Review article: the iron overload syndromes

Transcription

1 Alimentary Pharmacology and Therapeutics Review article: the iron overload syndromes A. Siddique* & K. V. Kowdley *Department of Hepatology, Virginia Mason Medical Center, Seattle, WA, USA. Center for Liver Disease, Virginia Mason Medical Center, Digestive Disease Institute, Seattle, WA, USA. Correspondence to: Dr A. Siddique, Virginia Mason Medical Center, th Ave, Seattle WA 98101, USA. Publication data Submitted 8 June 2011 First decision 26 June 2011 Resubmitted 5 February 2012 Accepted 8 February 2012 EV Pub Online 4 March 2012 This commissioned review article was subject to full peer-review. SUMMARY Background Iron overload syndromes encompass a wide range of hereditary and acquired conditions. Major developments in the field of genetics and the discovery of hepcidin as a central regulator of iron homeostasis have greatly increased our understanding of the pathophysiology of iron overload syndromes. Aim To review advances in iron regulation and iron overload syndrome with special emphasis on hereditary haemochromatosis, the prototype iron overload syndrome. Methods A PubMed search using words such as iron overload, hemochromatosis, HFE, Non-HFE, secondary iron overload was undertaken. Results Iron overload is associated with significant morbidity and mortality. Sensitive diagnostic tests and effective therapy are widely available and can prevent complications associated with iron accumulation in end- organs. Therapeutic phlebotomy remains the cornerstone of therapy for removal of excess body iron, but novel therapeutic agents including oral iron chelators have been developed for iron overload associated with anaemia. Conclusions Iron overload disorders are common. Inexpensive screening tests as well as confirmatory diagnostic tests are widely available. Increased awareness of the causes and importance of early diagnosis and knowledge of the appropriate use of genetic testing are encouraged. The availability of novel treatments should increase therapeutic options for patients with iron overload disorders. Aliment Pharmacol Ther 2012; 35: doi: /j x

2 Review: iron overload INTRODUCTION The term iron overload can be used to describe a condition resulting in increased total body iron stores, with or without organ dysfunction. 1 Iron homeostasis depends on a complex feedback mechanism between body iron requirements and intestinal absorption. Humans lack a physiological mechanism for excretion of excess iron. The hormone hepcidin, a 25-amino acid peptide, is produced mainly in the liver and secreted into the blood and is now recognised as the key regulator of iron homeostasis; dysregulation of hepcidin is a cause of many disorders of iron homeostasis. 2, 3 Haemochromatosis was first described by Armand Trousseau in 1865 as a case of bronze diabetes and cirrhosis. 4 Von Recklinghausen in 1889 named this condition hemochromatosis after discovering that these patients had an iron- containing pigment in the liver cells. 5 In 1935, Sheldon recognised the inherited nature of this disorder and the association with abnormal iron metabolism. 6 However, the specific gene defect was not discovered until 1996 when Feder et al. identified the HFE mutation. 7 Primary iron overload syndromes are now known to be caused by mutations in several iron regulatory genes and characterised by inappropriately low levels of hepcidin resulting in increased dietary iron absorption and iron overload. Iron overload syndromes are broadly divided into two groups: Inherited or Primary iron overload and Secondary iron overload syndromes, as described in Table 1 and Table 2. NORMAL IRON METABOLISM (FIGURE 1) Dietary iron is present in two forms: heme (10%) and nonheme (90%). Nonheme iron is primarily present in the ferric form (Fe 3+ ). Duodenal cytochrome b (Dcytb), a ferric reductase, which is expressed in the duodenal brush border, plays an important role in dietary iron absorption by reducing ferric to ferrous iron (Fe 2+ ) Table 1 Inherited iron overload syndromes HFE related hemochromatosis (Type 1) C282Y/C282Y C282Y/H63D Other HFE mutations Non-HFE related hemochromatosis Juvenile Hemochromatosis (Type 2) Type 2A Hemojuvelin mutations Type 2B Hepcidin mutations Transferrin receptor 2 hemochromatosis (Type 3) Ferroportin diseases (Type 4) Classical Nonclassical Table 2 Secondary iron overload syndromes Iron-loading anaemias Thalassemic syndromes (b Thalassaemia) Sideroblastic Anaemias Chronic Hemolytic Anaemia Aplastic Anaemia Pyruvate Kinase Deficiency Chronic liver disease Hepatitis C infection NAFLD Alcoholic liver disease Porphyria Cutanea Tarda Iatrogenic Red Blood cell transfusion Long-term hemodialysis Miscellaneous Aceruloplasminaemia African iron overload Neonatal iron overload which is then taken up by divalent metal transporter1 (DMT1) The DMT1 expression is regulated by body iron requirements. The mechanism for absorption of heme-iron remains unclear. Heme iron is absorbed into the enterocytes by a carrier protein; heme oxygenase in enetrocytes releases the iron bound to heme in the Fe 2+ form which then likely enters a common pathway with nonheme iron. 11 Following uptake by enterocytes, iron is either stored intracellularly as ferritin or is released into the plasma by the iron transport protein ferroportin 1 (FPN). 12 FPN is the only iron exporter identified thus far and plays a 12, 13 critical role in regulating plasma iron concentration. FPN is expressed in the duodenal mucosa, macrophages, hepatocytes and syncytial trophoblasts of the placenta. 12 Once in the circulation, iron binds to transferrin (Tf) and is transported to various sites for its utilisation or storage. Prior to its binding to Tf, Fe 2+ is oxidised to Fe 3+ by the multicopper oxidase hephaestin expressed on the basolateral surface of intestine, and by ceruloplasmin present in the plasma. 14, 15 Fe 3+ bound to Tf is then delivered to the bone marrow, liver and immune cells where it binds to transferrin receptor 1 (TfR1). Tfr1 is highly expressed in the erythroid precursors to ensure increased iron uptake for erythropoiesis. The interaction of transferrin-bound iron (TBI) with TfR1 results in invagination of the cell membrane and formation of an endosome containing the TBI-TfR1 complex. 16 Acidification of the endosome, likely by a Na+-H+-ATPase, causes conformational changes and iron is released from Tf. 17 The released iron which is present in the Fe 3+ form is converted back to Fe 2+ by STEAP3, a metalloreductase Aliment Pharmacol Ther 2012; 35:

3 A. Siddique and K. V. Kowdley (a) (b) Figure 1 Iron regulation by Hepcidin. A model for HFE-mediated signalling to hepcidin in hepatocytes. (a) At low plasma iron concentration, HFE is bound to TfR1 and other proteins involved in signalling to hepcidin remain silent; (b) An increase in plasma iron levels results in displacement of HFE from TfR1, followed by iron uptake. This triggers the assembly of a putative iron-sensing complex, comprising of HFE, TfR2, BMPs (such as BMP-2, BMP-4 and BMP-9) and their receptor BMPR, and Hjv, which mediates signalling to activate hepcidin transcription via Smad proteins. Thus, the hepatocyte integrates signals for regulation of iron metabolism at the cellular and systemic level. Published in World J Gastroenterol 2008; 14: protein present in the endosome membrane, and is transported from endosome to the cytoplasm by DMT1. 12 Subsequently, Tf is returned to the circulation and TfR1 is recycled back to the cell membrane allowing both molecules to start the cycle again. 17 Intracellular iron content is tightly controlled by the iron responsive elements (IRE) and the IRE-binding proteins, IRP1 and IRP IREs are components of the mrna present in the untranslated region of the mrnas of ferritin and TfR1. Stabilisation or inhibition of mrna is dependent upon the site of IRP binding to IRE. IRP1 acts as an iron sensor in high oxygen environments whereas IRP2 acts at physiological oxygen tensions. 21 When intracellular iron level is low, IRP binding to IRE at the 3 UTR of TfR1 mrna causes stabilisation, increased synthesis, and up-regulation of TfR1 levels in the duodenum thereby increasing dietary iron absorption. 22 Conversely, IRP binding to IRE in 5 UTR of ferritin mrna inhibits translation and blocks synthesis of ferritin. Therefore, in iron depleted states the IRP binding causes increased expression of TfR1 and decreased ferritin. Conversely in iron replete states there is less IRP1 and IRP2 for IRE binding resulting in increased ferritin and decreased TfR1. The most important regulator of iron homeostasis, however, is the hormone hepcidin. Hepatic hepcidin 878 Aliment Pharmacol Ther 2012; 35:

4 Review: iron overload expression is elegantly regulated by a number of proteins expressed in the hepatocytes, including hereditary haemochromatosis protein HFE, transferrin receptor 2 (TfR2), haemojuvelin (HJV), bone morphogenetic protein 6 (BMP6), matriptase-2 and Tf. Hepcidin serves as the signal from the principal iron storage site (the liver) to the iron absorptive site (the duodenum). The hepcidin gene (HAMP) has been identified on chromosome 19q The gene product, preprohepcidin, a 84 amino acid protein, is synthesised mainly in the hepatocytes. 3 Hepcidin expression is increased in response to iron overload and inflammation and is decreased in response to iron deficiency, hypoxia and ineffective erythropoiesis. Hepcidin binds to FPN and causes its phosphorylation, internalisation and degradation. 12 This results in reduced iron export from enterocytes and macrophages resulting in decreased serum iron. Conversely, when hepcidin expression is decreased, iron absorption and cellular iron export is up-regulated, resulting in increase in serum iron. The USF2 gene knockout mouse which does not express hepcidin, develops iron overload resembling human HH and Hfe / mice. 24 Hepcidin regulation depends upon signalling through the bone morphogenetic protein (BMP)/Smad pathway. 25 BMPs are cytokines in the TGF-beta superfamily. BMP6, which is highly expressed in the liver, has a predominant role in the activation of the Smad signalling pathway. 26 BMPs bind to type I and type II cell serine/threonine kinase receptors forming a BMP receptor complex. The activated complex induces phosphorylation of a subset of intracellular Smad proteins (Smad1, Smad5 and Smad8). The receptor activated Smads in turn form a complex with the common mediator Smad4 and these complexes are translocated into the nucleus where they mediate gene transcription. 27 Haemojuvelin (HJV) has also been shown to act as a BMP coreceptor and facilitate the activation of the BMP receptor complex. 28 Finally, in iron overload states, as the Tf is saturated, the excess iron may also be found as nontransferrin-bound iron (NTBI). NTBI is toxic and is quickly cleared from plasma by the liver. 29 Hfe / show increased plasma NTBI and increased hepatocyte NTBI uptake suggesting that NTBI has an important role in hepatocyte iron loading. 30 HFE- RELATED HAEMOCHROMATOSIS (TYPE 1 OR CLASSICAL HAEMOCHROMATOSIS) (TABLE 3) HFE-related haemochromatosis (HH) is the most common inherited disorder of iron metabolism. It is inherited in an autosomal recessive pattern, with variable penetrance. 31 It usually manifests in the fourth or fifth decade and the phenotypic features are more common and more pronounced in males. A homozygous mutation in the hereditary haemochromatosis gene, HFE is responsible for HH. HFE, a nonclassical major histocompatibility class I like molecule, binds to b2-microglobulin and this association enables it to be moved to the cell surface where it binds to TfR1. 7 The normal function of HFE has been difficult to elucidate and the mechanisms by which HFE affects hepcidin transcription are only now beginning to emerge. Under normal physiological conditions TBI binds with high affinity to TfR1 expressed on the cell membrane. HFE protein competes with Tf for binding TfR1 to modulate cellular iron uptake. 32, 33 The presence of HFE in Table 3 Characteristics of hereditary hemochromatosis Type Gene Frequency Inheritance Iron Indices Mechanism Severity Characteristics Type 1 or Classic HFE Common AR Elevated TS and SF Type 2/JH 2A 2B HJV Relatively rare AR Elevated TS and SF Low hepcidin ++ Onset 4th 5th decade. Parenchymal iron accumulation Reduced HAMP activation ++++ Onset 3rd decade. Hypogonadism and cardiomyopathy common HAMP Very rare Low/absent hepcidin Type3/TfR2 TfR2 Very rare AR Elevated?iron sensing +++ Similar to HFE-HH TS and SF Type 4 Classical Nonclassical SLC40A1 Relatively rare AD + Reticuloendothelial iron overload Normal or low TS in classical Reduced iron export from macrophages. Hepcidin resistance + mild, ++moderate, +++severe, ++++very severe: TS, transferrin saturation; SF, serum ferritin. Aliment Pharmacol Ther 2012; 35:

5 A. Siddique and K. V. Kowdley duodenal crypt cells and the interaction between HFE andtfr1 led to the crypt programming model to explain the systemic iron homeostasis. 34 Crypt enterocytes take up iron from plasma Tf at their basolateral surfaces and migrate to the villus region where they differentiate into absorptive cells. 35 Hfe / mice were found to have significant impairment in the duodenal uptake of circulating TBI. 36 This led to the hypothesis that crypt cell HFE may act as an iron sensor and, in the presence of an abnormal HFE protein, iron uptake into the enterocyte does not occur resulting in iron-deficient crypt cells. When these cells then migrate to the villus region, there is an inappropriate up-regulation of iron transport proteins and increased dietary iron absorption to correct 36, 37 the erroneously perceived deficit. The discovery of the hormone hepcidin, a negative regulator of iron absorption, has shifted the focus to the liver as the main regulatory site for dietary iron absorption. 38 Hfe / mice have decreased hepatic hepcidin mrna expression and increased hepatic iron deposition similar to a human haemochromatosis phenotype. 39 Moreover, when Hfe / mice were crossed with transgenic mice that over express hepcidin, there was prevention of the iron overload phenotype, implicating an overriding role of hepcidin in iron homeostasis. 40 Furthermore, ablation of HFE expression in mouse enterocytes does not disrupt normal physiological iron metabolism. 41 These observations argue for a more important role for HFE in the liver rather than the intestinal crypt cells. The three molecules, HFE, HJV, TfR2, all of which are expressed in the liver likely play a major role in hepcidin regulation. According to the hepcidin hypothesis, in iron overload states that there is an appropriate increase in hepcidin expression, resulting in reduced cellular expression of FPN and decreased iron efflux from duodenal enterocytes, macrophages involved in the recycling of iron from senescent erythrocytes and from hepatocytes, the site of most storage iron. Conversely, in iron depleted states, hepcidin expression is reduced and FPN expression is high, resulting in increased iron absorption and transport. Hepcidin also likely affects the expression and activity of other iron-transport molecules, such as DMT1 by altering the labile iron pool (LIP) in enterocytes and thereby indirectly affecting the gene transcription of the iron transport proteins. 42 A number of HFE mutations have been identified. The most common clinically relevant mutations, however, are the C282Y and H63D. The C282Y mutation is a missense mutation on the short arm of chromosome 6 that causes the amino acid tyrosine to replace cysteine at position 282 in the HFE protein. 7 The H63D mutation is characterised by a histidine to aspartic acid substitution at amino acid Approximately 85 90% of patients with the typical phenotype of HH are C282Y homozygotes whereas 3 5% are C282Y/H63D compound heterozygotes. 31 The prevalence of C282Y homozygosity is 1 in 250 persons in the general population and about 1 in 200 persons of northern European ancestry. 43 However, iron overload features do not manifest in many C282Y homozygotes, suggesting incomplete penetrance and the possibility that there may be other genes that act as modifiers of the HH phenotype. 43 The H63D mutation is more common compared to C282Y with a carrier frequency of 10 20% of population of European descent. The clinical significance of the H63D mutation remains unclear in the absence of C282Y; only 0.5 2% of subjects with compound heterozygosity for C282/H63D develop iron overload. 44 H63D homozygosity is unlikely to cause clinical disease in the absence of other factors, such as 45, 46 viral hepatitis and alcohol. Data from a large population-based 12-year follow-up study involving over persons of Northern European ancestry revealed that 28% of C282Y homozygous males but only 1% of female C282Y homozygotes developed iron-overload related disease. 47 In comparison, among the compound heterozygotes only one in 82 men and no women had iron overload-related disease. The two main factors associated with increased phenotypic expression of the homozygous C282Y mutation are male gender and increased alcohol consumption. 48 Other factors that have been suggested are excessive dietary heme consumption, protective effect of tea drinking and noncitrus fruit It has also been proposed that calcium channel blockers by prolonging DMT1 activity and PPI by its effect of gastric acid suppression may decrease 51, 52 nonheme iron absorption. NON-HFE RELATED HAEMOCHROMATOSIS Juvenile Haemochromatosis or type 2 haemochromatosis Juvenile haemochromatosis (JH) is an autosomal recessive disease characterised by massive hepatocellular iron deposition as well as iron deposition in endocrine glands. Unlike classical HH, patients with JH present with clinical disease early in life, usually by the third decade. This syndrome affects both genders equally and has a more rapid and severe course. 53 Depending on the gene involved, JH is divided into two subtypes, although the 880 Aliment Pharmacol Ther 2012; 35:

6 Review: iron overload clinical presentation is indistinguishable. Type 2A is due to mutations in the haemojuvelin (HJV) gene encoding protein haemojuvelin on the long arm of chromosome 1 (1q21). 54 A number of mutations have been identified, of which the G320V is the most frequent. Type 2B is due to mutation in the hepcidin (HAMP) gene on chromosome 19 (19q31). 55 HJV is expressed in the liver, heart and skeletal muscles, and is considered as an upstream regulator of hepcidin. The mutant HJV proteins inhibit hepcidin expression as confirmed by low urinary hepcidin excretion in patients with JH. 53 HJV acts as a BMP co-receptor and enhances the phosphorylation of the Smads (Smad1, 5 and 8), thereby increasing hepcidin expression. 56 Transferrin receptor 2 or type 3 haemochromatosis Type 3 Haemochromatosis, a rare disorder, is due to a mutation in the TfR2 gene located on the long arm of chromosome 7 (7q22). 57 Although this condition is reported worldwide, most reports are from Italy. 58 There have also been reports from France, Portugal, Taiwan and Japan Type 3 haemochromatosis manifests at an earlier age compared with HH. 57 Although some individuals present in the second decade, others may manifest as adults with fatigue and arthralgia and/or organ involvement including cirrhosis, diabetes mellitus and arthropathy. 63 In a small proportion of patients, the condition appears nonprogressive. 63, 64 TfR2 mutant mice exhibit a phenotype similar to HH with low levels of hepcidin mrna in the liver, indicating that TfR2 acts upstream of hepcidin. 65 Several mutations of TfR2 have been described; the first mutation identified was the nonsense mutation (Y250X) that truncates TfR2 at amino acid TfR2 is expressed mainly in the liver and has a lower affinity for iron uptake compared with TfR1, but has a higher 66, 67 capacity to transport TBI to the hepatocyte. Ferroportin iron overload or type 4 haemochromatosis Type 4 haemochromatosis, the second most common inherited iron overload syndrome after HH, has distinct genetic, clinical, biochemical and histological features. 12, 13 Unlike the other inherited iron overload syndromes, this condition is inherited in an autosomal dominant pattern; the hepatic phenotype is characterised by iron accumulation in liver macrophages with relative sparing of hepatocytes. 68 Classical ferroportin disease is characterised by hyperferritinaemia, a normal to low transferrin saturation (TS) and macrophage cell iron accumulation. A number of mutations (A77D, V162del, D157G, N174I, Q182H, Q248H and G323V mutation) are associated with classical ferroportin disease These mutations cause loss of FPN activity and thereby result in decreased cellular iron export and retention of iron within macrophages, resulting in tissue iron accumulation which manifests as elevated serum ferritin (SF). The decreased availability of iron for the circulating Tf manifests as a low transferriniron saturation and mild anaemia. Due to the anaemia these patients may not tolerate phlebotomy. 73 Nonclassical ferroportin disease, on the other hand, is characterised by hyperferritinaemia, increased TS and iron accumulation preferentially in hepatocytes, with some macrophage cell iron. 74 This condition is associated with gain-of-function (N144H, Y64N, C326Y, S338R, Y501C) mutations that result in normal FPN activity, but resist internalisation and degradation by hepcidin resulting in a FPN that is always switched on SECONDARY IRON OVERLOAD Haematological disorders Erythroid disorders associated with ineffective erythropoiesis may manifest a haemochromatosis phenotype. Four main groups of haematological disorders may cause iron overload: (i) The thalassaemia syndromes (thalassaemia major and intermedia) (ii) Sideroblastic anaemias, both acquired and congenital (iii) congenital dyserythropoietic anaemias which include pyruvate kinase deficiency, chronic pernicious anaemia, hereditary spherocytosis and sickle cell anaemia (iv) acquired myelodysplastic syndrome. The thalassaemia syndromes represent the most common causes of ineffective erythropoiesis and secondary iron overload. 79 Individuals with haematological disorders, especially those with thalassaemia and myelodysplastic syndrome, eventually become transfusion dependant. One unit of packed red blood cells (RBCs) contains mg of elemental iron. Therefore, significant iron overload may develop over time secondary to multiple transfusions. Apart from transfusional iron overload, various other mechanisms may also be involved in the pathogenesis of iron overload in these patients, including downregulation of hepcidin. Hepatocyte cell lines exposed to hypoxia demonstrate a down-regulation of hepcidin production. 80, 81 Furthermore, the soluble form of HJV (shjv) by competing with membrane form of HJV (mhjv) may suppress BMP signalling resulting in downregulation of hepcidin. 82, 83 Therefore, any stimulus resulting in increased shjv, including iron deficiency and hypoxia, Aliment Pharmacol Ther 2012; 35:

7 A. Siddique and K. V. Kowdley may lead to decreased hepcidin expression. Tissue hypoxia also increases erythropoietin (EPO) expression, which directly down-regulates hepcidin expression in vitro. 84 Finally, two molecules (GDF15 and TWSG1) have been identified that may serve as signals for iron regulation. Serum from patients with beta thalassaemia and other congenital anaemias were found to contain high levels of GDF15 which, by virtue of its inhibitory effect of hepcidin, may contribute to iron overload in these individuals TWSG1 acts by inhibiting the BMP-dependent activation of Smad-mediated signal transduction resulting in iron overload. 88 Unlike primary haemochromatosis, these conditions are commonly associated with anaemia and iron accumulation is predominantly in liver macrophage cells. 89 Because of the associated anaemia, these patients do not tolerate phlebotomy and chelation therapy is required to prevent longterm sequelae. African iron overload African Iron Overload is prevalent in a number of Sub- Saharan African countries and has been attributed to consumption of large quantities of traditionally fermented home brewed beer rich in iron. The iron content in this beer may be as high as mg/l, in comparison with standard commercial beer that has an iron content of only 0.5 mg/l. 90 Although patients with African Iron Overload may have a history of heavy alcohol consumption, overt histological features of alcoholic liver disease are usually not evident. In fact, the pathophysiology is probably more complex and it has been suggested that African Iron Overload may have a genetic component. A Q248H mutation of ferroportin gene SLC4OA1 has been frequently reported in families with African iron overload. 91 African Americans in the US have a lower prevalence of primary iron overload compared with Caucasians. However, the Q248H mutation has also been reported in African Americans; iron overload among African-Americans is associated with a pattern of hepatic iron deposition similar to African Iron Overload, with iron deposition in both hepatocytes and macrophages. 92 Chronic liver disease Chronic viral hepatitis, most commonly Hepatitis C virus infection (HCV), may be associated with iron overload. 93, 94 The pathophysiology of iron overload in HCV is likely a combination of release of iron from necrotic hepatocytes, a direct effect of HCV on iron homeostasis, presence of HFE mutations and/or dysregulation of hepcidin. 95 In a transgenic mice model of HCV, reactive oxygen species associated with the virus caused inhibition of C/EBPa resulting in downregulation of hepcidin and hepatic iron accumulation. 96, 97 Furthermore, release of proinflammatory cytokines by liver macrophages may activate hepatic stellate cells causing up-regulation of alpha smooth muscle actin and procollagen a-1 resulting in increased fibrogenesis. 98 Alcoholic liver disease is associated with iron deposition initially in the hepatocytes, but with advanced disease, iron accumulates in both hepatocytes and macrophages. 99 Iron and alcohol act synergistically in causing liver injury. Alcohol consumption is associated with elevated serum iron and abstinence of just 2 6 weeks significantly reduces these elevated serum iron indices. 100 The mechanism by which alcohol induces hepatic iron overload is likely similar to HCV. Alcohol and its metabolites generate reactive oxygen species and lipid peroxidation products, which cause cellular damage in hepatocytes. 101 Thioredoxin, an indicator of oxidative stress, is significantly higher in patients with ALD compared with healthy subjects. 102 Similar to HCV, hepcidin expression may be depressed due to inhibition of C/EBPa leading to iron accumulation and subsequent activation of macrophages. Non-alcoholic fatty liver disease (NAFLD) is now considered the most common cause of chronic liver disease with a spectrum of disease ranging from benign fatty liver to non-alcoholic steatohepatitis (NASH); the latter may predispose to cirrhosis and hepatocellular carcinoma. 103 Mild to moderate elevation of iron indices and hepatic iron concentration are frequently observed in patients with NASH. 104 Oxidative stress, iron overload and insulin resistance act synergistically resulting in liver injury. The pathogenesis of NASH has been proposed to be caused by a multiple-hit hypothesis. The first hit results in accumulation of hepatic fatty acids leading to steatosis. A second hit then leads to progression of simple steatosis to steatohepatitis. 105 Hepatic iron may enhance oxidative stress and may be the second hit contributing to the pathogenesis of NASH, at least in some 106, 107 patients. The pathogenesis of increased hepatic iron in NAFLD and NASH is likely multifactorial. The role of HFE mutations in the pathogenesis of NASH remains controversial. Some studies have shown an increased prevalence of HFE mutation in patients with NASH although data from groups with a low prevalence of HFE mutations have not shown the same association. Recent studies have shown that FPN and 108, 109 HJV are both down regulated by increased expression of TNF-alpha, which may occur in NASH. 110 The role of hepcidin in NASH remains uncertain. Although hepcidin 882 Aliment Pharmacol Ther 2012; 35:

8 Review: iron overload expression by adipose tissue may be increased, increased insulin levels may have the opposite effects. 111 Further studies are needed to elucidate whether hepcidin expression is increased or decreased in NAFLD and NASH. Porphyria Cutanea Tarda (PCT) is an iron-dependent condition characterised by vesiculo-bullous eruption on the hands and face. Mild to moderate iron overload maybe present in 60 70% of patients with PCT. 112 Several studies have shown a high prevalence of C282Y and H63D mutation in patients with PCT, and these mutations maybe present in up to 70% of individuals with PCT. 113 Aceruloplasminaemia Aceruloplasminaemia is an autosomal recessive condition due to a mutation in the ceruloplasmin gene. 114 Ceruloplasmin (CP) is a multicopper ferroxidase synthesised in the hepatocytes. 115 CP is required for mobilisation of iron and oxidation of Fe 2+ to Fe 3+ which is essential for release to transferrin. Deficiency of ceruloplasmin results in iron deposition in liver, pancreas, basal ganglia and other organs. The classic triad of aceruloplasminaemia is comprised of retinal degeneration, neurological symptoms and diabetes mellitus. 116 Patients with aceruloplasminaemia have normal hepatic copper content, but markedly elevated hepatic iron concentration and SF concentrations equivalent to those observed in persons with haemochromatosis. 117 CLINICAL MANIFESTATIONS OF IRON OVERLOAD Type 1 or HFE-HH can be classified into four groups (i) presence of a genetic mutation which predisposes to iron overload (ii) biochemical evidence of iron overload without any symptoms or end organ damage (iii) iron overload with early nonspecific symptoms like fatigue, malaise and arthralgia (iv) iron overload associated with end organ damage, such as cirrhosis or diabetes. 118 The classic triad of cirrhosis, bronze discoloration of the skin and diabetes mellitus is rarely seen, likely due to increased awareness and earlier diagnosis. Liver disease The liver being the primary organ of iron storage is frequently involved. Asymptomatic patients may present with only an enlarged liver on physical examination or elevated serum aminotransferase levels. 119 Approximately 38 97% of patients develop significant hepatic iron but only 10 25% progress to hepatic fibrosis whereas 4 6% develop cirrhosis. 120 Progression to cirrhosis depends on the duration of iron overload, severity as evidenced by a SF level of over 1000 lg/l and presence of other risk factors, such as chronic viral hepatitis or alcohol abuse. 121 Patients who consume 60 g alcohol per day or more are approximately nine times more likely to develop cirrhosis. 122 Once established, cirrhosis is irreversible with phlebotomy, although rare causes of regression have been reported. 123 Studies have also shown increased fibrosis and cirrhosis in patients with HCV 104, 124 and NASH who have HFE mutation. Endocrine disease Diabetes is common in patients with iron overload and may be seen in >70% of patients with cirrhosis. 125 Diabetes in iron overload is likely due to either decreased insulin secretion from accumulation of iron in the beta cells of pancreas and/or increased insulin resistance. 126 The SF level is not predictive for diabetes mellitus development. 127 Diabetes may be partially reversible with phlebotomy, especially if phlebotomy is initiated prior to the development of cirrhosis. Hypogonadism in iron overload is due to hypothalamic, pituitary or gonadal dysfunction and may be the presenting manifestation in both genders with males presenting with decreased libido and impotence and women with amenorrhea. 128 Thyroid dysfunction is less common and both hypothyroid and hyperthyroidism may occur. 129 Joint Involvement Arthropathy develops in 25 50% of patients. 130, 131 A predilection for disease in the second and third metacarpophalangeal joints (MCP) is observed. Other joints that may be involved include proximal interphalangeal (PIP) joints, wrist, elbows, shoulder and hips causing significant disability. Arthropathy is generally symmetrical and polyarticular and patients present with chronic pain, joint stiffness, bony enlargement and minimal signs of inflammation. 132 Acute episodes of inflammatory arthritis secondary to calcium pyrophosphate crystal deposition and rarely a syndrome of septicaemia with monoarticular or oligoarticular septic arthritis caused by Yersinia species have been described. 132 A higher incidence of joint replacement has been observed in patients with HH. 133 Furthermore, up to 25% of patients with HH are affected with osteoporosis. Osteoporosis is independently associated with hypogonadism, lower body weight and the severity of iron overload. 134 Cardiac involvement Accumulation of iron in the heart can result in cardiomyopathy (both restrictive and dilated), arrhythmias (sick sinus syndrome, atrial fibrillation) and heart failure. 135 Aliment Pharmacol Ther 2012; 35:

9 A. Siddique and K. V. Kowdley Reversal of left ventricular dysfunction with therapy has been observed. 136 Sudden death due to cardiac dysrhythmias and cardiomyopathies can occur among patients 137, 138 with advanced iron overload. Skin involvement Hyperpigmentation may be one of the earliest signs of disease and has been reported in up to 90% of patients in one study. 139 Skin pigmentation occurs due to melanin and/or iron deposition in the basal layer of the epidermis and around sweat glands. Skin involvement is most pronounced on sun-exposed skin, particularly on the face, neck, extensor aspects of the lower forearms, dorsum of the hands, lower legs and old scars. 140 The skin appears brownish bronze or at times, slate grey. Cutaneous atrophy, flattening of the nails and loss of body hair are also common. 140 The clinical presentation in non-hfe-haemochromatosis and secondary iron overload is similar to HH in many aspects. The spectrum of clinical manifestations may extend from nonspecific fatigue and arthralgia to end organ damage. Patients with JH present at an earlier age and frequently have cardiac and/or endocrine manifestations; mortality is usually due to congestive heart failure and arrhythmias. 141 Hepatosplenomegaly is common in iron overload anaemias like beta thalassaemia. Patients with iron overload anaemias are also at an increased risk of cirrhosis which may develop as early as third or fourth decade. Other complications, such as diabetes, dilated cardiomyopathy and hypogonadism, are also common in beta thalassaemia. In patients with African Iron Overload the initial presentation commonly is complication of iron overload, such as cirrhosis and diabetes or might involve indirect sequelae such as osteoporotic fractures, scurvy or tuberculosis to which these patients have increased susceptibility. 142 IRON OVERLOAD AND CANCER RISK Conflicting results have been reported regarding iron overload and hepatic and extrahepatic malignancies. Patients with HH are at an increased risk of hepatocellular carcinoma (HCC) and are 20 times more likely to develop liver cancer than all other cancers combined. 143 HCC usually occurs in those with established cirrhosis, although rarely may also occur in the absence of cirrhosis. 144 In HH patients, 6% of men and 1.5% of women may develop liver cancer. 145 Epidemiologic studies have indicated that the risk of HCC may be higher in HH-related cirrhosis than in comparable cirrhosis due to other chronic liver disease. 145 The risk of HCC is further increased in the presence of other co-factors, such as alcohol abuse, viral hepatitis and age over 55 years. 146 Female patients have a markedly lower risk than men. Childbirth and menstrual blood loss decrease excessive iron and may postpone the development of cirrhosis and subsequent liver cancer. 143 Although some studies have shown an increased risk of extra-hepatic cancer including colorectal cancer, breast cancer in women, oesophageal cancer, lung and malignant melanoma, others have not found the same association. 147 C282Y heterozygotes are not at an increased risk for breast, colorectal, or prostate cancer. 147 An increased prevalence of HCC and oesophageal cancer has been reported in patients with African Iron Overload; possible risk factors include iron overload, concomitant hepatitis B infection and possible prolonged dietary exposure to aflatoxin 142, 148 B1. DIAGNOSIS OF IRON OVERLOAD (FIGURE 2) Serological tests for iron overload The initial approach to diagnosis in patients with suspected iron overload should include indirect markers of iron stores which include TS, SF and unsaturated iron binding capacity (UIBC). TS is considered the initial screening test and elevated initial TS should be confirmed with a second test. The earlier recommendation of fasting TS to exclude circadian and postprandial variation of TS is no longer necessary; a recent study has shown no improvement in sensitivity or specificity of fasting over random TS tests. 149 ATSof 45% identifies % of C282Y homozygotes. 150 A small proportion of patients with HH, especially younger individuals at an early stage of disease may have TS < 45%, necessitating close follow-up. 151 SF is an excellent predictor of advanced fibrosis and cirrhosis, but lacks specificity as a screening test when used alone. 152 Hyperferritinaemia is present in a number of other conditions including ALD, HCV, neoplastic disease and NA- FLD among others. 153 Irrespective of age and gender, a SF > 1000 lg/l is associated with a greater risk of cirrhosis. In C282Y homozygotes, a SF over > 1000 lg/l, elevated aminotransferase level and a low platelet count predicts cirrhosis in over 80% of patients. 154 A normal SF (less than 200 lg/l in premenopausal women and 300 lg/l in men and postmenopausal women) in combination with a TS < 45%, has a negative predictive value of 97% for excluding iron overload. A recent study by Gurrin et al. demonstrated that male C282Y homozygotes with a baseline SF between 300 and 1000 lg/l had a 25% chance of progressing to SF > 1000 lg/l on an average after 12 years with the 884 Aliment Pharmacol Ther 2012; 35:

10 Review: iron overload Transferrin saturation (TS)/Serum ferritin (SF) TS < 45%, normal SF TS > 45%, elevated SF TS < 45%, elevated SF Iron overload excluded HFE genotype Alcohol, inflammation, malignancy, NAFLD, Ferroportin Disease C282Y/C282Y C282Y/H63D C282Y heterozygote or non C282Y Ferritin > 1000 µg/l or abnormal liver enzymes or risk factors e.g. alcohol Ferritin < 1000 µg/l and normal liver enzymes Exclude secondary iron overload/consider non-hfe Liver biopsy to assess cirrhosis Watchful waiting if ferritin normal or modestly elevated Phlebotomy Figure 2 Algorithm for management of iron overload. greatest risk of progression among men with a baseline TS > 95%. In contrast, women with SF lg/l had only an 18% chance of progressing to SF > 1000 lg/l during the same time period. 155 However, iron overload may be present with an elevated SF level and a normal TS level, particularly in non-hferelated iron overload. 156 Therefore, a significant elevation in SF with no obvious explanation, especially if greater than 1000 lg/l, may require a liver biopsy to determine whether iron overload is present. The UIBC is the inverse of TS and can be obtained as a one-step automated test. In large-scale population screening studies, UIBC has been shown to be comparable or slightly better than TS and maybe used as a low cost alternative screening test for detecting HH. 156 Genetic testing The HFE mutation analysis should be performed in individuals with abnormal iron studies. Genotype testing involves determination of the common HFE gene mutations C282Y and H63D. S65C occurs in less than 1% of clinically significant HH and is not routinely tested. 157 Presence of TS > 45% and C282Y homozygosity or C282Y/H63D compound heterozygosity may be considered diagnostic of HH. The genetic tests available include targeted mutation analysis and sequence analysis. Although targeted mutation analysis tests for the two common C282Y and H63D mutations, sequence analysis identifies less common mutant alleles and is available only in a few clinical and research laboratories. 7, 158 Evidence of increased hepatic iron concentration based on either a liver biopsy or MRI imaging in the absence of C282Y homozygosity or C282Y/H63D compound heterozygosity may indicate iron overload from non-hfe related haemochromatosis or secondary iron overload. Testing for Non-HFE gene mutations in 159, 160 HJV, TfR2, FPN and hepcidin are not generally commercially available. Aliment Pharmacol Ther 2012; 35:

11 A. Siddique and K. V. Kowdley Liver biopsy Liver biopsy was considered the gold standard for the diagnosis of HH prior to the availability of genetic testing and continues to have a role in diagnosis and prognosis. 161 A liver biopsy should be considered in the following situations among patients with HH: (i) a SF > 1000 lg/l for assessment of degree of fibrosis in HFE-HH. (ii) Elevated liver enzymes and/or increased alcohol consumption (>60 gm/day) to evaluate for other concomitant liver diseases, such as NAFLD, alcoholic liver disease or HCV in patients with iron overload. (iii) Diagnosis of non-hferelated haemochromatosis, as genetic testing in these conditions are not widely available. In HFE-HH, liver histology shows a characteristic pattern with iron accumulation predominantly in the periportal hepatocytes with absent or minimal iron in the reticuloendothelial cells. In contrast, predominant reticuloendothelial cell iron maybe present in patients with ferroportin disease and secondary iron overload 89 (Figure 3). Liver tissue is also used to measure hepatic iron concentration (HIC) and calculation of hepatic iron index (HII). The normal HIC is less than 1800 mg/g dry weight (equivalent to 32 lg/g). A threshold HIC of >71 lg g dry weight along with a HII 1.9 lmol/g/year was previously suggested to be helpful in identifying phenotypic HH vs. secondary iron overload. 162 However, it is now evident that phenotypic HH can occur at a lower HII and certain secondary iron overload states, such as thalassaemia may have a HII comparable to HH, and therefore HII is no longer routinely used. Considerable effort is being made to develop non-invasive markers for detecting cirrhosis in HH. Serum type IV collagen concentration is elevated in HH and a level >115 ng/ml is sensitive, although less specific in detection of cirrhosis. 163 A recent study has shown that serum concentration of Hyaluronic acid >46.5 ng/ml showed 100% sensitivity and specificity in detecting cirrhosis in HH. 164 Other fibrosis markers, such as serum laminin and tissue inhibitor of metalloproteinase (TIMP-I) levels, seem to be of little value for fibrosis prediction. 163 Imaging Transient elastography (FibroScan) is a non-invasive, rapid method, allowing assessment of liver fibrosis by measuring liver rigidity. However, this technique to measure liver stiffness is often difficult especially in the presence of obesity. 165 Non-invasive imaging tests, CT and MRI, are useful for diagnosis, determination of severity and for monitoring therapy. CT noncontrast imaging demonstrates diffuse increased attenuation of the liver, usually greater than 75 Hounsfield units. 166 However, MRI is more sensitive and specific for the detection of abnormal hepatic iron. MRI T2* is now gaining popularity as a non-invasive method for liver iron estimation. The iron in the liver causes local distortion in the magnetic fields and relaxation of the spins resulting in loss of signal intensity in the liver. The loss of signal intensity is proportional to the iron deposition 167 (Figure 4). The hepatic iron deposition is then quantified by measuring the ratio of the signal intensity of the liver and of a reference tissue (e.g. paraspinous muscle). 168 In addition, MRI can also detect complications of iron overload, such as cirrhosis and HCC. Patients with thalassaemia and other anaemias requiring multiple transfusions demonstrate abnormal iron overload in both liver and the spleen, unlike patients with HH who usually do not have splenic iron. Patients with thalassaemia may also exhibit cardiac iron overload; there is no direct correlation between liver (a) (b) Figure 3 Liver histology in iron overload. (a) Periportal iron deposition in patient with hereditary hemochromatosis, (b) Extensive Iron in Kupffer cells in a patient with Thalassemia. 886 Aliment Pharmacol Ther 2012; 35:

12 Review: iron overload Figure 4 Magnetic resonance imaging of the liver in iron overload. T1-weighted out of phase sequence (left) consistent with focal fatty infiltration. Low signal on T2-weighted sequences compatible with iron deposition from hemochromatosis. Small islands of low signal in the spleen secondary to iron deposition. Nodularity of liver capsule consistent with cirrhosis. and cardiac iron overload. Therefore, in these individuals MRI of both the liver and heart should be obtained. 169 Low values of cardiac T2* reflect high cardiac iron levels and predict heart failure and arrhythmia. 170 SCREENING FOR HH Population screening There is presently no clear consensus regarding screening for the general population. The relatively high prevalence, existence of sensitive screening tests, significant morbidity and mortality in untreated patients and effective treatment which improves survival has led to a consideration of population screening. However, low clinical penetrance, potential disadvantage of detecting asymptomatic persons with normal iron parameters and possibility of social and psychological stress are cited as reasons against population screening. 119 Delatycki et al. undertook genetic testing in over asymptomatic individuals in a work place setting and found that one in 221 were homozygous and one in 8.4 were heterozygous for C282Y mutation. Moreover, they were able to demonstrate that screening carried a low risk of anxiety and more importantly asymptomatic subjects who were identified to be at risk were motivated to take steps to prevent disease. 171 However, at present population screening for HH is not recommended. 161 Family screening Once the proband case is identified and confirmed, family screening is strongly recommended. 172 Siblings of an affected proband with HFE-HH have a one in four risk of homozygoisty and screening should be offered. By contrast, the estimated prevalence of HH in children of identified proband is low and approximately 1 in Ideally, the spouse of affected individual should be tested and if the spouse is wild type for HFE, no further testing of the offspring is needed. Children considered to be at risk for adult-onset HH should not have testing in the absence of symptoms. 161 Other first degree relatives of probands should be tested to detect early disease. 173 TREATMENT OF IRON OVERLOAD Phlebotomy is the mainstay of treatment in both symptomatic and asymptomatic individuals with HH and clinical or biochemical evidence of iron overload (Figure 2). The main reason for institution of therapy is to prevent development of cirrhosis as the survival in treated patients before development of cirrhosis is similar to that of the general population. Therapeutic phlebotomy includes two phases, an initial induction phase to induce iron depletion followed by maintenance phase to prevent excess iron reaccumulation. 174 There is general agreement that premenopausal women with a SF <200 lg/l and postmenopausal women and men with SF <300 lg/l do not require treatment. There is no clear consensus in asymptomatic subjects especially with mild to moderate elevation of SF (i.e. SF lg/ L, but below 1000 lg/l) and normal liver tests. These individuals are at a low risk of developing HH-associated signs and symptoms. 175 Watchful waiting is an option although volunteer blood donation or prophylactic phlebotomy given its low risk and potential benefit is suggested. 156 In symptomatic patients, however, therapy should be initiated to try and ameliorate symptoms and prevent progression to organ damage. Although most Aliment Pharmacol Ther 2012; 35:

13 A. Siddique and K. V. Kowdley symptoms including fatigue, increased skin pigmentation and insulin requirements in diabetic patients respond to therapy, arthropathy is less responsive and may in fact worsen as iron may persist in the synovial and cartilage even after adequate phlebotomy. 176 Furthermore, HCC, the most dreaded complication of cirrhosis continues to be a threat even after adequate iron depletion. In patients in whom therapeutic phlebotomy is indicated, therapy should be initially started once or twice a week as tolerated until iron stores are reduced to the desired end point. One unit ( ml) of blood removes about mg of iron. The SF should be followed while on phlebotomy and measured after removal of every 1 2 g of iron. 177 Once the SF reaches below the target level 100 lg/l or lower, the SF should be checked more frequently to prevent iron deficiency. 174 After the goal SF 50 lg/l has been achieved, maintenance phlebotomy is typically needed 2 4 times per year. A secondary goal is to reduce the TS below 50%. The hematocrit should be checked prior to each venesection and should be within 10 points or no lower than 20% below the initial hematocrit level. If phlebotomy results in anaemia prior to iron depletion, the frequency of phlebotomies may need to be reduced to once every 2 weeks. Although phlebotomy treatment does not generally improve established cirrhosis, the degree of fibrosis may improve. 178 Pharmacological doses of vitamin C accelerate mobilisation of iron and this rapid mobilisation may precipitate sudden death from cardiac dysrhythmias. 179 Therefore, supplemental vitamin C should be avoided by patients undergoing phlebotomy. Therapeutic erythrocytapheresis (TE) wherein iron is removed as haemoglobin, is useful in cases of massive iron overload to achieve the required SF level in a shorter period of time. 180 A pilot study comparing HH treated with TE treated with phlebotomy showed a reduction of almost 70% in both the total number and the duration of treatments in the TE group. 181 ORTHOTROPIC LIVER TRANSPLANTATION Orthotropic liver transplantation (OLT) is indicated in patients with haemochromatosis who develop decompensated cirrhosis and/or HCC. Previous studies suggest post-olt survival in patients with iron overload is poor and iron depletion prior to transplant may improve survival. 182 One study in patients with HH reported a 1-year survival of 54% compared with 79% survival in other liver recipients. 183 However, a recent study has suggested survival following liver transplantation due to HH has improved in recent years and may be similar to other causes of liver disease. 184 Increased infections (especially fungal) in the first year after transplantation and cardiac complications after 1 year were the most common causes of posttransplantation deaths in HH. 185 TREATMENT OF SECONDARY IRON OVERLOAD Phlebotomy may also have a role in secondary iron overload. In certain conditions, such as PCT, phlebotomy and iron depletion remains the cornerstone of therapy. Similarly, iron chelation therapy has been shown to prevent complications and early death in iron loading anaemias, such as in patients with thalassaemia. The indications for phlebotomy in other conditions, such as HCV and NAFLD remain controversial. In patients with HCV, phlebotomy and iron depletion has been shown to improve biochemical and to a lesser extent histological outcomes. 186 However, phlebotomy is not associated with improved virological response to antiviral therapy. 186 Similarly in NAFLD, phlebotomy may reduce elevated ALT and improve insulin resistance; however, improvement in liver histology or prevention of progression to cirrhosis has not been demonstrated. 187 Phlebotomy is therefore not recommended in mild secondary iron overload associated with conditions, such as HCV and NAFLD. CONCLUSION Haemochromatosis is the most common genetic disorder among Caucasians. A number of proteins are involved in the regulation of iron homeostasis and mutations in the genes encoding these proteins are implicated in the various forms of haemachromatosis. A number of other diverse conditions, such as HCV, NA- FLD and thalassaemia, can also result in iron overload. Target organs affected by iron overload include the liver, heart, joints, skin, endocrine organs and pancreas resulting in complications, such as cirrhosis, cardiomyopathy, diabetes and possible increased risk of hepatic and extrahepatic cancers. HFE gene testing is widely available for the diagnosis of HFE-related haemochromatosis, but liver histology is required for the diagnosis of less common non-hfe related haemochromatosis and secondary iron overload. Early diagnosis and prompt treatment with phlebotomy with or without iron-chelation therapy (among patients with iron-loading anaemias) can prevent complications and improve survival in patients with iron overload. ACKNOWLEDGEMENT Declaration of personal and funding interests: None. 888 Aliment Pharmacol Ther 2012; 35: