When untreated, hemochromatosis is associated with significant CLINICAL GENOMICS. Hemochromatosis: Genetic Testing and Clinical Practice.

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1 CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2005;3: CLINICAL GENOMICS Hemochromatosis: Genetic Testing and Clinical Practice HEINZ ZOLLER and TIMOTHY M. COX Department of Medicine, University of Cambridge, Cambridge, United Kingdom 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. When untreated, hemochromatosis is associated with significant morbidity and even premature death. Because effective treatment is available, early diagnosis is the key to preventing or ameliorating hemochromatosis and, it is believed, most of the complications associated with iron storage. Recently, a profusion of genes implicated in iron transport and storage has come to light largely as a result of investigations to characterize several unique syndromes of pathologic iron storage (Table 1). Genetic testing has become an integral part of the diagnostic approach to the patient with suspected iron overload, but clinical interpretation of genetic tests is confounded by the polygenic nature of hemochromatosis and the strong environmental co-factors that determine its clinical expression. Therapeutic decisions are therefore based on the integration of all available information including the results from clinical examination, biochemical, radiologic, and genetic analysis; in case of doubt, liver biopsy with histopathologic examination might still be required for diagnosis or staging of disease. The aim of this article is to review the role of genetic tests in the clinical management and the molecular understanding of this disease as well as their future implications. Diagnosis Clinical Presentation Hemochromatosis is a systemic disorder mainly affecting the liver, heart, pancreas, and endocrine system. Symptoms of hemochromatosis include fatigue, darkening of the skin, hair loss especially in the axillae, impotence, and arthralgia caused by arthropathy typically affecting the metacarpophalangeal joints. Heart failure, arrhythmias, and diabetes are less common but are associated with a worse prognosis. 1 Hemochromatosis might further come to light as liver fibrosis or cirrhosis with complications including hepatocellular carcinoma, which has been reported in the noncirrhotic stages of liver disease. Hepatocellular carcinoma accounts for about one third of deaths in patients with hemochromatosis. 2 Finally, impairments of the endocrine system, including hypogonadism and adrenocortical failure, are rare manifestations in adults with hemochromatosis, but they occur frequently in young subjects with pathologic iron storage (Table 2). Alcohol is a key environmental factor in determining clinical expression of hemochromatosis, and alcohol consumption should be assessed when patients with suspected iron overload are investigated. A comprehensive interpretation of clinical, biochemical, and histologic findings also Abbreviations used in this paper: CT, computed tomography; DMT1, divalent metal transporter 1; MRI, magnetic resonance imaging; OMIM, online Mendelian inheritance in man; TfR2, transferrin receptor by the American Gastroenterological Association /05/$30.00 PII: /S (05)

2 946 ZOLLER AND COX CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 3, No. 10 Table 1. Recently Identified Proteins Implicated in Iron Storage and Transport Gene Protein (Proposed) Function DCYTB duodenal cytochrome b Intestinal brush border ferrireductase DMT1 divalent metal transporter 1 Apical enterocyte/erythroblast carrier protein HCP1 heme carrier protein 1 Intestinal heme transporter HEPH hephaestin Membrane bound ceruloplasmin homologue FPN1 ferroportin 1 Basolateral enterocyte/ macrophage carrier protein HFE HFE Non-classic HLA class I molecule TFR2 transferrin receptor 2 Low affinity hepatic irontransferrin receptor HFE2 hemojuvelin Putative GPI anchored membrane protein HAMP hepcidin Iron regulatory peptide NGAL lipocalin-2 Putative siderophore-binding protein GPI, glycosylphosphatidylinositol. requires a complete history including a record of blood transfusions, the use of iron and other supplements, family history, ethnic origin, and the precise nature of the diet. Biochemical Abnormalities When iron overload is suspected, serum concentrations of ferritin, transferrin, serum iron, and transferrin saturation or total iron binding capacity should be measured. Increased serum ferritin concentrations are also found in inflammatory or malignant diseases, where C-reactive protein and blood sedimentation rate are elevated; under these circumstances, serum iron and transferrin saturation are usually reduced. In contrast, elevated transferrin saturation predicts the presence of hemochromatosis with confidence. 3 A full blood count will exclude iron-loading anemias such as sideroblastic anemia, thalassemia, and other hemoglobinopathies, particularly in patients of African or Southern European origin. Serum albumin, bilirubin, and coagulation studies, as well as the activities of ALT and AST, will provide information about liver function and reveal the presence of hepatocellular injury. Elevated serum ferritin with normal or low transferrin saturation in the absence of inflammation is typically found in patients with hyperferritinemia-cataract syndrome or hemochromatosis type 4 (ferroportin syndrome, see below). Genetic Testing In 80% 90% of hemochromatosis patients of Northern European ancestry, the disease is associated with homozygosity for the so-called C282Y polymorphism or compound heterozygosity for the C282Y and H63D polymorphism of the HFE gene on chromosome In patients with clinical and biochemical iron overload, genetic testing for hemochromatosis is the rule. The presence of the following indicates predisposition to hemochromatosis: (1) homozygosity for C282Y or (2) compound heterozygosity for C282Y and H63D. Absence of these hemochromatosis genotypes in patients with suspected iron overload poses a diagnostic challenge. In a subgroup of such patients, compound heterozygosity for the C282Y and the S65C mutation has been found, and this mutation seems to be particularly common in certain geographic regions such as Brittany, where testing for the S65C 8 mutation in C282Y heterozygotes with suspected hemochromatosis is recommended. Several less common mutations in the HFE gene including V59M, 9 R66C, 10 G93R, I105T, 9,11 E168Q, 12 R224G, 10 and V295A 13 have been identified in patients with iron storage and heterozygosity for the C282Y mutation on the other parental allele. Among these mutations, E168Q has exclusively been found in association with the H63D mutation, both of which are thought to occur in cis on the same chromosome. A reverse-hybridization assay that allows simultaneous identification of 11 known mutations of the HFE gene is commercially available (ViennaLab, Vienna, Austria; This method also allows genetic testing for a few known mutations in the ferroportin 1 (FPN1) and transferrin receptor 2 (TFR2) gene simultaneously. In summary, genetic testing in patients with suspected hemochromatosis has a high positive predictive value, but genotypes that are not associated with hemochromatosis do not exclude the disease and require further investigation by using magnetic resonance imaging (MRI) and/or liver biopsy. There are a few private HFE mutations including the splice site mutation, IVS3 1G T 14 and intronic variants such as IVS5 1 G/A 15 reported, the latter of doubtful clinical significance. 12,13 Iron storage disease in rare and particularly consanguineous pedigrees in the absence of HFE mutations should prompt molecular analysis of other genes including TFR2, HFE2, and the HAMP gene. Table 2. Signs and Symptoms of Hemochromatosis Symptoms Abdominal pain Darkening of the skin Fatigue Hair loss (axillary) Arthralgia Loss of libido/impotence Signs Liver cirrhosis a Hepatocellular carcinoma Cardiomyopathy and arrhythmias Diabetes mellitus Arthropathy (metacarpophalangeal joints) Hypopituitary hypogonadism a Portal hypertension is not a prominent feature of hemochromatosis.

3 October 2005 HEMOCHROMATOSIS 947 Imaging Studies Imaging studies contribute greatly to the evaluation of hemochromatosis. MRI is the preferred modality 16 (Figure 1). Noninvasive quantification of iron storage by MRI has a clear advantage over chemical quantification of liver biopsy specimens. The procedure moreover allows repeated measurements, and sampling errors are minimized (Figure 2). Absolute iron quantification is an innovative development using software to capture sequential information from particular T2 weighted relaxation proton signals (FerriScan; Inner Vision Biometrics Pty Ltd, Claremont, Perth, Australia; Superconducting quantum interference device (SQUID) represents an alternative tool for noninvasive magnetic measurements of nonheme hepatic iron content, which has a lower sensitivity but higher accuracy when the concentrations of iron in liver tissue are high; this is particularly useful in monitoring chelation therapy in thalassemic patients. Availability of this method is restricted to a few academic centers worldwide. 17 The sensitivity of computed tomography (CT) scanning for quantification of hepatic iron is lower than that of MRI, but x-ray hypodensity of the liver might be a crude sign of iron overload. Contrast-enhanced triphasic CT scanning and MRI are both gold-standard techniques for the diagnosis of suspected hepatocellular carcinomas, 18 which are a complication of hemochromatosis, even in the noncirrhotic livers. Although sonography has a poor sensitivity for iron, liver ultrasound contributes importantly in the staging of liver diseases and portal hypertension as well as the surveillance of malignant tumors. 18 Histologic Findings Figure 1. T2-weighted MRI images of healthy control (A), a patient with hemochromatosis type 1 (B), and a patient with hemochromatosis type 4 (ferroportin syndrome; C). In both hemochromatosis patients, the liver shows a decreased signal intensity; in hemochromatosis type 4, the signal intensity of the spleen is also markedly decreased. Figures are courtesy of Reto Bale, Innsbruck, Medical University, Austria. Liver biopsy might provide valuable diagnostic and prognostic information in patients iron overload. Besides histologic distribution and grading of the iron storage, biopsy reveals the severity of fibrosis and might indicate coexistent viral, autoimmune, alcoholic, metabolic, or even drug-related liver disease. Hepatic iron concentration is an indicator of total body iron status and is used to calculate the hepatic iron index. This index (Hepatic iron [ mol/g dry liver tissue] Age [y]) corrects for the physiologic increase in hepatic iron with age and might differentiate between hemochromatosis and excess iron associated with chronic hepatic inflammation or heavy alcohol consumption. 19 Numerous studies confirm the value of the hepatic iron index in diagnosis of hemochromatosis; an index of 1.6 is a strong diagnostic criterion for hereditary hemochromatosis Recently, clinical evaluation of end-organ injury followed by biochemical studies and genetic testing

4 948 ZOLLER AND COX CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 3, No. 10 Figure 2. Proton transverse relaxation rate (R2) images and distributions for the liver of a subject hereditary hemochromatosis homozygous for C282Y at intervals during venesection therapy. Images were obtained by using the method of St Pierre et al. 134 Higher R2 values correspond to higher tissue iron concentrations. The liver images illustrate the spatial variation of liver iron concentrations (LICs) throughout the liver, whereas the R2 distributions give quantitative information on the mean LIC and its variation in the organ. The mean LICs are derived from a calibration curve relating R2 to LIC, enabling noninvasive measurement of LIC with high sensitivity and specificity. 16 Measurement of LIC in combination with measurement of body mass enables the number of blood units required for iron depletion by venesection to be estimated. Figure is courtesy of Dr Tim St Pierre, The University of Western Australia. has reduced the number of patients in whom liver biopsy is required. Liver biopsy remains valuable as a diagnostic tool in patients with pathologic serum iron parameters without causal mutations in the HFE gene. In one study, serum ferritin of greater than 1000 g/l with decreased platelet counts ( /L) and increased ALT was associated with cirrhosis in up to 90% of C282Y homozygotes. 25 Although these criteria have been considered indications for liver biopsy, it might be argued that they should be taken to demand immediate therapeutic intervention, leaving biopsy for patients with more ambiguous manifestations of disease. Hemochromatosis is characterized by fibrosis in association with cellular iron deposition in various organs, most notably the liver, heart, pancreas, and pituitary and adrenal glands (especially the zona glomerulosa). In contrast, hemosiderosis can be regarded as tissue iron deposition in the absence of tissue damage. 26 In genetic hemochromatosis, stainable iron is found in hepatocytes 27 (Figure 3A). Iron storage in macrophages and Kupffer cells might be a his-

5 October 2005 HEMOCHROMATOSIS 949 Figure 3. Histologic appearances in hemochromatosis liver. (A) Perls stain of liver section from a cirrhotic patient with hemochromatosis type 1: intrahepatic iron deposition. (B) Hematoxylin-eosin stain of a liver section from a patient with hemochromatisis type 4 (ferroportin syndrome): hepatic hemosiderin deposits without affecting the hepatocytes. tologic finding at advanced stages of the disease. A little noted feature of hemochromatosis is the absence of necroinflammatory changes in the affected tissues. Ultrastructural studies in livers from patients at early stages of the disease showed that iron initially accumulates in lysosomes, 28 whereas in later stages, iron-rich aggregates are found in the cytoplasm. 29 Kupffer cell siderosis suggests that the iron overload is related to chronic liver inflammation, repeated blood transfusions, heavy alcohol consumption, and iron-loading anemias. 30 However, mutations in the gene encoding ferroportin 1 on chromosome 2q32 (FPN1, IREG1, MTP1, SLC40A1) were identified in families with autosomal dominant transmission of primary reticuloendothelial iron overload, ie, iron storage principally affecting cells of the macrophage system (Figure 3B). Stainable iron in macrophages on histologic examination and iron storage affecting liver and spleen on MRI in the absence of coexisting diseases are an indication for genetic investigation of the ferroportin 1 gene, especially in cases with affected first-degree relatives. Nomenclature and Classification Attempts to unify the classification and nomenclature of human iron storage disorders by international experts have hardly penetrated the canon of medicine and have been bedevilled by the rapid identification of novel genetic defects implicated in these diseases, which has required constant revision. 34 The most up-to-date classification of iron storage disorders is provided by online Mendelian inheritance in man (OMIM) ( ncbi.nlm.nih.gov/) and is based on the underlying genetic defect. However, this classification fails to include bigenic variants or take into account the rare disorder of neonatal hemochromatosis. Hereditary hemochromatosis now includes at least 5 defined genetic entities, each with different clinical and histopathologic features (Table 3). Adult Hemochromatosis The most common forms of hemochromatosis become manifest in the third to fifth decades of life and are therefore referred to as adult hemochromatosis. Hemochromatosis type 1 is the most common form of adult iron overload and is associated with mutations of the HFE gene on each parental allele (see above). A rare variant of adult hemochromatosis is bigenic hemochromatosis, in which the association of C282Y heterozygosity with a heterozygous mutation in the hepcidin gene is associated with adult iron overload. 35 In contrast, digenic inheritance of heterozygous mutations in the HFE gene and a heterozygous mutation in the

6 950 ZOLLER AND COX CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 3, No. 10 Table 3. Classification of Hereditary Hemochromatosis Genetic defect Clinical classification OMIM classification (gene name) Homozygous or compound heterozygous for mutations in the gene Heterozygous for mutations in the gene Inheritance Frequency Adult hemochromatosis HFE HFE Autosomal recessive 60% 95% of all HH cases HFE3/TFR2 TFR2 Autosomal recessive 50 cases a FPN1/SLC40A1 FPN1 Autosomal dominant 200 cases Not classified HFE HFE2 Digenic 3 cases Not classified HFE HAMP Digenic 2 cases Juvenile hemochromatosis HAMP HAMP Autosomal recessive 3 cases HFE2 HAMP Autosomal recessive 50 cases Neonatal hemochromatosis NH/NHC Unknown Immunity against Paternal antigen(s)/ Autosomal recessive 100 cases OMIM, Online Mendelian Inheritance in Man. a Mutations in the TFR2 gene are associated with a relatively early onset of iron overload. hemojuvelin gene was not associated with pathologic iron storage. 36 Homozygosity for the C282Y mutation in the HFE gene and heterozygosity for hemojuvelin gene mutations are associated with a more severe phenotype when compared with C282Y homozygosity alone. 37 There are other rare forms of genetic hemochromatosis in which patients have classic clinical features of hepatocellular iron overload, hypogonadism and (latent) diabetes, but lack mutations in the HFE gene. 38 In such patients, mutations in the gene encoding transferrin receptor 2 (TFR2, HFE3) on chromosome 7q22 have been identified. 39 The molecular genetics of hemochromatosis associated with TFR2 are complicated by the private nature of most TFR2 mutations (Y250X, 39 R105X, 40 E60X, M172K, 41 AVAQ del, 42,43 and Q690P. 42 ) Therefore complete sequencing of the TFR2 gene, a laborious process, is usually required to identify pathologic mutations. Autosomal Dominant Adult Hemochromatosis This form of iron overload was termed hemochromatosis type 4 (or ferroportin syndrome) and is clinically distinct from classic hemochromatosis. 44 Patients with hemochromatosis type 4 rarely present with cirrhosis, but marked hyperferritinemia, usually 1000 g/l, occurs before the third decade of life, which might be detected fortuitously. In contrast to classic hemochromatosis (types 1 and 3), elevation of transferrin saturation is unusual Extrahepatic manifestations have been described in some cases, but no firm clinical association with heart disease, diabetes, arthropathy, or hypogonadism has yet been established. Again, the molecular genetics of the disease are complex; the most frequent mutation is the del169v mutation, and a growing list of mutations with distinct clinical and molecular features is being reported in association with primary macrophage iron storage (see Table 1 44,52 ). In contrast to the C282Y and H63D mutations of the HFE gene, which are almost exclusively found in whites, 3 FPN1 gene mutations have been identified in patients of various ethnic backgrounds including Africans and Asian Pacific Islanders. 53 Juvenile Hemochromatosis Clinical presentation of iron overload before the age of 30 years is arbitrarily defined as juvenile hemochromatosis. 54 A striking feature is the almost equal occurrence of inherited juvenile hemochromatosis in male and female patients, whereas in adult hemochromatosis, men are 9 times more likely to be affected than women. 26,55 This might reflect the severity of the metabolic abnormality, which overwhelms the potential modifying effects of diet, menstruation, and pregnancy on clinical expression and severity of iron storage in adult hemochromatosis. Furthermore, the heart and endocrine system are more severely affected in juvenile than in adult hemochromatosis (Figure 4). 56,57 Life-threatening cardiac disease is frequent and usually associated with infantilism as a result of gonadal failure. Genetic linkage of the disease with a locus on chromosome 1q was first reported in 1999, 58 and mutations in both alleles of a then newly identified gene termed HFE2 (HJV) were first reported in and later confirmed in other patient cohorts. 60 In cases of juvenile hemochromatosis not linked to chromosome 1, homozygous mutations in the gene encoding hepcidin (HAMP, chromosome 19q13) were found

7 October 2005 HEMOCHROMATOSIS 951 Figure 4. Histologic appearances in juvenile hemochromatosis. Sections from the heart of a 27-year-old woman who underwent heart transplantation for cardiac failure. Whole mount section of the heart muscle stained with (A) hematoxylin-eosin and (B) Perls stain. Both sections show iron deposits principally affecting the outer myocardial layer. (C and D) High-power view of the affected tissue showing iron deposits in cardiomyocytes in hematoxylin-eosin stained sections (C) and heart stained with Perls reagent. 135 The distinct nosologic entity of juvenile hemochromatosis is, however, challenged by the recent identification of combined genetic defects in HFE (compound heterozygosity for C282Y and H63D) and TFR2 (Q317X) in patients with juvenile hemochromatosis in the absence of HAMP or HFE2 mutations. 64 Neonatal Hemochromatosis Neonatal hemochromatosis is a condition in which the fetus or neonate has hepatic injury and heavy iron deposition in the liver, as well as the extrahepatic tissues such as salivary glands and pancreas. 65 Stainable iron is a normal finding in the liver of the newborn, and abnormalities in iron metabolism occur in the context of several perinatal or neonatal infections or metabolic conditions affecting the fetus; these pose a particular challenge for the diagnosis of neonatal hemochromatosis. The occurrence of cirrhosis in the newborn combined with extrahepatic iron deposition in the absence of infections have been proposed as diagnostic criteria. 66 In suspected cases, mucosal biopsy of the lips with histologic examination of the minor salivary glands might be considered as a low-risk intervention of diagnostic value. 67 The prognosis of neonatal hemochromatosis is poor, and when untreated the outcome is usually

8 952 ZOLLER AND COX CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 3, No. 10 fatal within the first weeks of life. The most effective therapeutic option is liver transplantation 68 ; alternatively, a cocktail of different antioxidants combined with desferrioxamine has been suggested to improve outcome. 69 The high rate of reoccurring neonatal hemochromatosis in sibs is the basis for the hypothesis that neonatal hemochromatosis is a genetic disorder, but the welldocumented occurrence of the disease in maternal halfsiblings indicates the operation of strong maternal factors, including mitochondrial inheritance or combined environmental and genetic defects. Linkage of the disease with HFE or chromosome 1q (HFE2) was excluded. 65 Findings from a recent trial indicate that the risk for neonatal hemochromatosis can be reduced by treatment to modify immune responses during pregnancy. Weekly intravenous infusion of 1 mg/kg body weight pooled human immunoglobulin from the 20th gestational week to term resulted in a lower frequency and milder presentation of neonatal hemochromatosis in 16 newborns than expected, thus supporting the hypothesis that the pathogenesis of neonatal hemochromatosis involves immunologic factors of maternal origin. 70 Molecular Basis of Disordered Iron Homeostasis in Hemochromatosis Despite considerable progress in the identification of genetic defects, in patients with iron storage disorders, the molecular pathophysiology of hemochromatosis remains incompletely understood. Hemochromatosis arises from an imbalance between iron absorption and excretion. Because there is compelling evidence that in human beings iron excretion is greatly restricted, 71 it is generally accepted that the pathologic iron storage in genetic hemochromatosis arises from inappropriately increased absorption of dietary iron. 72 Hepcidin: An Iron-Hormone The anatomic separation between the sites for iron uptake, storage, and utilization in duodenum, liver, and bone marrow, respectively, indicate the need for informational signalling about iron status and requirements between liver, bone marrow, and the small intestine. There is emerging evidence that hepcidin might serve as a signalling molecule because it is produced by the liver and released into the blood in response to iron loading or acute infections. 73 The link between hepcidin and iron metabolism initially arose from the observation that hepcidin is significantly up-regulated in response to parenteral induction of iron overload in mice. 74 Subsequently, iron storage was observed in a mouse model in which the hepcidin was disrupted serendipitously. 75 Identification of mutations in the human hepcidin gene in pedigrees affected with juvenile hemochromatosis (not linked to chromosome 1q) 61 demonstrates the importance of hepcidin in human iron metabolism. Furthermore, there is accumulating evidence from human studies as well as various animal and cell models 85 supporting a central role for hepcidin as the iron hormone, whose absence allows uncontrolled intestinal absorption of iron from the diet. Elevated concentrations of hepcidin in the blood and urine are found in conditions of macrophage iron storage and when intestinal iron absorption is reduced, such as acute inflammatory responses and transfusional iron overload. 77 Iron loading anemias such as congenital dyserythropoietic anemia or thalassemia intermedia, in which iron absorption is increased, are associated with low urinary hepcidin concentrations. Similarly, different forms of juvenile and adult hemochromatosis, with the exception of hemochromatosis type 4, are also associated with low concentrations of hepcidin in the urine. 78 For adult hemochromatosis and animal models of the disease, relatively decreased (or for the degree of iron overload not sufficiently increased) steady state mrna expression has been found. 80 It has been postulated that the molecular target of hepcidin is ferroportin 1, which appears to be inactivated after binding to hepcidin. Ferroportin 1 is a multi-transmembrane protein that is expressed in duodenal enterocytes and macrophages, where it is implicated in cellular iron export. Human hepcidin analogues specifically bind to ferroportin 1 in vitro and induce internalization and degradation of the complex. This mechanism accords well with functional data showing that hepcidin reduces iron export in macrophages and enterocytes. 86 The finding that patients with certain mutations in the gene encoding ferroportin 1 (hemochromatosis type 4) have increased urinary hepcidin excretion supports the hypothesis that hemochromatosis type 4 can be viewed as a form of hepcidin receptor resistance, thus allowing continued iron absorption despite increased hepcidin concentrations under conditions in which about one half of its receptors are inactivated by mutations in the FPN1 gene. Some but not all ferroportin mutations that are associated with hemochromatosis type 4 were recently shown to be nonfunctional in cellular iron export, 87 without a dominant negative effect on the functional ferroportin allele. The phenotypic heterogeneity of ferroportin syndrome might be explained by the different effect of certain mutations either on hepcidin binding or on cellular iron export. HFE: The Hemochromatosis Gene Product HFE is expressed in the liver, and although HFE antibodies mainly stain non-parenchymal cells, 88 reverse transcriptase polymerase chain reaction analysis of different isolated cell fractions from the liver indicates that HFE mrna expres-

9 October 2005 HEMOCHROMATOSIS 953 sion is most abundant in hepatocytes. 89 The role of HFE in iron homeostasis is not fully elucidated, but there is mounting evidence that HFE controls iron export from cells. 90 HFE is a non-classic major histocompatibility complex class 1 molecule, which can physically interact with transferrin-receptor 1, and this interaction has been shown to lower the affinity between transferrin and its receptor. 91 At the concentrations of iron saturated transferrin found in plasma, however, HFE has no effect on the interaction between transferrin receptor and its ligand. 92 The C282Y mutations in the HFE gene result in retention and degradation of the nascent protein in the Golgi complex, because the mutation prohibits assembly of the HFE protein with 2 microglobulin, which would be essential for targeting of HFE to the cell membrane. 93 The finding that 2 microglobulin knockout mice develop iron overload accords well with the proposed effect of the C282Y mutation. 94 The functional consequences of HFE gene mutations in vivo have been demonstrated in several knockout mice, which develop age-dependent iron accumulation in the liver but no cirrhosis. 95,96 Further studies have shown that the genetic background significantly contributes to the severity of iron overload in different HFE knockout mouse strains. 97 Genetic linkage analysis for cognate modifier loci led to identification of the murine hepcidin gene on mouse chromosome 7 as the major modifier gene. 98 Different degrees of iron loading on different genetic backgrounds are associated with variation in the expression of iron transporters in murine intestine. 99 This is in accord with human studies, in which intestinal iron transporters were found to be overexpressed. The basolateral duodenal iron transporter ferroportin 1 was unequivocally increased in hemochromatosis patients when compared with control subjects, but conflicting evidence for the regulation of the apical intestinal iron transporter divalent metal transporter 1 (DMT1) exists On the basis of clinical and pathologic similarities between hemochromatosis associated with mutations in the genes encoding transferrin receptor 2 and HFE (types 1 and 3), a functional relationship of transferrin receptor 2 and HFE for iron sensing has been proposed. The reported co-localization between HFE and transferrin receptor is awaiting further confirmation, and functional studies have excluded a physical interaction between those molecules at neutral ph. 107 In summary, the molecular pathogenesis of adult hemochromatosis and the consequences of HFE deficiency in various cell types are still unclear, and hepcidin is emerging as an important regulator of iron metabolism. With the exception of hemochromatosis type 4, low hepcidin expression and secretion is a unifying feature of all primary iron overload syndromes, but the molecular mechanisms underlying the functional interaction between hemojuvelin, HFE, and transferrin receptor 2 are still unresolved. Treatment Treatment for hemochromatosis should be considered when a patient presenting with biochemical indication of iron storage has an HFE genotype associated with the disease (homozygous for C282Y or C282Y/ H63D heterozygous). Diagnosis of hemochromatosis requires the finding of elevated fasting transferrin saturation on at least 2 occasions and in the absence of other known causes of elevated transferrin saturation, 108 such as transfusional iron overload or iron loading anemias. It should be mentioned that serum transferrin saturation is subject to diurnal variation, and a fasting morning specimen is required for confident measurement. Phlebotomy provides in most cases an effective strategy to remove iron, but in anemic patients and in patients with ascites or severe heart disease with hypotension and arrhythmias, it might not be tolerated or might even be contraindicated. In such patients, treatment with parenteral desferioxamine (2g/day subcutaneous infusion during at least 8 hours per day) usually achieves a negative iron balance and might be lifesaving. 109 Phlebotomy is usually carried out at weekly intervals until mild anemia (hemoglobin, g/dl) develops. The amount of iron mobilized is about 250 mg of iron per unit of blood removed or calculated by the following formula: Fe [g] hemoglobin concentration [g/dl] blood removed [L]. The amount of iron removed before development of anemia provides a reasonably accurate estimate of the degree of iron overload. In normal men, the mobilizable iron pool is 5 g, and in women it is less than 3 g; in patients with hemochromatosis, up to 20 g of iron can be removed before anemia develops. 110,111 After successful iron removal, serum ferritin concentrations should be kept below 50 g/l, which is usually achieved by maintenance therapy with 4 6 phlebotomies per year. Although the efficacy of phlebotomy has never formally been tested in a controlled trial, the incidence and prevalence of liver carcinomas, liver cirrhosis, heart disease, and diabetes are lower in treated hemochromatosis patients than expected. 2, In selected cases, phlebotomy has even been reported to reverse portal hypertension and liver cirrhosis, 109,114 but in cirrhotic patients phlebotomy is associated with a significant risk of decompensation of the liver disease. It should be noted that the incidence of liver tumors is lower during phlebotomy, but the development of hepatomas during phle-

10 954 ZOLLER AND COX CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 3, No. 10 botomy has been reported, and surveillance for liver carcinomas during treatment is therefore mandatory. 114 Arthritis and impotence symptoms are common in individuals with hemochromatosis and can have a significant impact on their quality of life, but in contrast to liver and heart disease, arthropathy and diabetes do not improve on venesection. Sporadic reports indicate that hypogonadotrophic hypogonadism might resolve completely after institution of iron depletion. 55,115,116 Finally, liver and heart transplantation have been reported as effective measures for end-stage organ failure; hemochromatosis generally is a rare indication for liver transplantation, and no evidence-based advice on phlebotomy treatment after liver transplantation can be given. Accidental transplantation of organs from donors with hemochromatosis has been reported, but conflicting data on the development or regression of iron storage in such organs exist. 120,121 The association between cirrhosis and hemochromatosis type 4 is not clearly established, and phlebotomy, which is not well tolerated in those patients, should therefore be carried out with caution. In summary, patients with abnormal serum iron parameters (male: serum ferritin 300 g/l and transferrin saturation 50%; female: ferritin 200 g/l and transferrin saturation 45%) and a hemochromatosis-associated HFE genotype (C282Y homozygosity or C282Y/H63D heterozygosity) should be treated by weekly phlebotomy (540 ml), and the mobilizable iron calculated by quantification of the iron removed before development of anemia (1 ml of blood, hemoglobin 14 g/dl, is estimated to contain 0.49 mg of elemental iron). A liver biopsy is of diagnostic importance in patients with suspected iron overload and normal HFE genotype, heterozygosity for C282Y or H63D, as well as H63D homozygotes. The availability of facile treatments renders early diagnosis of iron overload syndromes mandatory. Because the morbidity associated with hemochromatosis can be reduced by phlebotomy, it is clearly important to identify at-risk individuals early. 1,2,112 Screening Family Screening Most authorities agree that family screening should be initiated once the diagnosis of hemochromatosis has been made. 122 Different screening strategies have been proposed, and the most practical and cost-effective in middle-aged patients seems to be genotypic screening in siblings and spouse before screening in offspring. 123 Although prospective data on the outcomes of patients with C282Y homozygosity and normal serum iron parameters are lacking, the expressivity of HFE gene mutation seems to be higher in family members of patients with hemochromatosis 124 than HFE gene mutations in the general population 125 or among blood donors. 126 This difference was attributed to common modifier genes and similarities in lifestyle within families. 127 Serum iron parameters should be determined in individuals identified with hemochromatosis-associated genotypes, and according to the iron status either active treatment or surveillance should be initiated. Screening in families of patients with hemochromatosis in the absence of HFE mutations should be carried out on a phenotypic basis by determination of serum transferrin saturation. This does not apply to hemochromatosis type 4 (ferroportin syndrome), which is the only known autosomal dominant variant of adult iron overload syndromes. In those cases, serum ferritin determination will reveal affected family members. We also have evidence that serum pro-hepcidin is significantly elevated in such patients, which is helpful in establishing the diagnosis. 128 In contrast to urinary hepcidin, serum pro-hepcidin can be measured by using a commercially available enzyme-linked immunosorbent assay (DRG International, Inc, Mountainside, NJ; Family screening in patients with juvenile hemochromatosis requires genetic diagnosis in the index case, where the genes encoding hemojuvelin and hepcidin should be sequenced and genotyping offered to family members. Finally, family screening and in particular antenatal diagnosis would be extremely desirable in families affected with neonatal hemochromatosis, but genetic testing is impossible, and fetal iron overload develops very late in pregnancy, which renders diagnosis by MRI difficult. Population Screening Although complications of hemochromatosis are widely stated to be prevented by early phlebotomy, population screening for hemochromatosis is not generally recommended at present. One problem has been the assignment of a category termed pre-cirrhotic hemochromatosis, especially by investigators who have usefully evaluated the outcome of venesection therapy in patients with iron storage disease and in populations in which a high frequency of HFE C282Y-related hemochromatosis might be predicted (Niederau, northern Italian Fargion). 2,128 We submit that such an assignment presumes that an individual with abnormal iron parameters indicating increased storage at one point will inevitably develop tissue iron injury and full-blown hemochromatosis, with or without cirrhosis. Recently,

11 October 2005 HEMOCHROMATOSIS 955 studies of the expressivity and penetrance of the C282Y genotype (see below) have shown that C282Y homozygosity is not tantamount to a diagnosis of hemochromatosis or absolutely predictive of its development, even though iron accumulation in the liver correlates with age. Long-term follow-up studies would be necessary to determine the course of iron storage over time and might be crucial for identifying those environmental and other co-factors that influence expression and progression of disease with accompanying tissue injury. With the low and variable penetrance of this genotype and the presence of other genetic determinants of iron storage, homozygosity for C282Y appears to be neither necessary nor sufficient for the development of hemochromatosis. The penetrance of homozygosity for the C282Y mutation has been estimated at 1% in a populationbased and about 25% in a family-based screening study. 124,125 Defining the penetrance of C282Y homozygosity is clearly of material importance for the introduction of mass population screening for the homozygous C282Y genotype and has been the objective of numerous studies. 124,126, Variation in the inclusion criteria and the different definitions for hemochromatosis might explain the inconsistency of the results, with estimated penetrance ranging from 1% 50%. 130 Formally, it is also possible that differences in the genetic structure of the populations studied and their dietary iron content might materially affect the rate of iron loading Population screening for HFE-associated hemochromatosis has lately been described in an Australian study as practicable and acceptable, but here its effectiveness may be questioned because 46 of the 47 homozygotes detected by screening more than 11,000 individuals had already taken steps to prevent iron storage before they were identified. 132 Phenotypic screening has also not yet found wide acceptance because of lack of specificity and diagnostic yield. 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