Non-HFE hemochromatosis
Authors: Antonello Pietrangelo
Journal: Hepatology (2004)

The term 'hemochromatosis' refers to an autosomal recessive disorder of iron metabolism associated with two mutant alleles of the HFE gene (usually leading to a Cys282Tyr mutation in the gene product) and characterized by a slow and progressive increase in plasma iron content, which, in adults, may lead to systemic iron loading of parenchymal cells (particularly hepatocytes) and, eventually, to organ disease. In rare cases, mutations in other iron-loading genes may result in a similar syndrome. The term was coined by von Recklinghausen in 1889 to describe the association at autopsy of widespread tissue injury, usually cirrhosis, with increased tissue staining for iron.1 Sheldon, in his review of all published cases, linked this term to an inherited disorder of iron metabolism that is overwhelmingly more common in males and sometimes has a familial incidence.2 A breakthrough in the history of the disease came with the recognition of its autosomal recessive nature and the location of the pathogenic gene on the short arm of chromosome 6.3, 4 These observations preceded the identification of an iron-regulating gene, now named HFE,5 that is mutated in hemochromatosis. Once the HFE gene was identified, it immediately appeared to be clear that not all patients with an inherited hemochromatosis-like phenotype carried pathogenic mutations in the HFE gene. This was particularly evident in southern European countries.6, 7 Therefore, the term 'non-HFE hemochromatosis' was coined to define hereditary iron overload in patients without pathogenic mutations in the HFE gene.8 Like 'non A-non B hepatitis', 'non-HFE hemochromatosis' was mainly a negative term, based on the ignorance of the genetic basis of the underlying disorders. Since then, unprecedented progress in animal and human iron genetics has led to the identification of new forms of primary iron overload caused by mutations in other genes involved in iron metabolism. For one of these disorders (i.e., ferroportin-associated iron overload, or ferroportin disease), epidemiology, natural history, and molecular pathogenesis are different from the corresponding characteristics in HFE hemochromatosis. These differences also may suggest a different approach to diagnosis and screening. Other disorders share pathogenic mechanisms, clinical presentation characteristics, and an autosomal recessive genetic nature (e.g., TFR2 and juvenile-associated iron overload) with classic hemochromatosis. In this context, these disorders might be considered different forms of the same clinicopathologic syndrome. Therefore, the term 'non-HFE hemochromatosis' probably should be restricted to TFR2 and juvenile-associated iron overload. In addition to classic hemochromatosis and ferrorportin disease, they define the group of 'primary iron-overload disorders', i.e., disorders due to a defect in a gene primarily involved in iron homeostasis. Other primary iron-overload diseases are the rare forms of aceruloplasminemia9, 10 and atransferrinemia.11 In the former, the lack of ferroxidase activity of plasma ceruloplasmin leads to defective iron release from tissues; the clinical picture is dominated by a neurological syndrome. In the latter, the lack of transferrin leads to increased iron absorption and excessive iron influx in parenchymal cells; severe iron-deficiency anemia is the main clinical manifestation. A unique pedigree in which iron overload is associated with a mutation in the iron-regulatory element of the H-ferritin gene also has been described.12 A distinction should be made between primary iron-overload disorders and secondary iron-overload disorders, in which iron overload is secondary to specific diseases (e.g., thalassemia or other iron-loading anemias, porphyria, etc.) or acquired factors (e.g., alcohol exposure, liver diseases, etc.). RE, reticuloendothelial; mRNA, messenger RNA; IRE, iron-responsive element. Ferroportin disease (also known as ferroportin-associated iron overload) is an autosomal dominant inherited disorder of iron metabolism that results from pathogenic mutation of the SLC40A1 gene, previously called SLC11A3. The first description of the disease was published in 1999, when an autosomal dominant form of hereditary iron overload similar to classic hemochromatosis but not linked to chromosome 6p was reported.13 Distinctive features included tissue iron accumulation predominantly in reticuloendothelial (RE) cells, steadily increasing serum ferritin levels, which were disproportionately high compared with transferrin saturation, marginal anemia, and mild organ disease.13 In 2001, a genomewide screen in the original pedigree provided evidence of linkage with respect to markers on 2q32.14 A candidate gene, SLC40A1, was identified in that region, and all affected patients were heterozygous for a 230C→A substitution; which resulted in replacement of alanine 77, a small, hydrophobic amino acid, with aspartate, a large, negatively charged amino acid; within a predicted myristylation site of the ferroportin protein. In the same year, another group reported that a form of non-HFE hereditary iron overload was associated with heterozygosity for another ferroportin mutation (N144H) in a Dutch pedigree.15 Ferroportin disease now has been reported in many countries, and, at variance with the distribution of the C282Y mutation in the HFE protein, it is distributed worldwide regardless of ethnicity. A common mutation involves three sequential bases in exon 5 and predicts the loss of one of three valine residues at positions 160–162. It probably is due to a slipped-strand mispairing and has been found in pedigrees from the United Kingdom, Australia, Italy, and Greece.16-19 Other ferroportin mutations have been reported in French, French Canadian, and Asian families with hyperferritinemia.20-22 In 2000, ferroportin 1/IREG1/MTP1 protein was identified independently by three different laboratories and was shown to play a role in the export of iron in frog oocytes and in cell lines.23-25 It is expressed in cells that play a critical role in mammalian iron metabolism, including placental syncytiotrophoblasts, duodenal enterocytes, hepatocytes, and RE macrophages. In the human intestine, this protein is expressed strongly under conditions of enhanced iron absorption, such as anemia or hemochromatosis.26, 27 It is involved in the pathogenesis of the hypoferremia associated with anemia of chronic disease, characterized by iron trapping in reticuloendoethelial (RE) cells and reduced intestinal iron transfer.28 Ferroportin expression is responsive to iron and inflammatory stimuli.25, 26, 29-31 Its messenger RNA (mRNA) possesses an iron-responsive element (IRE) in the 5′ untranslated region that binds iron-regulatory proteins and may confer iron-dependent regulation. In agreement with this model, the ferroportin promoter is responsive to iron in HepG2 and CaCo2 cells: deletion of various promoter fragments does not eliminate iron-dependent regulation, whereas removal of the IRE leads to loss of iron control.32In vivo, additional mechanisms may be important in controlling ferroportin expression, particularly in the duodenum, in response to stimuli such as hypoxia or erythron demands.33 According to consensus structural predictions, ferroportin has 9 or 10 transmembrane helices.16, 23, 24 Although the reported mutations span the entire protein, the majority involve the region between the first and fourth transmembrane domains (Fig. 1). This region may be involved in iron binding and/or transport activity or may define a functional binding site for a circulating protein (possibly serum ceruloplasmin or hepcidin) that is important for export of iron from the cell. Positions along the protein backbone of known ferroportin mutations associated with hereditary iron overload. The software prediction of ferroportin membrane organization is adapted from Devalia et al.16 Copyright American Society of Hematology, used with permission. The proposed function of the gene product of SLC40A1 in iron export is consistent with the phenotype of the disease and with the original hypothesis of a selective disturbance of iron recycling in RE cells.13 The finding that different mutations in the same protein lead to the same disorder, characterized by iron accumulation in macrophages, is more consistent with a loss of protein function14 than with a gain of protein function.15 Nonetheless, no experimental proof of the functional effects of various ferroportin mutations has been provided yet. A loss-of-function mutation might cause impairment of iron export from cells, and mainly RE cells, which normally must process and release a large quantity of iron derived from the lysis of senescent erythrocytes (Fig. 2). This leads to tissue iron accumulation (which is responsible for high serum ferritin levels) but also to the decreased availability of iron for circulating transferrin (reflected in low transferrin saturation), which may be responsible for marginal anemia. Progressive tissue iron loading may result from both impaired release of iron from macrophages and hepatocytes and from increased iron influx following the compensatory activation of iron absorption due to marginal anemia. Lack-of-function mutations may be more relevant pathophysiologically in the context of macrophage iron metabolism but less important in iron export from the intestine and from hepatocytes, for which other systems may overcome the functional deficiency. Nonetheless, it is possible that different mutations throughout the protein may affect iron transfer capability differently or involve protein domains that are important in interactions with cell-specific molecular partners. These mutations also may have different effects on protein function depending on the specific cellular context. At the molecular level, it is likely, but not proven, that a mutated allele exerts a dominant negative effect over the wild-type allele. Pathogenesis of primary iron-overload diseases. Inactivation of genes primarily involved in iron metabolism, such as HFE, TFR2, and hepcidin, leads to uncontrolled release of iron from intestinal and macrophage cells and expansion of the circulating iron pool (transferrin-bound and non-transferrin-bound iron). Subsequently, increased iron influx into parenchymal cells is responsible for cell damage and organ toxicity. If the rapidity and extent of expansion of the circulating iron pool are high, as in the case of hepcidin-associated iron overload, endocrine and cardiac damage will dominate clinical presentation. The opposite occurs in the case of inactivation of HFE or TFR2. In ferroportin disease, defective release of iron from storage sites (particularly, reticuloendothelial macrophages) is primarily responsible for tissue iron overload. In ferroportin disease, reduced transferrin saturation may lead to inadequate iron supply to bone marrow and marginal anemia. Late in the disorder, progressive saturation of transferrin with iron also occurs, while the continuing iron retention in organs progresses. The earliest biochemical abnormality, appearing in the first decade of life, is high serum ferritin levels with normal or low transferrin saturation.13 Serum ferritin levels, as well as tissue iron loading, increase with age. In young females, hypochromic anemia may be reported and may require oral iron supplementation. In the fourth and fifth decades, the level of transferrin saturation also may increase, but it rarely reaches 100%, as it does in classic HFE hemochromatosis (Table 1). The biochemical penetrance of the genetic defect is complete, as all documented patients to date have exhibited hyperferritinemia. Clinically, according to available reports, the picture appears more heterogeneous, ranging from simple biochemical abnormality to the full spectrum of symptoms and signs that are typical of hemochromatosis.15 In general, the phenotype appears to be mild, and in spite of severe iron burden, liver disease is limited to signs of fibrosis, primarily sinusoidal. This is consistent with the typical pattern of hepatic iron distribution (Fig. 3) and with the notion that non-parenchymal cell (Kupffer cell) iron overload is better tolerated and less fibrogenic than parenchymal cell iron overload.34 In fact, histologically, early Kupffer cell iron overload is a characteristic feature of the disease; however, other studies have confirmed the original finding that some degree of parenchymal iron overload also is present (Fig. 3B).16, 17, 35 Hepatocellular iron overload has a homogeneous lobular distribution without the periportal-central iron gradient that is typical of hemochromatosis. Due to the mixed pattern of iron accumulation in parenchymal and nonparenchymal cells, a decrease in both liver and spleen signal intensity (due to a decrease in T2 relaxation time) can be observed on magnetic resonance imaging study (Pietrangelo A, unpublished observation, 2002). This differs from classic hemochromatosis, in which decreased signal intensity is evident only in the liver. It is likely that additional environmental factors (e.g., alcohol abuse, hepatitis infection, etc.) and genetic factors (e.g., HFE and non-HFE gene status) may influence the final clinical manifestation of the disease, as in classic hemochromatosis. Hepatic histologic pattern of iron accumulation in primary iron-overload disorders (Perls' Prussian blue stain). (A) Hemocromatosis. Cirrhotic-stage disease in a patient (age, 58 years) who is homozygous for C282Y. Inset: granular iron deposits in hepatocytes. (B) Ferroportin disease. Advanced-stage disease with massive iron overload characterized by coalescent iron deposits in Kupffer cells and macrophages (patient age, 65 years). Inset 1: early-stage disease with typical iron accumulation predominantly in Kupffer cells (patient age, 25 years). Inset 2: late-stage disease, viewed at high magnification; both Kupffer cells and hepatocytes accumulate iron. (C) TFR2-associated hemochromatosis (patient age, 32 years). Iron is observed in Zone 1 and in parenchymal cells (inset), as in early HFE hemochromatosis. (D) Hepcidin-associated hemochromatosis (patient age, 20 years). Massive iron overload of hepatocytes, with panlobular iron distribution, can be seen. Although phlebotomy is an effective therapeutic tool, in some individuals, a weekly phlebotomy program is not tolerated and slight anemia and low transferrin saturation rapidly occur despite still-elevated serum ferritin levels. With a less aggressive phlebotomy regimen, these patients also can become iron depleted, although a therapeutic target of serum ferritin concentration < 30 ng/mL, adopted for classic hemochromatosis, should be avoided, due to the risk of anemia. Adjuvant therapy with erythropoietin may be beneficial. Discontinuation of phlebotomy treatment is followed by a rapid rise in serum ferritin levels. Ferroportin disease should be suspected in all cases of familial hyperferritinemia and in sporadic cases in the absence of known secondary causes (e.g., infection, dysmetabolism, inflammation, or malignancy). Differential diagnosis also should consider the rare form of familial hyperferritinemia—congenital cataract syndrome—which is not associated with tissue iron overload,36, 37 aceruloplasminemia,9, 10 or dysmetabolic hepatosiderosis,38 which are present in individuals with dyslipidemia. Patients with autosomal recessive iron loading disorders similar to hemochromatosis may carry mutations in the TFR2 gene.39 Until recently, only one type of transferrin receptor, transferrin receptor 1 (TfR1), had been identified. Then, in 1999, Kawabata et al.40 cloned a second human transferrin receptor gene, TFR2, which mapped onto chromosome 7q22, had significant sequence homology with TFR1, and mediated the cellular uptake of transferrin-bound iron. The frequency of TFR2 mutations is low, and to date, they have been detected in four Italian, one Portuguese, and one Japanese pedigree.39, 41-43 Two transcripts of the TFR2 gene have been found in humans: an α form (TFR2-α), which is 2.9 kilobases long, and a β form (TFR2-β), which is 2.5 kilobases long. TfR2-β probably is a soluble intracellular form of TfR2. The tissue distribution and expression level of TFR2 mRNA are distinctly different from the corresponding characteristics in TFR1 mRNA: the former is highly expressed in liver and normal erythroid precursor cells and erythroleukemic cells44; the latter is expressed at low levels in the liver and in all types of cells except for mature erythroid cells. The regulation of expression of these two genes also differs. TFR1 is regulated by cellular iron level through the IRE-iron-regulatory protein system, as well as by the proliferation and differentiation states of cells.45TFR2 mRNA does not contain an IRE,44 and its expression is not regulated by cellular iron levels, but it is cell cycle dependent.46TFR2 expression and TFR1 expression also differ during erythroid differentiation and embryonic development. The presence in the TFR2 promoter of consensus sequences for GATA-1 transcription factors and for the liver-enriched transcription factor C/EBP may be important in the regulation of tissue-specific expression of TfR2 in the liver and in erythroid cells.47 TfR2 mediates the uptake of transferrin-bound iron,40 possibly via receptor-mediated endocytosis, similar to TfR1, but the affinity of TfR2 for transferrin is 25–30 times less than that of TfR1.48 Still, TfR2-mediated transferrin-iron uptake may be of major importance in hepatocytes, which express low levels of TfR1. In fact, an alternative pathway of transferrin-bound iron uptake involving energy-dependent endocytosis of transferrin has been demonstrated to be operative when TfR1 expression is inhibited.49 It is difficult to reconcile the preceding information, which would predict defective tissue iron uptake following TfR2 inactivation, with the finding that pathogenic mutations of TFR2 in humans and Tfr2 gene deletion in mice50 lead to a hemochromatosislike phenotype. In vitro experiments involving soluble recombinant forms of both TfR2 and HFE failed to detect a direct interaction between these two proteins.48 In one study, TfR2 was not found in intestinal crypts,51 where TfR1-HFE interactions may be important for hemochromatosis pathogenesis.52 In contrast, Griffiths and Cox53 colocalized TfR2 and HFE in specific subcellular compartments of crypt cells. Hypothetically, because TfR2 and hepcidin both are synthesized in the hepatocytes, TfR2 expression or iron uptake by a TfR2-mediated process may regulate the expression of hepcidin and indirectly affect the rate of tissue iron retention (Fig. 2). Clinical descriptions of hereditary iron overload associated with TFR2 are rather scant. Considering the available information, the clinical phenotype appears to be similar to that of hemochromatosis. In all human diseases characterized by enteral iron loading, iron accumulates preferentially in periportal hepatocytes (Fig. 3C). In one study, disease with unusually early onset was observed in a patient age 14 years, while another patient had severe hypogonadism; consequently, suspicion of 'juvenile hemochromatosis' was raised.54 Surprisingly, in two female patients, spontaneous reduction of hepatic iron stores was observed over time, with no signs of fibrosis. In another pedigree, affected individuals presented with marked hepatic iron overload; mild hypochromic anemia (which is not a typical feature of classic hemochromatosis); and, in one young male patient, hypogonadism.42 Finally, in a Japanese family, massive hepatic iron overload and cirrhosis were observed in a 50-year-old proband.43 TfR2 is an important protein in iron homeostasis; however, the epidemiologic impact of hereditary iron overload associated with TfR2 mutations is low. The TFR2 gene is relatively large, spanning 21 kilobases and including 18 exons; thus, detection of new TFR2 mutations in individual patients remains cumbersome. Analysis of TFR2 mutations should receive special consideration for individuals with non-HFE hepatic iron overload who come from families with high levels of consanguinity. In recent years, a rather vague term, 'juvenile hemochromatosis', has been used to refer to a form of hereditary iron overload that affects men and women equally and develops in a pattern resembling that of hemochromatosis, but at a greatly accelerated rate. The disorder first was recognized in 1979.55 Because of the rate of iron loading and the increased susceptibility of tissue to the toxic effects of iron, affected individuals are more likely to present with cardiomyopathy and endocrinopathy, including reduced glucose tolerance, than with severe liver disease (Fig. 2). Despite what initially was believed to be true, it now is clear that juvenile hemochromatosis is genetically heterogeneous. It is likely that pathogenic mutations in proteins that are of primary importance in the regulation of iron homeostasis may give rise to a similar clinical picture. In fact, homozygous mutations in hepcidin have been reported in individuals with a clinical condition that resembles the historical definition of juvenile hemochromatosis.56 Another gene (or multiple genes) responsible for a similar syndrome is located on chromosome 1q.57 Recently, a candidate gene named HFE2 was cloned in this region.58 The possibility also exists that a second gene is somehow linked to hepcidin metabolism or activity. Conceptually, if both TfR2 and HFE influence the synthesis and/or function of hepcidin, combined mutations in TFR2 and HFE should give rise to a similar juvenile iron loading syndrome. Hepcidin, the product of the HAMP gene, is a circulating antimicrobial peptide produced in the hepatocytes in response to inflammatory stimuli and to iron.59-61 Mice with alterations in the transcription factor upstream stimulatory factor 2 or in C/EBPα, both of which are required for hepcidin transcriptional control, have a hemochromatotic phenotype.62, 63 Transgenic animals that overexpress hepcidin die perinatally, due to severe iron-deficiency anemia occurring in the context of RE cell iron overload.64 In addition, hepcidin appears to be the main mediator of hypoferremia associated with chronic diseases, characterized by iron sequestration in macrophages and decreased intestinal iron absorption.65 Therefore, hepcidin is the primary negative regulator of iron release from intestinal, macrophage, and placental cells, and possibly from other cells as well. In HFE hemochromatosis, production of this peptide appears to be abnormally low,66, 67 and it may be responsible for the chronic release of iron from macrophages and intestinal cells (Fig. 2). It is noteworthy that hepcidin overexpression in rodent HFE hemochromatosis is not associated with hepatic iron overload.68 This finding demonstrates that hepcidin may blunt the iron-loading effect of mutated HFE, but it does not prove that underexpression of hepcidin plays a direct role in the pathogenesis of hemochromatosis. Nonetheless, hepcidin plays a dominant role in cellular iron trafficking. Its absence may give rise to a dramatic release of iron from storage sites and from the intestine into the bloodstream. This release leads to expansion of the plasma iron pool and to the overflow of iron in parenchymal cells via receptor-dependent or -independent mechanisms (Fig. 2). The most common symptom at presentation is hypogonadism, which, at the end of the second decade, may be present in all cases (Fig. 2; Table 1).69-71 In sporadic cases, abdominal pain and cardiac disease also are common findings, and cirrhosis seems a delayed event in the course of the disorder. Patients with juvenile hemochromatosis syndrome typically die of heart failure before reaching the fourth decade of life. The onset of hypogonadism or cardiopathy before cirrhosis suggests that endocrine organs and the heart have a particular susceptibility to iron toxicity. Rapid iron accumulation in these organs may be less tolerated than in the liver, which is physiologically more protected against iron toxicity. The liver holds a central position in the regulation of iron metabolism, as the site in which the main iron-carrier and storage proteins (i.e., transferrin and ferritin) and the iron hormone hepcidin are synthesized and in which the iron regulator HFE and the iron transporters TfR2 and ferroportin are preferentially expressed (Fig. 4). Consequently, the liver also has a central role both in the pathogenesis of and as a targeted organ in hemochromatosis and other primary iron-overload disorders (Fig. 5). Genetic defects that are responsible for the inactivation of these proteins may cause enhanced influx (HFE, TfR2, and hepcidin) or reduced export (ferroportin) of iron in the liver. This invariably will lead to hepatic iron accumulation and, potentially, organ disease. In the case of hemochromatosis, the faulty protein, HFE, is not an iron carrier, nor is it involved directly in intestinal iron absorption; instead, it modulates the function of other iron-related proteins, such as the receptors for transferrin and hepcidin.52 Therefore, it is not surprising that in hemochromatosis, several decades are needed for the genetic defect to translate into a clinically evident disorder. In contrast, in other primary iron-overload disorders in which a direct iron carrier or a major regulator of body iron trafficking is involved, the phenotypic expressivity appears at earlier stages (in ferroportin- and hepcidin-associated iron overload) and, in the case of hepcidin-associated iron overload, is far more severe than in classic hemochromatosis (Figs. 2, 3; Table 1). The liver in iron homeostasis. Key proteins in iron metabolism are either synthesized (hepcidin and TfR2 in hepatocytes) or preferentially expressed (ferroportin and HFE in Kupffer cells) in the liver. HFE is depicted in Kupffer cells, but expression in hepatocytes also has been reported. The most important iron compartment in humans (the 'erythron compartment') also is represented. In this compartment, iron continuously recycles through reticuloendothelial macrophages, where it is freed by the breakdown of senescent red cells, to the circulating iron carrier transferrin and to bone marrow, where it is incorporated into the hemoglobin of erythroid precursors. A limited amount of iron is absorbed daily to compensate for losses and to maintain a constant total iron pool. The liver as the central organ in the pathogenesis of primary iron-overload disorders. The main pathogenic pathways involved in various primary iron-overload disorders, with emphasis on the role of liver-specific or preferentially expressed iron genes, are listed on the right-hand side. Ferroportin, TfR2, and hepcidin are important proteins in metabolism, and their recognition has been greatly informative in understanding the regulation of iron trafficking; however, with the possible exception of ferroportin-associated disease, the global epidemiologic impact of hereditary iron overload associated with mutations in the genes that encode these proteins is low. Nonetheless, it is believed that polymorphisms or heterozygote modulation of these genes may have a significant impact on the phenotypic penetrance of other diseases, such as classic hemochromatosis. Individuals with heterozygote mutations in HFE may have an unusually severe phenotype. These individuals could carry additional abnormalities in genes that share common pathogenic pathways with HFE (Figs. 2, 3), such as hepcidin and TFR2. On the other hand, ferroportin, the main protein responsible for supplying iron to circulating transferrin from RE cells and the intestine, may aid in setting the individual levels of circulating iron and (indirectly) hemoglobin. In this regard, it is noteworthy that a polymorphic change in ferroportin recently was found to be associated with a tendency toward low hemoglobin and high serum ferritin levels in African and African American populations.72 During the past few years, we have witnessed dramatic progress in the field of iron research—progress that has led to the recognition of new genes, proteins, and diseases. The liver, which is the main storage organ for iron, the main source of iron regulators, and the main site for fundamental homeostatic function in iron metabolism, stands at the center of the complex pathways that govern iron metabolism and trafficking in health and in disease.