be differentiated: genetic (adult or juvenile) hemochromatosis characterized by elevated TfSat, and hereditary hyperferritinemias where TfSat is normal (or only slightly modified). Adult genetic hemochromatosis (GH) is related mainly to mutations of the HFE gene, and exceptionally to mutations of the TFR2 gene. Juvenile GH is a rare condition related principally to mutations of the HJV gene coding for hemojuvelin, and rarely to mutations of the HAMP gene coding for hepcidin. Hereditary hyperferritinemias are linked to mutations of three genes: the L-ferritin gene responsible for the hereditary hyperferritinemia cataract syndrome (without iron overload), the ferroportin gene leading to a dominant form of iron overload, and the ceruloplasmin (CP) gene corresponding to an iron overload syndrome with neurological symptoms. The proposed strategic approach may change with the identification of other genes involved in iron metabolism.

Iron deposits in the liver and abnormalities in serum iron biochemistry are frequently observed in patients with chronic liver diseases, but data for patients with hepatitis B virus (HBV) infection are scarce. Moreover, the role of HFE mutations in iron deposits in this condition remains unknown. The aim of the present study was to determine the prevalence of serum iron biochemical abnormalities and iron deposits in the liver of chronic HBV patients, and to evaluate the consequences for the activity and severity of liver disease. Additionally, we studied the role of HFE gene mutations in iron deposits.

Eighty-one male non-cirrhotic HBV patients were studied. Serum iron biochemistry, liver enzymes and C282Y/H63D mutations were investigated. Liver biopsies were scored for necroinflammatory activity (histological activity index [HAI]), fibrosis and iron deposits.

Elevated transferrin saturation (TS) was found in 27.1% of patients and liver iron deposits in 48.7%; these deposits were mild in 68.4% and moderate in 31.6%. Patients with liver iron deposits exhibited significantly higher scores for HAI and fibrosis than those without iron deposits. HFE mutations were identified in 23.4% of patients (14 H63D heterozygotes, four H63D homozygotes, one compound mutation). No difference in the prevalence of C282Y and H63D mutations was observed between HBV patients (1.2% and 23.4%, respectively) and the general population (4.1% and 27.8%, respectively). No association was detected between HFE mutations and elevated TS or liver iron deposits.

Elevated TS and liver iron deposits were frequent in non-cirrhotic HBV patients. Iron deposits were mainly mild and associated with higher activity and severity of liver disease, but not with HFE mutations.

To determine the relationship between hepatic stellate cell (HSC) populations and severity of liver disease and liver iron deposits in patients with chronic hepatitis C virus (HCV). We also studied the relationship between iron cellular distribution and HSC population and the role of HFE mutations in the determination of iron deposits.

Forty-nine chronic HCV patients with varying degrees of liver damage and liver iron deposits were studied. A liver biopsy was scored for histology activity index (HAI), fibrosis and iron deposits. The number of HSC in the liver was evaluated by an immunohistochemical double-staining method to identify glial fibrillary acid protein (GFAP) and smooth muscle alpha-actin (alpha-SMA).

The HSC population was significantly higher in HCV patients than in normal controls and was predominant in zones 1 and 3. Liver iron deposits were observed in 49% of patients and were mild/moderate in most cases. We found a significantly higher number of GFAP and alpha-SMA positive cells in patients with liver iron deposits compared with those without iron deposits, and a positive correlation between liver iron scores and number (%) of GFAP and alpha-SMA positive cells. We observed a significantly higher number of GFAP and alpha-SMA positive cells in moderate/severe hepatitis than in minimal/mild hepatitis, and a positive correlation between GFAP and alpha-SMA positive cells and HAI and fibrosis scores.

Liver iron deposits in chronic HCV are common and are associated with activation of HSC. Thus, even mild iron deposits might stimulate HSC and contribute to liver damage.

The HFE gene contains two main missense mutations: C282Y and H63D. Individuals with these mutations carry a risk of developing hereditary haemochromatosis (HH). The common form of this disease is due to homozygosity for the C282Y mutation. Population studies have shown the variation of the prevalence of these mutations in different countries and ethnic groups. The purposes of this current study were to determine the prevalence of the C282Y and H63D mutations in the Balearic Islands and the genotypic characterization of patients diagnosed with HH, as well as those with iron overload and liver diseases. A total of 1330 Balearic chromosomes were analyzed. The results showed that the populations of the Balearic Islands were not homogeneous. No C282Y carriers were observed in a group of descendants of Majorcan Jews (Chuetas) and the frequency was very low in Minorca (1.2%) in comparison with the other islands of Majorca (4.7%) and Ibiza (6.5%). The carrier frequency of the H63D mutation was similar in the three islands and very high (43.1%) in the descendants of Majorcan Jews. The study of patients was carried out in 129 individuals. The homozygous C282Y genotype was the principal one involved in hereditary haemochromatosis (90%), whereas the other HH patients were C282Y/H63D compound heterozygous and H63D homozygous.

More than 80% of the patients affected by hereditary hemochromatosis, a common inherited iron disorder, are homozygotes for the 845G --> A (C282Y) mutation of the HFE gene. However, depending on the population, 10-20% of hereditary hemochromatosis can be linked either to other HFE genotypes, particularly the compound heterozygous state for C282Y and the 187 C --> G (H63D) mutation, or to mutations of new other genes. Recently, Camaschella et al. (Nat. Genet. 25, 14-15, 2000) identified a stop mutation (exon 6 nt 750 C --> T, Y250X) on the transferrin receptor-2 (TFR2) gene in two unrelated Sicilian families with hereditary hemochromatosis. The TFR2 gene is a transferrin receptor gene homologue that seems to be involved in iron metabolism. Moreover, one of the patients described by Camaschella et al. was a H63D homozygote. H63D homozygosity can be associated with various phenotypes from asymptomatic subjects to patients with a typical form of hereditary hemochromatosis. Thus, the Y250X mutation could be the molecular defect responsible for hereditary hemochromatosis in subjects with atypical HFE genotypes. We have searched for the Y250X mutation in 63 unrelated French subjects. Forty-three had a diagnosis of hereditary hemochromatosis based on classical criteria. This group included 12 H63D homozygotes, 3 C282Y heterozygotes, and 3 patients with none of the two most prevalent HFE mutants. These 18 patients had no other HFE sequence change and were subsequently subjected to DNA sequencing of the 15 last exons and flanking sequences of the TFR2 gene. The 25 remaining hereditary hemochromatosis patients who were tested for the Y250X mutant were compound heterozygotes for the C282Y and H63D mutations. Finally, we also tested for this TFR2 mutation 20 H63D homozygotes with milder manifestations of iron overload and no acquired cause of iron overload. None of the 63 tested subjects had the Y250X mutation. Concurrently, none of the 18 hereditary hemochromatosis patients who had their TFR2 gene sequenced had any deleterious mutation. Thus, TFR2 mutations are not responsible for hemochromatosis in non-C282Y homozygous patients of our area.

First considered as a polymorphism of the HFE gene, the H63D mutation is now widely recognised as a haemochromatosis associated allele. But few H63D homozygotes with clinical manifestations of hereditary haemochromatosis (HH) have been reported. Concurrently, an increasing number of genes have been shown to interact with HFE in iron metabolism.

To describe the clinical expression of iron overload (IO) associated with H63D homozygosity, and search for potential genetic modifiers (within the HFE or other genes) that could explain the variability of the phenotypes.

We retrospectively analysed the clinical phenotype of 56 H63D homozygotes referred for a personal or family history of IO. For each subject we examined intragenic HFE haplotypes and transferrin receptor (TfR) gene polymorphisms and searched for the Y250X mutation on the TFR2 gene. Additionally, we sequenced the HFE gene of H63D homozygotes with HH.

Fifty of 56 subjects had biological and/or clinical abnormalities of iron metabolism. Up to two thirds of patients (n=34) had no acquired cause of IO. Among these, 12 had a phenotypic diagnosis of HH. In the iron loaded group there was a strong prevalence of male patients. No correlation was found between the potential genetic modifiers and phenotypes. No additional mutation of HFE was identified.

The variable phenotypes associated with H63D homozygosity do not appear to be linked to other HFE mutations, to the TFR2 Y250X mutation, or to HFE or TfR gene intragenic polymorphisms. The exact role of H63D homozygosity in IO and HH needs to be further investigated in unselected populations.

We evaluated the iron status and searched for mutations C282Y and H63D in the hereditary hemochromatosis gene (HFE) in 34 pyruvate kinase (PK)-deficient patients from 29 unrelated families. Nine had received multiple transfusions. Thirteen of the 25 nontransfused patients displayed increased serum ferritin concentration, in the absence of conditions known to raise this parameter. HFE genotype was abnormal in 9 of 34 patients. The allele frequency was 1.8% for mutation 845G--> (C282Y) and 16.1% for mutation 187C-->G (H63D). Nontransfused subjects with abnormal genotype had serum ferritin and transferrin saturation values significantly higher than those with wild-type genotype. Of the 12 adult nontransfused patients with increased iron status parameters, 1 was C282Y homozygous, 1 compound heterozygous for C282Y and H63D, 3 H63D heterozygous, and 7 had a normal HFE genotype. Serum ferritin and transferrin saturation were not related to hemoglobin, reticulocytes, and bilirubin concentration. At multivariate analysis serum ferritin was independently associated with age and gender, but not with splenectomy and HFE genotypes. The retrospective evaluation of the iron status profile of 10 patients (3 with abnormal and 7 with wild-type HFE genotype) with at least 10 years follow-up showed that overt iron accumulation requiring iron chelation had occurred only in the 3 patients (2 of whom were splenectomized) with the mutated HFE gene.

The actual prevalence of the main hemochromatosis (HFE) mutations in the Italian adult population and their phenotypic expression have not yet been established. This information is key to advocate a mass-screening program.

Two thousand one hundred adults were tested for the C282Y/H63D HFE gene mutations by an automated genotyping assay as well as transferrin saturation (TS) and serum ferritin levels.

No homozygotes for the C282Y mutation were found. Heterozygosity for the C282Y mutation was 3.1%, while heterozygosity and homozygosity for the H63D mutation were 21.5% and 2.5%, respectively. TS was significantly higher in C282Y heterozygotes and H63D homozygotes as compared to wild-type individuals (P < 0.01). Interestingly, of the HFE wild-type subjects 5.9% had a TS value above the 45% threshold.

This study shows that (i) the predicted prevalence for C282Y homozygosity in Italy is 1:3900; (ii) the C282Y/H63D wild-type population has an increased baseline of iron parameters possibly due to genetic factors not linked to the C282Y/H63D mutations; (iii) since in the latter population the actual tissue iron burden cannot be assessed, phenotypic (TS) screening in Italy is not recommended until the true prevalence of all mutations in the HFE gene and in other hemochromatosis genes will be established.

Porphyria cutanea tarda (PCT) is associated in most cases with iron overload, which may participate in decreased activity of uroporphyrinogen decarboxylase in the liver. The aetiology of this iron overload remains unknown; however, it has been demonstrated that mutations of HFE, the genetic haemochromatosis gene, might be present in a significant proportion of Anglo-Saxon and Italian patients. Furthermore, transferrin receptor polymorphism may influence the affinity of this receptor to its ligand with a subsequent increase of cellular iron absorption and storage.

To evaluate the incidence and spectrum of HFE mutations and the relative frequency of the two main alleles of transferrin receptor in patients with PCT originating from southern France, and to evaluate the relationship of these genetic data with iron status, and with hepatitis B and C and human immunodeficiency virus (HIV) infections.

Thirty-six consecutive patients with either sporadic or familial PCT were prospectively included between 1997 and 2000. Search for the presence of the three main mutations of the HFE gene and identification of the transferrin receptor alleles were performed using polymerase chain reaction followed by enzymatic digestion. Iron parameters and viral status for hepatitis B and C viruses and HIV were determined.

Seven patients (19%) showed heterozygous C282Y mutation, but no C282Y homozygote was present; five patients (14%) carried homozygous H63D mutation, while eight (22%) were heterozygous for this mutation. One patient was heterozygous for the S65C mutation (3%). Iron parameters demonstrated overload in all patients, without a clear difference between patients with and without deleterious mutations of the HFE gene. Infection by hepatitis C virus was documented in 20 patients (56%), and was significantly less frequent in patients with deleterious HFE mutations. The profile of transferrin receptor alleles in PCT patients did not show significant variation compared with the general population.

This study confirms the high frequency of HFE mutations in patients with PCT and supports the hypothesis that HFE gene abnormalities might play a significant part in the PCT pathomechanism, probably through iron overload; by contrast, transferrin receptor polymorphisms do not appear to play a significant part in iron overload in PCT.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder causing inappropriate dietary iron absorption that affects North Europeans. HH is associated with the C282Y mutation of the HFE gene, and the H63D mutation to a lesser degree. Both mutations are abundant in Europe, with H63D also appearing in North Africa, the Middle East, and Asia. Emigration from Europe over the past 500 years has introduced C282Y and H63D to America, Australia, New Zealand, and South Africa in an essentially predictable fashion. The distinctive characteristics of the population genetics of HH are the confined racial distribution and high frequency in North European peoples. C282Y frequencies in North Europeans are typically between 5% and 10%, with homozygotes accounting for between 1/100 and 1/400 of these populations. The scarcity of the C282Y mutation in other populations accounts for the lack of HH in non-Europeans.

Over 90% of patients with hemochromatosis in the United Kingdom are homozygous for the C282Y mutation on the HFE gene. The Centers for Disease Control (CDC) in the United States has recommended that adults should be screened for HFE mutations to identify susceptible individuals before onset of disease. The aim of this study was to evaluate the polymerase chain reaction using sequence-specific primers (PCR-SSP) as a method of large-scale population screening for the common HFE gene mutations, H63D and C282Y. A total of 10,583 consenting blood donors were tested using nonautomated procedures. Three alleles, termed HFE-1, -2, and -3, were detected with phenotype frequencies of 94.56%, 28.33%, and 15.79%, respectively, and gene frequencies of 0.76421, 0.15342, and 0.08237, respectively. All donors identified as homozygous for the C282Y mutation or heterozygous for both the H63D and C282Y mutations were confirmed by heterduplex analysis and/or PCR-SSP. The number of technical failures that affected the identification of donors homozygous for the C282Y mutation was 390 giving an overall repeat rate 3.7%, although this fell to 1% over the last quarter of the study. This study demonstrates that PCR-SSP may be used for large-scale population screening for the C282Y genotype associated with hemochromatosis.

Background: Hereditary hemochromatosis (HH) is a common autosomal recessive disease caused by an iron overload. Two mutations (C282Y and H63D) on the responsible HFE gene have been described. HH heterozygotes may have a slight iron overload that does not cause clinical disease. Compound heterozygosity may be associated with higher iron stores than C282Y heterozygosity. We studied biochemical iron parameters in HH C282Y and compound heterozygotes without a clinically significant iron overload. Methods: Data on hemoglobin, hematocrit, mean corpuscular volume, serum ferritin, serum iron, transferrin, and transferrin saturation were obtained from 40 C282 wild type controls (irrespective of H63D genotype), 61 C282Y heterozygotes, and 18 compound (C282Y/H63D) heterozygotes without clinical iron overload disease. Results: Serum ferritin levels were significantly higher in female HH heterozygotes, particularly in compound heterozygotes, than in normal women. In male heterozygotes, no difference in serum ferritin was found. We found higher mean serum iron and transferrin saturation levels in male and female HH heterozygotes than in normal controls, the highest in the group of compound heterozygotes. Conclusions: Mean serum ferritin (only in women), serum iron, and transferrin saturation are highest in compound heterozygotes and lowest in controls. C282Y heterozygotes seem to be an intermediate group between compound heterozygotes and the normal population.

Porphyria cutanea tarda (PCT) is commonly associated with iron overload and hepatitis C virus (HCV) infection. Association between hemochromatosis C282Y or H63D mutations and PCT has been observed, although not uniformly, and iron overload is also commonly found in chronic HCV hepatitis. The aim of the present study was to investigate the frequency of C282Y and H63D mutations and HCV infection in Brazilian patients with PCT and their relationship with iron overload.

Twenty-three patients (19 men) aged 39.6 +/- 11.1 yr were studied. All had dermatological lesions of PCT and high levels of urinary uroporphyrin. HCV infection and iron overload were investigated. DNA samples were analyzed for the presence of HFE mutations.

The frequency of C282Y was significantly higher in PCT patients than in 278 healthy individuals (17.4% vs 4%, odds ratio = 5.1, 95% confidence interval 1.5-17.6, p = 0.02), whereas no difference was observed regarding the H63D mutation (30.4% vs 31%, odds ratio = 1, 95% confidence interval 0.4-2.4, p = 1). Biochemical tests in PCT patients showed iron overload with transferrin saturation = 47.3 +/- 20.7% and ferritin = 566.8 +/- 425 ng/ml. Fifteen of 23 (65.2%) patients had HCV infection and alcohol ingestion was observed in 17 of 23 (73.9%).

PCT patients exhibited evidence of iron overload, a high frequency of HCV, and an association with C282Y mutation. These data further support the notion that both acquired and inherited factors contribute to the occurrence of PCT, and indicate that screening for C282Y may be justified in PCT patients.

Two common mutations of the haemochromatosis associated gene (HFE) (cys282tyr (C282Y) and his63asp (H63D)) have been implicated in haemochromatosis and as modulators in cardiovascular disease.

To investigate the role of these mutations in the pathogenesis of idiopathic dilated cardiomyopathy.

Case-control and prospective cohort study of patients attending a cardiomyopathy unit in a tertiary referral cardiac centre.

207 unrelated white patients with dilated cardiomyopathy, followed up for 259 patient years, and 200 controls were tested for HFE C282Y and H63D mutations by polymerase chain reaction and restriction digestion.

31/207 patients (15%) v 24/200 controls (12%) carried C282Y (adjusted odds ratio (OR) 1.2 (95% confidence interval 0.7 to 2.2)), 74/207 (36%) v 53/200 (27%) carried H63D (OR 1.6 (1.1 to 2.5)), and 10/207 (4.8%) v 4/200 (2%) were compound heterozygotes (OR 2.6 (0.8 to 8.5)). Four patients and six controls were H63D homozygous and one was C282Y homozygous. There was a progressive increase in mean serum iron ([Fe]) and transferrin saturations from patients with no mutation ([Fe] = 16.3 micromol/l, transferrin saturation = 23.7%) through H63D heterozygotes (17.5 micromol/l, 25.8%), C282Y heterozygotes (17.1 micromol/l, 26.6%), H63D homozygotes (20.0 micromol/l, 33.5%), compound heterozygotes (26.8 micromol/l, 41.7%), and C282Y homozygotes (34 micromol/l, 71%). At follow up (median 90 months) the rate of death or cardiac transplantation was 52/207 (25%). C282Y heterozygotes had less ventricular dilatation (mean (SD): 59.9 (1.7) mm v 64.9 (0.9) mm, p < 0.05), better fractional shortening (24 (1. 7)% v 18.8 (1.4)%, p < 0.01), and a trend towards improved survival without transplantation. [Fe] and transferrin saturation did not correlate with disease severity and were not associated with reduced survival.

The frequency of the H63D mutation is significantly increased in patients with idiopathic dilated cardiomyopathy. As H63D has a relatively minor effect on iron status, the mechanism of this association may be unrelated to iron metabolism.

Hereditary hemochromatosis is a fairly common disease in the Caucasian population, with a prevalence estimated at between 1.5 to 3/1,000 inhabitants. Over the past few years, its symptomatology has altered; at present, its clinical aspect with diabetes mellitus, cirrhosis, and darker skin pigmentation only constitutes 10% of new cases of this disease.

In 1996, the discovery of the C282Y mutation in the HFE gene radically altered the diagnostic approach to hereditary hemochromatosis. At present, any patient admitted with an isolated case of asthenia, or with arthralgia or hypertransaminasemia should be examined via transferrin-saturation testing: if the transferrin saturation coefficient is > 45%, then the presence of the C282Y mutation should be investigated to confirm the diagnosis of hemochromatosis. A liver biopsy is no longer necessary to establish the diagnosis, but this is still useful in cases of possible cirrhosis, which is the main risk factor for hepatocellular carcinoma. Phlebotomy remains the sole recommended treatment, and should be undertaken in a case-specific manner. Family screening should be carried out for all first-degree relatives for every new case that is diagnosed.

The discovery of the HFE gene has permitted hereditary hemochromatosis to be easily differentiated from other forms of hepatic iron overload including a new syndrome, dysmotabolic hepatosiderosis. Casos of homozygotic C282Y without hepatic iron overload have been described, but the clinical outcome of some of these cases requires further study, and adds to the controversy on whether systematic population screening should be made available.

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.

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.

It has been proposed that iron overload may adversely affect liver disease outcome. The recent identification of 2 mutations in the HFE gene related to hereditary haemochromatosis (Cys282Tyr and His63Asp) provided an opportunity to test whether they are associated with hepatic iron accumulation and the activity and severity of liver disease in hepatitis C virus (HCV) infection. We investigated the prevalence of HFE mutations in 135 male patients with chronic HCV hepatitis, and correlated genotype distribution with different parameters of iron status and the activity and severity of liver disease. Of these 135 patients, 6 (4.4%) carried Cys282Tyr and 32 (23.7%) carried His63Asp, frequencies which were similar to those observed in healthy controls. Serum iron levels and transferrin saturation (but not ferritin levels or liver iron content) were significantly higher in carriers than in non-carriers of HFE mutations. No difference was observed in serum ALT, AST and GGT levels between carriers and non-carriers. Finally, scores for necroinflammatory activity and fibrosis in the liver were significantly higher in HFE carriers than in non-carriers. Patients with chronic HCV infection carrying HFE mutations tend to present more evident body iron accumulation and a higher degree of necroinflammatory activity and fibrosis in the liver. HFE gene mutations might be an additional factor to be considered among those implicated in the determination of a worse prognosis of the liver disease in chronic HCV infection.

Hepatic iron overload has been reported in various metabolic conditions, including the insulin-resistance syndrome (IRS) and nonalcoholic steatohepatitis (NASH). The aim of this study was to show that such hepatic iron overload is part of a unique and unrecognized entity.

A total of 161 non-C282Y-homozygous patients with unexplained hepatic iron overload were included. We determined the age; sex; presence of IRS (1 or more of the following: body mass index of >25, diabetes, or hyperlipidemia); serum iron tests and liver iron concentration (LIC; reference value, <36 micromol/g); liver function test results; C282Y and H63D HFE mutations; and liver histological status.

Patients were predominantly male and middle-aged. Most (94%) had IRS. Transferrin saturation was increased in 35% (median, 42%; range, 13%-94%). LIC ranged from 38 to 332 micromol/g (median, 90 micromol/g), and LIC/age ratio ranged from 0.5 to 4.8 (median, 1.8). Allelic frequencies of both HFE mutations were significantly increased compared with values in normal controls (C282Y, 20% vs. 9%; H63D, 30% vs. 17%), only because of a higher prevalence of compound heterozygotes. Patients with no HFE mutations had similar degrees of iron overload as those with other genotypes, except for compound heterozygotes, who had slightly more iron burden. Steatosis was present in 25% of patients and NASH in 27%. Portal fibrosis (grades 0-3) was present in 62% of patients (grade 2 or 3 in 12%) in association with steatosis, inflammation, and increased age. Sex ratio, IRS, transferrin saturation, and LIC did not vary with liver damage. Serum ferritin concentration, liver function test results, and fibrosis grade were more elevated in patients with steatosis and NASH than in others, but LIC and allelic frequencies of HFE mutations were similar.

This study shows that patients with unexplained hepatic iron overload are characterized by a mild to moderate iron burden and the nearly constant association of an IRS irrespective of liver damage.

Mutations in the haemochromatosis (HFE) gene cause most of the cases of hereditary haemochromatosis among people of Northern European ancestry while remaining a rare cause of iron overload among indigenous persons of the Asia-Pacific region. Advances in understanding of the role of the HFE protein product and other recently cloned iron transporters signify an exciting period, as previously unknown components of the iron metabolism pathway are revealed one by one. Epidemiological studies have shown that this gene is more widespread than its phenotypic expression would suggest and that the heterozygous state may be implicated in the expression of other diseases of the liver such as porphyria cutanea tarda, hepatitis C virus infection and non-alcoholic steatohepatitis. The diagnosis, management and ethical implications for clinical practice in the aftermath of this discovery are discussed.

Hereditary haemochromatosis (HH) is an autosomal recessive disease involving mutations in the recently characterised HFE gene linked to HLA-A in the major histocompatibility complex. The known HFE polymorphisms include the wild-type allele, a G-->A substitution at base 845 (845A) and a C-->G substitution at position 187 (187G). Although most cases of HH are accountable by homozygosity of the 845A allele the exact risk of other HFE genotypes, especially those involving the 187G allele has not been determined. We have compiled estimates of disease risk for all known HFE genotypes by re-analyzing published studies. The data show a hierarchical risk calculated as odds ratio (OR) for each genotype 845A/ 845A (OR=2101); 845A/187G (OR=24); 187G/187G (OR=9); 845A/Wt (OR=5); 187G/Wt (OR=2). Interestingly, the disease risk of 187G-genotypes suggests that subtle functional changes in the HFE product can interact with other genetic factors (e.g. trans allele, gender) and environmental factors (e.g. diet) to manifest either as clinical disease, altered iron stores or a normal phenotype. This paradigm is potentially useful in understanding the contribution of HLA alleles to risk of various disorders especially autoimmunity.

Mutation analysis was performed on DNA samples of 965 individuals from four different ethnic groups in South Africa, in an attempt to determine the spectrum of sequence variants in the haemochromatosis ( HFE ) gene. This population screening approach, utilizing a combined heteroduplex and single-strand conformation polymorphism (HEX-SSCP) method, revealed three previously described and four novel missense mutations. Novel variants V53M and V59M were identified in exon 2, Q127H in exon 3 and R330M in exon 5. The exon 5 variant was identified in one of 13 patients referred for a molecular diagnosis of hereditary haemochromatosis (HH), who tested negative for the known C282Y and H63D mutations. Mutation Q127H was detected in exon 3 of the HFE gene together with mutation H63D in an apparently severely affected patient previously shown to carry the protoporphyrinogen oxidase ( PPOX ) gene mutation R59W, which accounts for dominantly inherited variegate porphyria (VP) in >80% of affected South Africans. The mutant allele frequency of the C282Y mutation was found to be significantly lower in 73 apparently unrelated VP patients with the R59W mutation than in 102 controls drawn from the same population ( P = 0.005). The population screening approach used in this study revealed considerable genotypic variation in the HFE gene and supports previous data on the involvement of this gene in the porphyria phenotype.

Hereditary haemochromatosis shows a wide variation in phenotypic expression, which is thought to be due, in part, to genetic factors. A single missense mutation in HFE, leading to an amino acid substitution (C282Y) has been shown to be the causative mutation, clearly responsible for clinical expression of the disorder. Since homozygosity for the C282Y mutation can give rise to a disorder which shows wide variation in clinical expression, we investigated the possibility that genetic modifiers of HFE may exist.

Linkage disequilibrium analysis was performed on chromosome 6p21.3 in 74 patients homozygous for the C282Y mutation using microsatellite markers spanning the haemochromatosis gene region. Phenotypic expression was evaluated based on transferrin saturation, serum ferritin, hepatic iron concentration and index, and iron grade.

Linkage disequilibrium (LD) analysis showed a predominant ancestral haplotype from D6S265 to D6S2236 covering a region of approximately 5 Mb. The overall LD distribution in this region showed two peaks of highly significant association at D6S105 (2 Mb proximal to HFE) and at D6S2239 approximately 50 kb distal to HFE. Male patients homozygous for D6S105 allele 8, had significantly higher hepatic iron indices than patients heterozygous or nullizygous for D6S105-8 (p<0.038).

This analysis indicates that modifying gene(s) or another mutation affecting HHC clinical expression may be located in the region of D6S105.

The HFE gene is a crucial candidate gene for hemochromatosis. The aims of this study were to assess the HFE genotypic profile in a large series of unrelated probands diagnosed as having phenotypic hemochromatosis, to characterize the sub-group of patients who were not homozygous for the major C282Y mutation, and to report the iron status of the detected HFE-identical siblings.

In 217 patients, the phenotypic diagnosis of hemochromatosis was based on strict bioclinical and/or histological criteria, and their genotypic profile (C282Y and H63D mutations) was determined.

1) 209 of the 217 probands were C282Y +/+. In 33 cases, an HFE-identical sibling was identified. Two of them had neither a clinical nor a biochemical phenotypic profile of hemochromatosis in the absence of any external factor which might have attenuated this expression. 2) Eight patients (seven males) were not C282Y +/+. Their genotypic profiles were: (C282Y +/-): six cases (four were H63D +/- and two H63D -/-); (C282Y -/-): two cases (one was H63D +/+, one H63D +/-). Phenotypic expression consisted of six cases of mild liver siderosis (among whom were the four compound heterozygotes and one case of alcoholic cirrhosis) and two severe cases of hepatic iron overload (one with alcoholic cirrhosis). Three HFE-identical siblings were identified, none of them presenting with iron excess.

In our population: 1) The classical phenotypic criteria fitted, in 96.3% of cases, with a homogeneous genotypic entity defined by homozygosity for the C282Y mutation. Incomplete penetrance of the homozygous status was shown by the absence of the hemochromatosis phenotypic profile in 6% of the HFE-identical siblings. 2) A minority (3.7%) were not homozygous for C282Y. These were essentially men with mild iron overload, and might present with distinct iron overload entity(ies) as suggested by the presence in three of an HFE-identical sibling with absence of iron overload.

It has been proposed that iron accumulation may contribute to atherogenesis by increasing free radical formation and oxidative stress. Epidemiological studies in which the association of iron status with atherosclerosis was assessed raised conflicting results. To test whether genetic haemochromatosis is associated with increased atherosclerosis, we determined the prevalence of two mutations in the HFE gene related to haemochromatosis (845G-->A; Cys282Tyr. and 187 C-->G, His63Asp) in 265 consecutive patients with premature (<50 years of age) angiographically-proven atherosclerotic disease (coronary and/or peripheral), and in 272 healthy controls. PCR amplification followed by RsaI (Cys282Tyr analysis) and BclI (His63Asp analysis) restriction digestion was employed to define the genotypes. The mutant Cys282Tyr allele had a frequency of 0.07 among controls and 0.04 among patients (carrier frequency of 14.0% and 8.3%, respectively). The frequency of the His63Asp mutant allele was 0.14 (28.6% of carriers) in controls and 0.11 (22.2% of carriers) in patients. Five of 265 patients (1.1%) and 9/272 controls (3.3%) were compound heterozygotes. In conclusion, a lower prevalence of the Cys 282Tyr mutation and a similar frequency of the His63Asp mutation was observed in patients with atherosclerotic disease in comparison with normal controls. These findings do not support an association between haemochromatosis and atherogenesis.

Hemochromatosis, the inherited disorder of iron metabolism, leads, if untreated, to progressive iron overload and premature death. The hemochromatosis gene, HFE, recently has been identified, and characterization of this gene has shown that it contains two mutations that result in amino acid substitutions-cDNA nucleotides 845 G-->A (C282Y) and 187 C-->G (H63D). Although hemochromatosis is common in Caucasians, affecting >=1/300 individuals of northern European origin, it has not been recognized in other populations. The present study used PCR and restriction-enzyme digestion to analyze the frequency of the 845 G-->A and 187 C-->G mutations in HLA-typed samples from non-Caucasian populations, comprising Australian Aboriginal, Chinese, and Pacific Islanders. Results showed that the 845 G-->A mutation was present in these populations (allele frequency 0.32%), and, furthermore, it was always seen in conjunction with HLA haplotypes common in Caucasians, suggesting that 845 G-->A may have been introduced into these populations by Caucasian admixture. 187 C-->G was present at an allele frequency of 2.68% in the two populations analyzed (Australian Aboriginal and Chinese). In the Australian Aboriginal samples, 187 C-->G was found to be associated with HLA haplotypes common in Caucasians, suggesting that it was introduced by recent admixture. In the Chinese samples analyzed, 187 C-->G was present in association with a wide variety of HLA haplotypes, showing this mutation to be widespread and likely to predate the more genetically restricted 845 G-->A mutation.

The discovery of the gene responsible for most cases of genetic haemochromatosis has made it possible to re-evaluate the role of haemochromatosis in causing iron accumulation in porphyria cutanea tarda and various inherited anaemias. Recent studies of these associations are reviewed.