non-HFE Hemochromatosis Research Articles

Clinical, Pathological, and Molecular Correlates in Ferroportin Disease. A Study of Two Novel Mutations.

Background: Mutations in the iron exporter Ferroportin (Fpn) lead to type IV hemochromatosis (Ferroportin Disease, FD), a dominantly inherited disorder with heterogeneous clinical and biochemical patterns. Some patients present with predominant macrophage iron overload (M), marked elevation of serum ferritin, normal-to-low transferrin saturation (TS), and, possibly, iron restricted erythropoiesis. Others present with a phenotype resembling classical HFE-related hemochromatosis, i.e. characterized by high TS and predominant hepatocyte iron overload (H). These differences are thought to reflect heterogeneity in the functional behaviour of Fpn mutant proteins.Methods: Two unrelated probands referring to the Centre for Iron Overload Disorders in Verona because of non-HFE hemochromatosis were screened for Fpn mutations by DHPLC (Cremonesi L, Br J Haematol 2005). The functional behaviour of mutants Fpn was studied by generating Fpn-GFP constructs transfected into different cell types (HEK293T, Cos7, and mouse bone marrow macrophages), and analyzing their cellular localization, as well as their capabilities to bind hepcidin and export iron (De Domenico I, PNAS 2005). The two mutations were also expressed in zebrafish, to evaluate their impact on iron-dependent erythropoiesis.Results: Patient 1, a 59 year old male, had clinical, biochemical (TS 74.8%, ferritin 9,000 μg/l), and pathological features (marked iron overload in either macrophages and hepatocytes, absence of overt cirrhosis) somewhat ambiguous, possibly suggesting a type M Fpn variant with late secondary hepatocyte overload. He was found to be heterozygous for the new L233P mutation. Functional studies revealed that Fpn L233P does not appropriately traffic to the cell surface, resulting in inappropriate inhibition by hepcidin. Fpn L233P expression in vivo in zebrafish resulted in iron limited erythropoiesis, consistent with a type M mutation leading to macrophage iron retention. Patient 2, a 59 year old female, had features more clearly suggesting a type M Fpn variant (TS 22.7%, ferritin 1,771 μg/l, macrophage iron load), but tolerated very well phlebotomies without developing signs of anemia. She was found to be heterozygous for the new I152F mutation. Functional studies revealed a unique pattern (never observed until now), since Fpn I152F localized appropriately on cell membrane, bound near normally to hepcidin, but showed a “primary” deficit of iron export capability. I152F expression in zebrafish resulted in a trend towards iron limited erythropoiesis, though quantitatively less clear than L223P.Conclusions: FD is a heterogeneous disease caused by generally “private” mutations in Fpn. The clinical, biochemical, and pathological features vary depending on the different behaviour of mutant Fpn. In vitro and in vivo molecular expression studies are very useful to clarify the pathophysiogical spectrum of this disease.

Non-HFE haemochromatosis

Non-HFE hereditary haemochromatosis (HH) refers to a genetically heterogeneous group of iron overload disorders that are unlinked to mutations in the HFE gene. The four main types of non-HFE HH are caused by mutations in the hemojuvelin, hepcidin, transferrin receptor 2 and ferroportin genes. Juvenile haemochromatosis is an autosomal recessive disorder and can be caused by mutations in either hemojuvelin or hepcidin. An adult onset form of HH similar to HFE-HH is caused by homozygosity for mutations in transferrin receptor 2. The autosomal dominant iron overload disorder ferroportin disease is caused by mutations in the iron exporter ferroportin. The clinical characteristics and molecular basis of the various types of non-HFE haemochromatosis are reviewed. The study of these disorders and the molecules involved has been invaluable in improving our understanding of the mechanisms involved in the regulation of iron metabolism.

Iron overload (with attention to genetic testing and diagnosis/management of HFE wild type patients)

The discovery of the HFE, HJV, HAMP, TfR2, and SLC40A1 genes and preliminary understanding of their roles in iron homeostasis have contributed tremendously to our understanding of the pathogenesis of genetic hemochromatosis. Although several new models of iron metabolism have been proposed, some key "sensor" steps of iron absorption in the enterocytes and of iron storage in hepatocytes and other cells remain unclear. A diagnosis of non-HFE genetic hemochromatosis should be considered in patients with unexplained iron overload who do not have the common mutations in the HFE genes. Phenotypic evaluation such as liver biopsy and measurement of hepatic iron concentration remain important in non-HFE hemochromatosis because mutations in other genes are rare and there are no other available noninvasive tests to confirm the diagnosis. Phlebotomy remains the mainstay of therapy also for non-HFE hemochromatosis. However, phlebotomy may not be well tolerated in certain forms of non-HFE hemochromatosis such as "ferroportin disease."

Hepatobiliary pathology

Publications concerning liver histopathology in fatty liver disease and chronic hepatitis C, iron and copper overload, and liver transplantation from the past year have been surveyed to highlight useful concepts and diagnostic information. Two microscopic forms of pediatric nonalcoholic steatohepatitis have been described: type 1 in which hepatocyte ballooning and/or pericellular fibrosis accompany the steatosis; and type 2 which has portal tract inflammation and/or fibrosis as the salient accompanying feature. In chronic hepatitis C, the ductular reaction appears to be a major factor associated with fibrosis. In patients transplanted for hepatitis C virus-related cirrhosis, immunostaining of post-transplant liver biopsies for alpha-smooth muscle actin (i.e. in activated hepatic stellate cells) may identify those individuals at risk for severe recurrence. Clinicopathological papers on several forms of non-HFE hemochromatosis were published and Wilson's disease was described in individuals of 60 years or more in age. Cholestasis in childhood was expertly reviewed and histopathologic precursor lesions of hepatocellular carcinoma were also examined in a comprehensive article. Recent publications with impact on liver biopsy interpretation include a morphologic classification of nonalcoholic steatohepatitis in childhood, the differential diagnosis of childhood cholestasis and pathogenetic factors involved in fibrogenesis in chronic hepatitis C.

Recurrence of Iron Deposition in the Cardiac Allograft in a Patient With Non-HFE Hemochromatosis

We report the case of a 36-year-old woman with a diagnosis of idiopathic dilated cardiomyopathy who underwent cardiac transplantation. The results of her initial iron studies were normal, but hemochromatosis was suspected after microscopy of the explanted heart revealed iron deposition. By 6 months post-transplantation, iron deposition was detected in her surveillance endomyocardial biopsy specimens and studies then confirmed the existence of non-HFE hemochromatosis. The patient has been stable on treatment with regular phlebotomies and a low vitamin C diet.

Hemochromatosis: Genetic Testing and Clinical Practice

The availability of a facile treatment for hemochromatosis renders early diagnosis of iron overload syndromes mandatory, and in many instances genetic testing allows identification of individuals at risk of developing clinical disease before pathologic iron storage occurs. Numerous proteins implicated in iron homeostasis have recently come to light, and defects in the cognate genes are associated with iron storage. Although most adult patients with hereditary iron overload are homozygous for the C282Y mutation of the HFE gene, an increasing number with hereditary iron storage have an HFE genotype not characteristic of the disease. Heterozygosity for mutations in the gene encoding ferroportin 1 (FPN1) is probably the second most common genetic cause of hereditary iron storage in adults; here the primarily affected cell is the macrophage. Rare defects, including mutations in the transferrin receptor 2 (TFR2) gene, have also been identified in pedigrees affected with "non-HFE hemochromatosis." Homozygous mutations in the newly identified genes encoding hemojuvelin (HFE2) and hepcidin (HAMP) cause juvenile hemochromatosis. At the same time, heterozygosity for mutations in these genes can modify the clinical expression of iron storage in patients predisposed to iron storage in adult life. Hemochromatosis might thus be considered as a polygenic disease with strong environmental influences on its clinical expression. As our mechanistic understanding of iron pathophysiology improves, our desire to integrate clinical decision making with the results of laboratory tests and molecular analysis of human genes poses increasing challenges.

The molecular genetics of haemochromatosis

The molecular basis of haemochromatosis has proved more complex than expected. After the 1996 identification of the main causative gene HFE and confirmation that most patients were homozygous for the founder C282Y mutation, it became clear that some families were linked to rarer conditions, first named 'non-HFE haemochromatosis'. The genetics of these less common forms was intensively studied between 2000 and 2004, leading to the recognition of haemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin-related haemochromatosis, and opening the way for novel hypotheses such as those related to digenic modes of inheritance or the involvement of modifier genes. Molecular studies of rare haemochromatosis disorders have contributed to our understanding of iron homeostasis. In turn, recent findings from studies of knockout mice and functional studies have confirmed that HAMP plays a central role in mobilization of iron, shown that HFE, TFR2 and HJV modulate HAMP production according to the body's iron status, and demonstrated that HAMP negatively regulates cellular iron efflux by affecting the ferroportin cell surface availability. These data shed new light on the pathophysiology of all types of haemochromatosis, and offer novel opportunities to comment on phenotypic differences and distinguish mutations.

Mutations of the HFE gene among Turkish hereditary hemochromatosis patients

Since the discovery of the HFE gene, C282Y and H63D mutations have been reported as significantly correlated with clinically manifested hereditary hemochromatosis (HH). As the other genes involved in iron metabolism have been described, non-HFE cases of HH have been identified. Since in the general Turkish population, the C282Y mutation is not found and the H63D mutation is of high frequency, we aimed to determine mutations in the HFE genes in our patients with HH. The HFE gene of the five patients with HH were sequenced. C282Y mutation was absent, and all HH patients were heterozygote for H63D mutation. No other mutation was found in HFE gene by sequencing. Although the higher allele frequency of the H63D mutation in Turkish HH patients than in the general population implies a role of the H63D mutation in iron overload, there is a strong possibility that Turkish HH patients have non-HFE hemochromatosis.

Non-HFE Hemochromatosis

The term "non-HFE hemochromatosis" (non-HFE HC) refers to several phenotypically similar but genetically distinct forms of hereditary hemochromatosis affecting individuals without pathogenic mutations of HFE. The involved genes are, sinsu strictu, transferrin receptor 2 (TfR2), hemojuvelin (HJV), and hepcidin (HAMP). Non-HFE HC share common pathogenic and clinical features with HFE HC. However, depending on the role of the affected gene in iron trafficking, the clinical onset may be earlier and phenotypic expressivity more severe than classic HC. Other forms of hereditary iron overload have distinct pathogenesis and phenotype. The most prevalent of these forms is "ferroportin disease," characterized by autosomal dominant trait, predominant reticuloendothelial cell iron overload, and mild organ damage. Non-HFE HC gene products, while responsible for rarer cases of HC as compared with HFE, are much more central than HFE in human iron homeostasis and understanding their function will greatly advance our comprehension of iron trafficking in health and disease.

Non-HFE hemochromatosis: Genetics, pathogenesis, and clinical management

Recent advances in our understanding of iron metabolism and the epidemiology of iron overload disorders have shown that hereditary forms of hemochromatosis can result from mutations in several iron metabolism genes other than HFE, including Hamp, HJV, TFR2, and SCL40A. These "non-HFE" forms of hemochromatosis are much rarer than HFE-related hemochromatosis but exhibit a similar phenotype, and with the exception of ferroportin disease, a similar pattern of inheritance and parenchymal iron accumulation. Therefore, these diseases can be thought of as variant forms of a primary hepatic iron overload syndrome; thus, a unified approach can be used for evaluation and diagnosis. Management generally consists of periodic phlebotomies until iron is depleted.

High prevalence of non-HFE gene-associated haemochromatosis in patients from southern Italy.

Hereditary haemochromatosis is an autosomal recessive disorder of iron regulation that results in abnormal intestinal iron absorption with progressive iron overloading of parenchymal cells. Two specific, single point mutations of the HFE gene (C282Y and H63D) have been described in haemochromatosis patients. Epidemiological studies have revealed a strict association between hereditary haemochromatosis and C282Y homozygosis or C282Y/H63D compound heterozygosis, suggesting that these mutations may provide a useful tool for diagnosis. However, recent investigations from southern Europe have reported lower allelic frequencies of the C282Y mutation among haemochromatosis patients, apparently depending on the geographical area of the population analysed. To assess the predictive value of the detection of the C282Y and H63D HFE mutations in our geographical area, we have evaluated their occurrence in 46 haemochromatosis patients from southern Italy. We found that only 19.6% of our patients were homozygous for the C282Y mutation and 21.7% were compound C282Y/H63D heterozygotes. Among the remaining 59%, approximately 40% did not display any of the known HFE mutations. We conclude that, in southern Italy, another genetic determinant/s must be responsible for many haemochromatosis cases and that a genetic screening for the C282Y and H63D HFE mutations is not sufficient for hereditary haemochromatosis diagnosis.

Duodenal cytochrome B and hephaestin expression in patients with iron deficiency and hemochromatosis

Background & Aims : An increased duodenal expression of the iron transporters, divalent-metal-transporter-1, and ferroportin is observed in patients with iron deficiency or hereditary hemochromatosis. Two oxidoreductases, termed duodenal cytochrome b and hephaestin, are proposed to co-operate with divalent-metal-transporter-1 and FPN1, respectively, to transfer iron from the duodenal lumen to the circulation. Methods : In the present study, we investigated the mRNA and protein expression of Dcytb and hephaestin in duodenal biopsies from patients with iron deficiency, HFE, and non-HFE-associated hemochromatosis and in control subjects by means of real-time polymerase chain reaction, Western blot, and immunofluorescence. Results : In iron deficiency a coordinated upregulation of the iron transporters divalent-metal-transporter-1 and ferroportin and of duodenal-cytochrome b and hephaestin was found, whereas in patients with HFE and non-HFE-associated hemochromatosis duodenal-cytochrome b and hephaestin protein and mRNA expression were not significantly different from control subjects. However, HFE but not non-HFE hemochromatosis patients presented with an increased duodenal ferric reductase activity. Spearman rank correlations showed that Dcytb, hephaestin, FPN1, and DMT1 mRNA expression are positively related to each other independently of the underlying disease, which ensures an efficient transepithelial transport of absorbed iron. Conclusions : Our data show that duodenal-cytochrome b activity in iron deficiency is stimulated via enhanced protein expression, whereas in HFE hemochromatosis it is up-regulated post-translationally. This points to different kinetics of intestinal iron uptake between iron deficiency and HFE hemochromatosis and also indicates that duodenal iron accumulation in HFE and non-HFE hemochromatosis is pathophysiologically different.

AVAQ 594-597 deletion of the TfR2 gene in a Japanese family with hemochromatosis

The majority of Caucasian patients with hemochromatosis are homozygous for C282Y mutation of the HFE gene. In contrast to its high prevalence in Caucasians, hemochromatosis is a rare disorder in Japan. This may be due to the low prevalence of the C282Y mutation of the HFE gene in Japanese. Recent reports suggest that the mutations of transferrin receptor 2 (TfR2) gene may be involved in non-HFE hemochromatosis. Therefore, we investigated the TfR2 gene of 6 sporadic and 5 familiar cases of Japanese hemochromatosis. Three siblings in one family were found to be homozygous for an AVAQ 594-597 deletion. All three had severe iron deposits in the hepatocytes and bile ducts, but none was affected by diabetes mellitus. This mutation was not detected in 100 control individuals. Further study was undertaken to investigate whether the large deletion of the TfR2 gene is the mutation responsible for some of the Japanese hemochromatosis cases.

Review article: haemochromatosis.

Hereditary haemochromatosis is the prototype disease for primary iron overload. The disorder is very common, especially amongst subjects of Northern European extraction. It is characterized by an autosomal recessive mode of inheritance, and most cases are homozygous for the C282Y mutation in the HFE gene. Haemochromatosis is now recognized to be a complex genetic disease with probable significant environmental and genetic modifying factors. The early diagnosis of individuals at risk for the development of haemochromatosis is important, because survival and morbidity are improved if phlebotomy therapy is instituted before the development of cirrhosis. The cost-effectiveness and utility of large-scale screening for haemochromatosis have been questioned given that many individuals with the homozygous C282Y mutation do not have iron overload or end-organ damage. However, the use of phenotypic tests, such as serum transferrin-iron saturation, for initial screening avoids the problem of the identification of non-expressing homozygotes. Liver biopsy remains important in management to determine the presence or absence of cirrhosis, particularly amongst patients with serum ferritin levels greater than 1000 ng/mL or elevated liver enzymes. Those with non-HFE haemochromatosis who cannot be identified on genotypic testing should have a liver biopsy to establish diagnosis. Patients with end-stage liver disease may develop liver failure or primary liver cancer, and liver transplantation may be required. Liver transplantation for haemochromatosis is associated with a poorer outcome compared with other indications because of infections and cardiac complications.

HFE and non-HFE hemochromatosis.

Hereditary hemochromatosis (HH) is a disorder of iron metabolism in which enhanced absorption of dietary iron causes increased iron accumulation in the liver, heart, and pancreas. Most individuals with HH are homozygous for a point mutation in the HFE gene, leading to a C282Y substitution in the HFE protein. The function of HFE protein is unknown, but the available evidence suggests that it acts in association with beta2-microglobulin and transferrin receptor 1 to regulate iron uptake from plasma transferrin by the duodenum, the proposed mechanism by which body iron levels are sensed. The identification of HFE has established the foundation for a better understanding of the molecular and cellular biology of iron homeostasis and its altered regulation in HH. Additionally, the ability to accurately diagnose iron overload disorders has been strengthened, family screening has been improved, and evaluation of patients with other forms of liver disease complicated by moderate-to-severe iron overload is now possible. However, the role of HFE testing in generalized population screening for HH is still controversial. Recently, other forms of HH have been described that are not related to HFE but are due to mutations in genes coding iron transport proteins.

Hereditary haemochromatosis: more causative genes

Hereditary haemochromatosis (HH) is a highly prevalent iron-overload disease that is mainly associated with recessive mutations in the gene encoding HFE, an MHC class I-like protein. Interestingly, non-HFE haemochromatosis has also been reported and two other loci have been recently identified. One maps to chromosome 1q and is responsible for a juvenile form of the disease. The other, mapped to 7q22, contains the gene encoding transferrin receptor 2, in which a C→G transversion introduces a nonsense mutation.

Hereditary haemochromatosis: more causative genes

Hereditary haemochromatosis (HH) is a highly prevalent iron-overload disease that is mainly associated with recessive mutations in the gene encoding HFE, a MHC class I-like protein. Interestingly, non-HFE haemochromatosis has also been reported and two other loci have been recently identified. One maps to chromosome 1q and is responsible for a juvenile form of the disease. The other, mapped to 7q22, contains the gene encoding transferrin receptor 2; a C→G transversion in this gene introduces a nonsense mutation. In addition, the genes encoding ferroportin and ferritin H are now to be added to the list. Ferroportin is a basolateral iron transporter involved in the movement of iron across the intestinal enterocytes to the bloodstream. Linkage analysis in a large Dutch family with inherited iron overload led to the identification of a single A→C transversion at position 734 (Ref. 1). This mutation substitutes an uncharged Asn residue at position 144, located within one of the predicted transmembrane domains of the protein, with a charged His residue. The expected reduction in the hydrophobicity of this domain could affect the folding of the molecule. Ferritin H is an iron-storage protein whose expression is essentially controlled at the translational level via the association of a protein trans-regulator with a cis-regulatory RNA element in the mRNA leader. A single mutation in this RNA structure has been found in a Japanese family 2. Strikingly, this mutation seems to increase the affinity of the RNA motif for the protein trans-regulator, resulting in reduced translation of the mutant mRNA.

Linkage to Chromosome 1q in Greek Families with Juvenile Hemochromatosis

ABSTRACT Hereditary hemochromatosis (HH) is a genetically heterogeneous disease. The HFE gene resides on chromosome 6 and its mutations account for the majority of HH cases in populations of northern European ancestry. Recently, two new types of hemochromatosis have been identified: Juvenile hemochromatosis (JH or HFE2), which maps to chromosome 1q21, and an adult form defined as HFE 3, which results from mutations of the TFR 2 gene, located at 7q22. We have performed a linkage study in five unrelated families of Greek origin with non-HFE hemochromatosis. Linkage at the chromosome 1q21 JH locus was detected in affected members with the use of polymorphic markers. Comparison of haplotypes between Greek and Italian JH patients revealed the presence of a common haplotype. However, the fact that many other haplotypes carrying the JH defect were observed in the two populations indicates that the respective mutations may have occurred in different genetic backgrounds. We suggest that hemochromatosis patients without HFE mutations should be evaluated for other possible types of hemochromatosis since hemochromatosis type 3 (HFE3) has a clinical appearance similar to HFE 1, and JH may have a late onset in some cases.

Expression of the duodenal iron transporters divalent-metal transporter 1 and ferroportin 1 in iron deficiency and iron overload

Imbalances of iron homeostasis are accompanied by alterations of intestinal iron absorption. The identification of divalent-metal transporter 1 (DMT1) and ferroportin 1 (FP1) has improved our understanding of transmembrane iron trafficking. To gain insight into the regulatory properties of these transporters in the duodenum, we studied their expression in patients with hereditary hemochromatosis (HFE-associated and non-HFE-associated), secondary iron overload, and iron deficiency. DMT1, FP1 messenger RNA (mRNA), and protein expression were analyzed in duodenal biopsy specimens from patients by means of TaqMan real-time polymerase chain reaction, Western blotting technique, and immunohistochemistry. DMT1 and FP1 mRNA levels are positively correlated with each other in all patient groups (P < 0.001). Moreover, DMT1 and FP1 mRNA levels were significantly increased in patients with iron deficiency, HFE and non-HFE hemochromatosis, whereas they were unchanged in patients with secondary iron overload. Alterations in DMT1 and FP1 mRNA levels were paralleled by comparable changes in the duodenal expression of these proteins. In patients with normal iron status or iron deficiency, significant negative correlations between DMT1, FP1 mRNA, and serum iron parameters were found, which were absent in subjects with primary hemochromatosis. DMT1 and FP1 are centrally involved in iron uptake/transfer in the duodenum and in the adaptive changes of iron homeostasis to iron deficiency and overload.

Iron and the Genetics of Cardiovascular Disease

The hypothesis that iron depletion protects against ischemic heart disease was proposed in 1981 as an explanation for the sex difference in heart disease rates.1 2 3 4 5 The “iron hypothesis” has generated significant debate, especially in the 1990s after publication of supportive findings from Finland.6 Confusion has been introduced into the debate by inappropriate study designs in tests of the hypothesis. A recurring weakness is that stored iron is measured by inappropriate methods. In addition, evidence against a particular mechanism may be erroneously taken as evidence against the core hypothesis. For example, in a particular study design, a finding of no association of stored iron with coronary atherosclerosis would not invalidate an association of stored iron with cardiovascular mortality. Recent studies relevant to the iron hypothesis have been reviewed.5 7 8 9 10 11 12 In 1994, Ascherio and Willett13 recognized that the iron hypothesis cannot be rejected on the basis of available data, stating that “[s]tronger evidence is needed before the hypothesis is rejected that greater iron stores increase the incidence of coronary heart disease or death from myocardial infarction.” Indeed, nothing in data made available since 1994 excludes the possibility that iron depletion has a large protective effect, large enough to explain the low incidence of myocardial infarction in menstruating women. A 1997 editorial14 on the iron hypothesis suggests that this once-controversial idea has become more acceptable to many scientists. Gillum14 noted that “this important hypothesis and related research fronts have led to many valuable studies like that of Kiechl et al [see Reference 99 ], which will greatly enhance our understanding of atherosclerotic vascular disease and may lead to innovations useful in clinical medicine and public health, perhaps in heretofore unanticipated ways.” Mechanisms of protection have not been …