Intracellular iron homeostasis: the iron regulatory proteins

To orchestrate intracellular iron traffic, cells contain proteins that have the ability to register the presence or absence of iron, identified in mammalian cells as two homologous iron regulatory proteins, IRP1 and IRP2. These discern cytosolic iron levels and alter the expression of proteins involved in iron movements, such as TfR1 (but not TfR2), DMT1 and ferroportin, or iron storage, such as ferritin. IRP1 and IRP2 share extensive sequence homology, but significant differences exist between the two. Sensing of iron levels is coupled to availability of oxygen and other oxidants. Many proteins involved in iron metabolism have motifs called iron-responsive elements (IREs), which are stem-loop structures in their 3′ or 5′ untranslated regions. When, for example, intracellular iron is scarce, the IRP binds to the 5′ untranslated region of the ferritin mRNA transcript, and ferritin synthesis is halted, to prevent iron storage, while IRP binding at the 3′ untranslated region of TfR1 stabilizes its mRNA, leading to higher TfR1 protein expression and increased iron uptake. It has been suggested that the molecular basis for the iron-sensing process in mammalian cells relies on stabilization or degradation of an iron- and oxygen-binding protein that target IRPs to the ubiquitin (Ub) proteasome.

Systemic iron homeostasis: hepcidin, the iron hormone

Systemic iron homeostasis is also tightly regulated to avoid iron starvation or excess, both responsible for disease states. The nature of the iron regulatory signals, mediators and targets are now being uncovered. Storage iron in hepatocytes and tissue macrophages must be mobilized in response to need, and the concept of store and erythroid ‘regulators’ was originally conceived by Finch. The store regulator would control duodenal iron uptake by means of a tightly regulated feedback mechanism to prevent iron overload. The erythroid regulator would enhance intestinal absorption in response to erythroid demand when there is an increased requirement for iron but the capacity of storage cells is insufficient. In addition, iron could be regulated at the level of the duodenum by the amount of iron recently consumed, thus invoking a dietary regulator, probably resulting from the buildup of intracellular iron. Even in systemic iron deficiency, a bolus of iron may cause a so-called mucosal block. Furthermore, iron homeostasis is also modified in conditions of hypoxia, by a humoral hypoxia regulator, and cellular iron may be retained and its absorption interrupted by infection, through the inflammatory regulator, presumably to withhold iron from the pathogen.

Such ‘regulators’ were largely theoretical until the discovery of hepcidin (hepatic bactericidal protein), or LEAP1 (liver-expressed antimicrobial peptide 1), the iron regulatory hormone. Hepcidin functions at the point of convergence of all humoral iron regulators, including the erythroid, the store and the inflammatory regulators. Originally identified as a type II acute-phase protein produced by the liver, hepcidin is a 25-amino acid peptide containing four disulphide bonds that forms a hairpin shape stabilized by these bonds. Hepcidin is synthesized as an 84-amino acid prepropeptide containing a typical N-terminal 24-amino acid, endoplasmic reticulum-targeting signal sequence and a consensus furin cleavage site immediately preceding the C-terminal 25-amino acid bioactive peptide. In addition to prohepcidin and hepcidin-25, carboxy-terminal 22- and 20-amino acid forms of hepcidin are found in the circulation and urine, but hepcidin-25 is the bioactive form. Hepcidin is a member of the cysteine-rich, cationic, antimicrobial peptide family and, in fact, has retained some antibacterial and antifungal effects in vitro . Of all known antimicrobial peptides involved in innate immunity, however, it seems hepcidin has evolved the unique capability of fighting pathogens by restraining serum iron that is necessary for their growth and proliferation during infection. This task is accomplished by binding to ferroportin. As a result of its interaction with circulating hepcidin, ferroportin is internalized and degraded, thereby diminishing the cells’ ability to transfer iron to the plasma compartment. Therefore low serum levels of hepcidin leads to increased iron flux from enterocytes and macrophages into the circulatory iron pool, whereas high serum hepcidin leads to low circulatory iron due to inhibition of its intestinal absorption or release from macrophages.

Hepcidin has evolved as part of the innate immune defence. As such, hepcidin is induced by infection and inflammation and plays a central role in the anaemia and hypoferraemia of chronic disease, alternatively called ‘anaemia of inflammation.’ Hepcidin responds to a variety of inflammatory signals and mediators, particularly interleukin-6 (IL-6), IL-1, IL-22, and activin B. However, hepcidin is in essence an acute-phase protein, and it senses, beyond inflammation, a number of intracellular and extracellular stress signals ( Fig. 4.1 A ). Endoplasmic reticulum (ER) stress is primarily associated with disruption of ER homeostasis and accumulation of unfolded or misfolded proteins in the ER. ER stress has been also involved in a number of pathophysiological states, including inflammatory response, nutrient disorders and viral infection. Exogenous and endogenous ER stressors can trigger hepcidin transcription through the cyclic adenosine monophosphate (cAMP) response element binding protein 3–like 3 (CREB3L3, also known as CREBH) and leads to perturbation of iron homeostasis in vivo . Also, metabolic stress can turn on hepcidin transcription. It has been recently reported that PPARGC1A, a transcriptional coactivator, cooperates with CREBH to activate hepcidin and regulate iron traffic in vivo during food deprivation. The latter finding has important implications during human disorders associated with insulin resistance and induced gluconeogenesis, such as obesity, diabetes and nonalcoholic fatty liver disease.

Stimulatory and inhibitory signals and pathways controlling hepcidin transcription. A, The main hepcidin stimulatory signals identified include iron, inflammation/infection, and endoplasmic reticulum (ER)/nutrient stress. The iron signals converge on a membrane-associated heterotetrameric signalling complex, composed of transferrin-iron, bone morphogenetic protein (BMP) ligands, two type I and two type II serine-threonine-kinase receptors, a co-receptor (haemojuvelin, HJV) and ancillary proteins (including HFE and TfR2) that trigger a common signal transduction cascade involving R-SMADs and co-SMADs and activate transcription of the hepcidin gene. Both HFE and transferrin receptor 2 (TfR2) act as sensors for the iron signal and are necessary for optimal induction of the BMP/SMAD pathway. A key mediator of hepcidin response to inflammation is IL-6, which stimulates hepcidin transcription through STAT3, possibly by interacting with the BMP/SMD pathway. Activin B likely uses the BMP/SMAD pathways to induce hepcidin during inflammation. Hormonal and nutrient signals during activated gluconeogenesis induce hepcidin through cyclic adenosine monophosphate (cAMP) and involve the transcriptional coactivator PPARGC1A and CREBH, a transcription factor also responsible for hepcidin regulation by a variety of ER stressors. The main signals for hepcidin inhibition arise in the bone marrow during active erythropoiesis and include growth differentiation factor 15 (GDF15), a member of the transforming growth factor beta (TGFβ) superfamily, twisted gastrulation protein (TWSG1), a BMP-binding protein, and erythroferrone (ERFE) . Three ‘negative modulators’ of the BMP/SMAD signaling pathway have also been identified: the soluble form of HJV, a membrane serine protease matriptase-2 (TMPRSS6) that possibly cleaves HJV and SMAD7. Neogenin, a membrane receptor for RGM, has been proposed to stabilize HJV and participate in HJV shedding, but its role is still controversial. B, The iron-sensing machinery in the liver. Iron transferrin from the portal vein enters the sinusoids and induces the local production of BMPs, such as BMP6, by sinusoidal cells and perisinusoidal cells. Both iron transferrin and BMPs engage the membrane-associated heterotetrameric signalling complex, as described in A .

From Pietrangelo A. Genetics, genetic testing, and management of hemochromatosis: 15 years since hepcidin. Gastroenterology 2015;149(5):1240–51.

Hepcidin induction in the presence of stress factors that perturb the internal homeostasis—our legacy to the innate immune response to pathogens—aims at preserving and retaining in the body (or within the cells) the precious iron needed for vital energy production.

During iron deficiency and anaemia, hepatic hepcidin transcription must be turned down so that more iron can be transferred from the intestine and storage sites to serum transferrin and to the bone marrow. Hypoxia and erythropoietin inhibit hepcidin synthesis and increase iron absorption. Circulating factors derived from maturing erythroblasts in the bone marrow have been reported to downregulate hepcidin transcription in the liver, including growth differentiation factor 15 (GDF15), a member of the transforming growth factor beta (TGF-β) superfamily; twisted gastrulation protein, a BMP-binding protein, and more recently erythroferrone, which mediates hepcidin suppression during stress erythropoiesis.

The main humoral signal for hepcidin synthesis is iron itself ( Fig. 4.1 B ). The iron-sensing system resides within the liver, and its disruption is usually responsible for human haemochromatosis. Iron-sensing involves transferrin-iron (i.e. the extent of Tf saturation) and a class of ligands of the TGF-β superfamily, the bone morphogenetic proteins (BMPs), which normally play a crucial role in embryonic development and in fundamental processes during postnatal life. Tf-iron takes part of a multiprotein complex at the hepatocyte plasma membrane made by BMPs, BMP receptors, a BMP co-receptor (haemojuvelin, HJV) and a number of ancillary proteins (including HFE and the second transferrin receptor, TfR2) ( Fig. 4.1 ). This interaction triggers the phosphorylation of SMAD1,5,8 complex (receptor-associated SMADs, R-SMADs), its subsequent binding to SMAD4 (common-partner SMAD, co-SMAD), the translocation of the SMAD complex to the nucleus and activation of hepcidin transcription. The BMP co-receptor HJV, which is present in either a soluble or a cell-associated form, provides specificity to the iron signal in the liver and functions as an enhancer for iron signalling to hepcidin. At least two other proteins are required for normal signalling of iron status to hepcidin via the BMP6/SMAD1,5,8 pathway, HFE and TfR2. In fact, functional loss of HFE in mice and humans leads to low hepcidin and haemochromatosis. Functional loss of TfR2 in mice and humans is also associated with blunted hepcidin expression and iron overload. The details of HFE and TfR2 function in the context of the BMP/SMAD signalling pathway are still not completely understood. HFE is a major histocompatibility complex (MHC) class I-like protein that interacts with TfR1, the receptor for serum Tf that mediates uptake of Tf-bound iron. The C282Y HFE mutation associated with human haemochromatosis disrupts a disulphide bond required for HFE binding to β2-microglobulin and transport to the cell surface and endosomal membranes, where it interacts with TfR1. The H63D mutation, a common HFE polymorphism, does not impair HFE-TfR1 interaction. HFE is not required for transcriptional regulation of BMP6 in response to dietary iron, but loss of HFE reduces BMP6 signalling, in vitro and in vivo . HFE interacts with the BMP type I receptor ALK3 to stabilize it and increase its expression, thereby inducing hepcidin expression. TfR2 mediates the uptake of Tf-bound iron by hepatocytes in vitro, but its in vitro affinity for Tf is much lower than that of TfR1. It has been postulated that also TfR2 interacts with HFE, forming a unique iron-sensing complex with TfR1-HFE that modulates hepcidin expression in response to Tf-iron. However, human studies in patients with combined TfR2 and HFE mutations and studies in HFE/TfR2 double-knockout mice have shown that the contemporary loss of HFE and TfR2 has additive phenotypic effects. This suggests that HFE and TfR2 regulate hepcidin and iron metabolism in an independent manner. Both proteins seem to be important for BMP signalling and necessary for an optimal response to BMPs.

The BMP-SMAD pathway is likely counter-regulated by a number of feedback regulatory signals that prevent overshooting of hepcidin expression in response to positive stimuli. A key role is likely played by a membrane serine protease 2 (also called TMPRSS6) that functions by inhibiting the BMP pathway, possibly by cleaving HJV, although this aspect is still debated. Loss of TMPRSS6 in humans causes a syndrome known as ‘iron deficiency, iron-refractory anaemia’ (IDIRA) characterized by hyperhepcidinaemia and microcytic iron deficiency anaemia resistant to oral iron supplementation. A second BMP-pathway inhibitor is SMAD7, induced by chronic dietary iron loading, and BMP-SMAD signalling pathway activity in the liver. Another player in the BMP/SMAD pathway is neogenin, a member of the DCC (deleted in colorectal cancer) family of tumour suppressor molecules. Neogenin appears to interact with HJV, but its role in HJV biology and hepcidin synthesis is still controversial. Interestingly, the livers of neogenin-mutant mice exhibit reduced BMP signalling, low levels of hepcidin and iron overload. BMPs and the BMP/SMAD pathway are therefore central in hepcidin transcription in the liver. BMP6 in particular seems to play a key role in this process. BMP6 is largely produced by hepatic sinusoidal cells and nonparenchymal cells (see Fig. 4.1 B ) and is directly induced by excess iron. In fact, blocking BMP6 in vivo inhibits hepcidin expression and increases serum iron, whereas genetic BMP6 ablation in mice leads to low hepcidin expression and haemochromatosis, indicating that BMP6 is an endogenous regulator of hepcidin expression and iron metabolism in vivo.

In summary, iron homeostasis requires specific transportation across membranes and meticulous intracellular storage. Diminution of iron stores caused by dietary deficiency, iron loss or infection gives rise to eventual iron restriction and progression to anaemia. The converse may arise, however, in which the large ionic iron molecule, in excess and in solution with oxygen, may generate free-radical formation through Fenton and Haber–Weiss chemistry, with hydrogen peroxide (H ₂ O ₂ ) being changed into its radical (HO ⁻ ). This leads to consequent damage to DNA, proteins and membranes.

The classic example of iron overload in human pathology is HFE -hereditary haemochromatosis, but numerous other entities are now identified to cause pathological iron overload. Some are well described and have a verified hereditary basis, whereas in others the genetic and hereditary basis is still speculative. Still others reflect iron overload on an inflammatory or infectious basis or as a reactive response to systemic or as-yet unknown disease processes.

Controversy surrounds nomenclature and classification of these iron overload states. Haemochromatosis (HC), as identified in the liver, has previously been related to iron loading in primary liver cells, hepatocytes and biliary epithelium, whereas haemosiderosis, iron staining in tissues, was employed by some as the terminology of choice when the overload was predominantly within Kupffer cells. Many overload states, however, may show elements of both HC and haemosiderosis. A further caveat is that current classifications, including the Online Mendelian Inheritance in Man (OMIM) database, are founded on the basis of single-gene defects and have significant shortcomings in that numbers of atypical cases have been identified that relate to multiple gene mutations. It appears now that failure to produce adequate amounts of the iron hormone hepcidin or its impaired activity, mostly from genetic changes but also from acquired factors, results in a syndromic entity that resembles the historical definition of HC. Thus hereditary HC can be defined and classified phenotypically, regardless of the underlying gene defect, as an inherited disorder resulting from an inborn error of iron metabolism causing hepcidin deficiency that leads to progressive loading of parenchymal cells of the liver, pancreas, heart and endocrine organs and leads to damage and disease states.

Historical perspective

Trousseau first described a diabetic patient with an ‘almost bronzed’ appearance in 1865. In 1871, Emile Troisier detailed the first autopsied case of a diabetic patient with cirrhosis and a red-brown liver containing clumps of pigment. Von Recklinghausen later termed the condition ‘haemochromatosis’. Two main theories emerged from subsequent reports: the primary disease was diabetes that then gave rise to cirrhosis and pigmentation, or alternatively, the pigment was primarily derived from the blood. After years of dispute as to its pathogenesis, Sheldon consolidated the existing knowledge of HC with its classic triad of diabetes, cirrhosis and melanin-based pigmentation. He described the entity as ‘an inborn error of metabolism, which has an overwhelming incidence in males and which at times has a familial incidence.’

The autosomal recessive inheritance pattern of classic hereditary HC was thereafter established, but it was only in 1976 that Simon et al. confirmed an association between the disorder and the human leukocyte antigens (HLAs) A3 and B14. The subsequent discovery that β2-microglobulin knockout mice developed iron overload analogous to human HC raised the postulate that the defective gene would be within an MHC molecule. Positional cloning experiments using linkage disequilibrium and haplotype analysis allowed the identification of the affected gene, an atypical class I HLA molecule originally designated HLA-H and subsequently redefined as HFE . Subsequent knockout of the mouse Hfe gene confirmed iron overload, thus confirming HFE to be the defective gene in classic hereditary HC.

As genetic testing for HFE mutations became more widespread, it rapidly became clear that the situation was more complicated than initially thought. Other iron genes were discovered whose mutations were associated with hereditary iron overload syndromes with some, or many, or apparently even all, of the phenotypic features of classic HC ( Table 4.1 ): ferroportin-associated iron overload ( FPN ), transferrin receptor 2-associated HC ( TfR2 ), hepcidin-associated ( HAMP ) and haemojuvelin-associated ( HJV ) ‘juvenile’ HC.

Sheldon almost certainly described patients having the characteristics of ‘juvenile’ HC. However, the entity was only formally recognized after the case study and literature review of Lamon et al. They described a 36-year-old woman with heart failure, diabetes mellitus, hepatomegaly and secondary amenorrhoea and reviewed another 52 cases published over the previous 85 years. Essential differences between these cases and classic hereditary HC are notable compared with the large series of Finch and Finch, namely, that it is a rare form of iron overload with rapid and severe progression of disease leading to significant complications before 30 years of age. Lamon et al. also noted the inherited nature of this rare disease and favoured an autosomal recessive inheritance. Subsequent genotyping identified the disorder to be distinct from HFE-linked iron overload. A high frequency of consanguinity is observed in patients with juvenile HC, with the disease occurring in siblings but not parents. A human-genome search demonstrated linkage between the disease and numerous markers on the long arm of chromosome 1q. Subsequent analysis of Greek, Canadian and French families carrying the disorder identified multiple deleterious mutations at LOC148738, whose protein product is identified as haemojuvelin (HJV), previously HFE2. In addition, the investigation of juvenile HC identified a second cohort that is even rarer, demonstrating a mutation, C70R, on 19q13, the gene encoding hepcidin. The concept and spectrum of juvenile HC has been further extended by the recent identification of two young patients presenting with typical features of the disease, severe endocrinopathy and cardiomyopathy, but testing positively for combined mutations for C282Y/H63D and TfR2 . In addition, there is evidence that selected HJV and HAMP mutations, when carried simultaneously with mutant HFE genes, may influence iron status in older patients being evaluated for a molecular diagnosis of HC because of increased transferrin saturation and/or serum ferritin. This further challenges the current classification system for hereditary HC in that ‘juvenile HC’ does not represent a distinct monogenic disorder but may be linked to the adult-onset form of hereditary HC.

Definition

The present definition of hereditary HC embraces the classic disorder related to HFE C282Y homozygosity (the prototype for this syndrome and by far the most common form) and the rare disorders more recently attributed to loss of TfR2, HAMP and HJV or, in very rare cases, FPN ( Table 4.1 ). This concept of HC stems from the idea that, beyond their genetic diversities, all known haemochromatoses belong to the same clinicopathological entity: they all originate from the failure to prevent unneeded iron from entering the circulatory pool as a result of genetic changes compromising the synthesis or activity of hepcidin, the iron hormone ( Fig. 4.2 ). Depending on the gene involved and its role in hepcidin regulation, the phenotype of HC varies, ranging from the rare HJV and HAMP juvenile forms characterized by massive iron loading with severe health and endocrine disorders, to the adult-onset phenotype dominated by liver disease and usually associated with HFE, TfR2 and rarely FPN mutations. In brief, the features that distinguish hereditary HC from all other iron-loading disorders listed in Table 4.1 are hereditary nature (usually autosomal recessive); early and progressive expansion of the plasma iron compartment (increasing transferrin saturation); progressive parenchymal iron deposits with potential for severe damage and disease that may involve liver, endocrine glands, heart and joints; nonimpaired erythropoiesis and optimal response to therapeutic phlebotomy; and inadequate hepcidin synthesis/activity.

The common genetic basis and phenotypic continuum of haemochromatosis (HC). The basic features of hereditary HC can be produced by pathogenic mutations of a number of iron metabolism genes ( HJV, HAMP, TfR2, FPN and HFE ). Depending on the gene involved and its role in hepcidin biology, the HC phenotype varies. If the altered gene plays a dominant role in hepcidin synthesis (e.g. HAMP itself or HJV ), circulatory iron overload occurs rapidly and reaches high levels. In these patients the clinical presentation will invariably be dramatic, with early onset (first to second decade) of full-blown organ disease (particularly affecting the heart and endocrine glands). If the HC gene is less critical to this process (e.g. HFE), a milder late-onset phenotype will arise. This continuum also includes ‘intermediate phenotypes’ (e.g. those caused by pathogenic mutations of TfR2 or by rare combinations of mutations). HCC, Hepatocellular carcinoma.

From Pietrangelo A. Hereditary hemochromatosis: pathogenesis, diagnosis, and treatment. Gastroenterology 2010;139:393–408.

Epidemiology

An autosomal recessive disease, classical HFE -associated hereditary HC is the most common monoallelic inherited disorder in Western society. The dominant missense polymorphism identified in 80% of cases, C282Y, is characterized by a single nucleotide change, G to A, resulting in a Cys→Tyr at position 282 of the unprocessed protein, occurring in a highly conserved region involved in the disulphide bridge in the MHC class I protein. A less common polymorphism is a C-to-G change resulting in a Hys→Asp substitution at amino acid 63, showing limited clinical effects, although compound heterozygosity for C282Y and H63D may cause disease expression. Of several other uncommon polymorphisms, S65C, in which cysteine replaces serine at position 65, has been associated with compound heterozygosity for either C282Y or H63D. It has been related to the development of mild to moderate hepatic iron overload but without clinical manifestations. Its significance is still controversial.

The C282Y polymorphism is most prevalent among populations of northern European origin, with more than 90% of patients with clinically penetrant HC in the United Kingdom being homozygous for C282Y. Worldwide allele frequencies are calculated at 1.9% for C282Y and 8.1% for H63D, with highest frequencies of 10% for C282Y in Irish chromosomes and 30.4% for H63D in Basque chromosomes. The origin of the genetic mutation was thought to represent a unique event in chromosome HLA-A3 and -B7 originating from the Celts in central Europe between 65 and 70 generations ago and spreading north and west by population migration. However, an alternative Viking origin has been postulated, and it is recently proposed that the initial C282Y polymorphism occurred in mainland Europe before 4000 bce , earlier than both the Celtic and the Viking period.

The genetic defect, which caused no serious obstacle to reproduction and may even have conferred some advantages, was passed on and spread through population migration. The distribution of the C282Y mutation coincides with its northern origin, with frequencies ranging from 12.5% in Ireland to 0% in southern Europe. In addition to C282Y, H63D, the ‘minor’ HFE polymorphism, has been found more frequently in HC patients than in the control population. The frequency of the H63D polymorphism shows less geographic variations with an average allelic frequency of 14 %, but its clinical impact appears to be limited. An additional HFE polymorphism is S65C, which can be associated with HC when inherited in trans with C282Y on the other parental allele. The prevalence of C282Y homozygosity among patients with liver disease is about 5%, 10-fold higher than the reported prevalence in the general population. This figure increases if patients with liver disease are preselected for increased transferrin saturation. Even higher C282Y frequencies can be found in patients with hepatocellular carcinoma, a known complication of HC.

Haemochromatosis is associated with homozygosity for the C282Y HFE mutation in approximately 80% of clinically characterized patients of European ancestry. Therefore almost 20% of such patients have the disease in absence of C282Y. Although compound heterozygosity (H63D/C282Y) appears to be disease associated, usually cofactors are implicated in the clinical expressivity.

Other genes associated with clinical HC are TfR2 , HJV , HAMP and FPN (see Table 4.1 ). None of these non- HFE HC appears to be restricted to Northern Europeans. Although the TfR2 mutation is rare, at least nine individual TfR2 mutations have been identified. With the exception of one patient of Portuguese descent with a c2069 A→C, Q690P mutation in the TfR2 gene mutation and two affected homozygous female siblings, most represent largely inbred families of Italian extraction with various mutations of the TfR2 gene and consanguinity a common feature. However, the AVAQ 594–597 deletion, originally described by Girelli et al. in their Italian cohort, has also been identified in three members of a Japanese family. Subsequent investigation of nine other, unrelated Japanese patients with HC of unknown origin described two more novel TfR2 mutations, L490R and V561X. The patient with the V561X mutation, age 58, was a member of a consanguineous family. Both these patients had cirrhosis and diabetes, one with associated skin pigmentation. HC is rare in the Far East, particularly among the Japanese. It would thus appear that TfR2 -hereditary HC is the most common form of hereditary iron overload among Japanese patients, although iron loading secondary to acaeruloplasminaemia is also identified in this population. However, in 2001, a Y231del mutation in the HFE gene was found in the Huh-7 hepatoma cell line (obtained from a Japanese donor) and was shown to prevent the translocation of HFE to the cell surface, similar to the C282Y mutation. More recently, this same HFE mutation has been reported in a Japanese pedigree affected by HC, indicating the occurrence of a HFE -related form of haemochromatosis also in Asian populations.

The juvenile form of HC is rare. Most cases are caused by mutations of HJV . One common HJV mutation, G320V, has been identified in all reported studies. It is present in half of juvenile HC families. Numerous other mutations have subsequently been identified. More recent analysis of patients from the central and northern parts of Europe, specifically Germany, Slovakia, Croatia and Ireland, has also identified the G320V mutation in the majority. A small proportion of patients with the juvenile form of HC carry mutations in the gene encoding hepcidin.

Although most FPN mutations give rise to a distinct form of hereditary iron overload called the ‘ferroportin disease’, unusual FPN mutations cause rare forms of HC similar to HFE -HC.

Pathogenesis

The first biochemical manifestation of HC is an increase of transferrin saturation that reflects an uncontrolled influx of iron into the bloodstream from enterocytes and macrophages. Except for menstruation, the body has no effective means of significantly reducing plasma iron levels. Without therapeutic intervention, overload in the plasma compartment will lead to the progressive accumulation of iron in the parenchymal cells of key organs, creating a distinct risk for oxidative damage. This stage is reflected in increasing serum ferritin levels. The time of onset and pattern of organ involvement in HC vary depending on the rate and magnitude of plasma iron overloading, which in turn depend on the underlying genetic mutation. For this reason, milder adult-onset forms (e.g. HFE and TfR2 related) and more severe juvenile-onset forms (e.g. HJV and HAMP related) are recognized. The extent of iron release from enterocytes and macrophages into the bloodstream in humans is under the control of the hepcidin–ferroportin axis (see Fig. 4.1 ). It now seems that HFE and TfR2 play a role in conveying the iron signal to hepcidin in the hepatocyte, although the details of this process are not fully uncovered.

In view of these findings, HC should be seen as a genetically heterogeneous disease that results from the complex interaction between genetic and acquired factors. If the altered gene plays a dominant role in hepcidin synthesis (e.g., HAMP itself or HJV ), circulatory iron overload occurs rapidly and reaches high levels (see Fig. 4.2 ). In these cases the modifying effects of acquired environmental and lifestyle factors will be negligible, and the clinical presentation will invariably be dramatic, with early onset (first to second decade) of a full-blown organ disease. In contrast, C282Y- HFE homozygosity results in a genetic predisposition that requires the concurrence of host-related or environmental factors to produce disease (see Fig. 4.2 ). Co-inherited mutations in other HC genes, such as HAMP and HJV , may have a role in disease penetrance of HFE -HC, but they are rare.

Clinical aspects

HFE -associated haemochromatosis

The clinical presentation in patients who are homozygous for C282Y is highly variable, with general signs of weakness (60% of patients), arthralgia/arthritis (30–40%), hepatomegaly/cirrhosis (13–60%), diabetes mellitus (10–30%), sexual dysfunction (10–40%) and cardiac symptoms with arrhythmia (20–29%) and cardiac failure (15–35%). Skin pigmentation is estimated to occur in 47% of proband cases. However, extrahepatic disease tends to be less prominent in modern studies, including diabetes mellitus, arthritis and heart disease. The initial presentation is often vague and nonspecific in many patients, and a high index of suspicion is required to diagnose the condition.

Furthermore, whereas patients with defined HFE -HC will test positively for the C282Y mutation, a significant proportion do not develop significant hepatic iron overload. Gene penetrance is not obligatory and recent large population-based studies confirm that penetrance is low in HFE -hereditary HC, implying that C282Y homozygosity is necessary but not sufficient on its own to cause clinically manifesting disease. Ethnicity, environmental factors such as blood loss (important modifier in females), dietary intake, alcohol and other, as-yet unknown genetic modulators may play a part in this differential expression. It is likely that these disease frequencies will change further with increased awareness of the disease and the development of screening techniques.

In proband patients in whom the diagnosis is suspected, serum testing for iron overload forms the cornerstone of initial identification: transferrin saturation, unbound iron-binding capacity (UIBC) and serum ferritin. Consensus is that Tf saturation >45%, UIBC <28 µmol/L and ferritin >300 µg/L (>200 in women) identifies hereditary HC, although these values may not be appropriate for all populations. Ferritin in isolation is highly sensitive and its normality rules out iron overload, but it is an acute-phase protein that is also elevated in inflammatory states.

Before the discovery of HFE gene mutations, liver biopsy confirmed diagnosis, identifying the typical histological features and allowing for grading and determination of the hepatic iron index (ratio of hepatic iron concentration divided by age), as discussed later. However, subsequent to the development of genotyping, the necessity for liver biopsy now depends on clinical status. If inflammation and other confounding factors are excluded, serum ferritin correlates well with the level of iron excess. Magnetic-susceptibility measurement or magnetic resonance imaging (MRI) may act as a second estimation of iron overload. In C282Y HFE homozygotes, if iron load is moderate and other diagnostic parameters are normal (e.g. serum transaminases, fasting blood glucose, hormonal evaluation, cardiogram, joint/bone x-ray films), therapy by venesection (phlebotomy) may commence without liver biopsy. In cases of heavy iron overload, when one or more of these parameters are abnormal, liver biopsy is mandatory, in particular to evaluate the presence of cirrhosis, other hepatic pathology or iron-free foci, and at this stage the biopsy is performed as a prognostic marker. The current gold standard in the case of hereditary HC is thus genetic testing. The relevance of the H63D mutation as well as C282Y/H63D compound heterozygosity is still uncertain because a small percentage may develop clinically significant HC, normally in the presence of damaging cofactors.

The macroscopic and microscopic features of penetrant HFE -HC are highly characteristic in the liver. The classic description is one of a brown to rusty colour, staining intense blue with Perls ( Fig. 4.3 ), with or without the gross appearance of cirrhosis. Histologically, the progression of the disease is again characteristic. Iron deposition begins in the periportal hepatocytes (zone 1) ( Fig. 4.4 ) and extends progressively to involve all zones of the liver ( Fig. 4.5 ). The iron has a characteristic pericanalicular pattern when observed under high power ( Fig. 4.6 ). With progression of the disease, there can be deposition of iron in the biliary epithelium ( Fig. 4.7 ) and transfer into Kupffer cells and portal macrophages with sideronecrosis ( Fig. 4.8 ). Progressive portal-based fibrosis may evolve to cirrhosis ( Fig. 4.9 ). Of importance is the identification of iron-free foci in histologically advanced liver disease, which may foretell the development of hepatocellular carcinoma, as discussed later.

Postmortem liver from a patient with hereditary haemochromatosis. The liver has an orange-brown colour that, when dipped in Perls solution, demonstrates the characteristic Prussian-blue reaction.

A 38-year-old man with abnormal liver function tests was subsequently identified as homozygous for the C282Y mutation based on biopsy findings. A, Liver biopsy is histologically unremarkable on routine staining (haematoxylin and eosin [H&E] stain). B, However, there is grade 1 iron deposition in periportal hepatocytes (Perls stain).

A 45-year-old man homozygous for C282Y with abnormal liver function tests. There is grade 3 iron overload. A hepatic iron gradient is still evident. (Perls stain.)

Pericanalicular pattern of iron distribution that is typical for hereditary haemochromatosis but not specific (Perls stain).

C282Y homozygote with grade 4 haemochromatosis. A, Iron accumulation can be appreciated on H&E staining. B, There is loss of the gradient pattern and marked iron overload. The bile duct (arrow) also has iron deposition. (Perls stain.)

A 45-year-old man confirmed to be homozygous for the C282Y mutation and with a history of alcohol abuse. Cirrhosis with grade 4 iron overload was confirmed histologically. Sideronecrosis is identified by large globules of haemosiderin associated with aggregates of heavily iron-laden Kupffer cells (PAS positive), both periportal and within the lobular parenchyma. (Periodic acid-Schiff [PAS] stain.)

Patient with esophageal varices and splenomegaly. Biopsy demonstrates established cirrhosis with haemosiderin deposition in hepatocytes, bile ducts and portal macrophages. (Perls stain.)

Venesection forms the cornerstone of successful therapy for hereditary HC. Ideally, this entails weekly phlebotomy (400–500 mL) until ferritin <20–50 µg/L and Tf saturation <30%, after which maintenance venesections are done to keep serum ferritin normal. If biopsy has been performed, the hepatic iron index can be calculated as well as a ‘body iron ratio’, which estimates total iron removed based on phlebotomies and related to age. In addition, the biopsy may be graded for degree of iron overload. Abstinence from iron-rich foods, supplements and vitamins is recommended. Chelation therapy is not warranted unless there are contraindications to venesection.

Fully penetrant hereditary HC carries with it significant potential for morbidity and mortality, including cirrhosis and hepatocellular carcinoma, diabetes mellitus, cardiac failure and impotence. The prevalence of alcohol abuse and chronic hepatitis C virus infections appears increased in patients with HC (see later), but sepsis secondary to bacterial infection does not appear to be a significant problem, with the exception of cases of Yersinia infections. Hepatocellular carcinoma is identified as a significant cause of mortality in HFE -hereditary HC, although its prevalence is not as high as was previously thought. The risk of developing other extrahepatic cancers is still debated. This anticipates the potential role of iron as a putative carcinogen.

However, quantification of these factors is confounded by the prevalence of the C282Y mutation when measured against the penetrance of the gene. Furthermore, many common entities classically identified in patients with hereditary HC, including diabetes and arthritis, also frequently occur in the general population. Earlier series demonstrated significant morbidity and mortality in both untreated and treated patients. Bomford and Williams identified poor survival amongst untreated patients of 18% and 6% at 5 and 10 years, respectively. Similarly, for treated cases, Niederau et al. identified a cumulative survival of 93% at 5 years and 77% at 10 years, significantly reduced compared with the expected survival rates for a normal population. However, more modern series identify that, although most C282Y homozygotes have increased levels of Tf saturation and serum ferritin, symptoms associated with iron overload are no greater in C282Y homozygotes compared with gender- and age-matched controls, and life expectancy is not significantly curtailed.

Ernest Beutler was the first to draw attention to the low clinical expressivity of the C282Y HFE mutation. Subsequent studies in which patients were followed for more than 20 years have then shown that disease progresses in only a minority of untreated C282Y homozygotes. As many as 38–50% of patients homozygous for HFE C282Y will develop iron overload, and 10–33% will eventually develop HC-associated morbidity. Penetrance is usually higher among male patients homozygous for HFE C282Y than female patients, probably because of menstruation and pregnancies. Apart from male gender, and obvious causes of blood loss, alcohol abuse is likely an important ‘modifier’ associated with HC-related cirrhosis. Combinations of mutations in HAMP , HJV and TfR2 have been associated with more severe phenotype in rare patients. Polymorphic variants of BMP2 are also reportedly associated with higher penetrance of HFE -HC. In recent years, novel loci affecting iron homeostasis in individuals at risk for HC have been reported, with some including known iron-related genes. Single-nucleotide polymorphisms (SNPs) at ARNT1, transferrin and TfR2 affect iron markers in HFE C282Y homozygotes at risk for HC. A genome-wide association study (GWAS) using DNA collected from 474 unrelated C282Y homozygotes indicated a link between the rs3811647 polymorphism in the transferrin gene and the phenotypic presentation of HFE-HH. More recently, an exome sequencing study reported that a p.D519G variant in the glyceronephosphate O -acyltransferase ( GNPAT ) gene showed the most significant association with severe iron overload, but the data have been challenged by others.

TfR2 -associated haemochromatosis

The clinical appearance of patients with TfR2 -associated HC mimics that of HFE -hereditary HC: high Tf saturation and serum ferritin levels and low penetrance in premenopausal women. The age range is somewhat younger, but with slow progression of iron overload, thus differing it from juvenile HC.

Furthermore, analysis of a family of southern Italian lineage with features of juvenile HC has recently demonstrated combined mutations for C282Y/H63D (compound heterozygosity) and TfR2 (Q317X) homozygosity while failing to show mutations for either HJV or hepcidin. Two siblings, male and female, diagnosed with severe endocrinopathy and cardiomyopathy at age 24 and 25 years, respectively, both showed cirrhosis with massive loading of iron in hepatocytes. A younger brother age 21 years, however, demonstrated a milder phenotype resembling the iron distribution of classic adult-onset hereditary HC but carrying only the Q317X serum TfR2 homozygote mutation.

Although relatively few TfR2 -HC cases have been documented, the liver pathology is again described as strongly resembling HFE -HC, with early iron deposition in periportal hepatocytes ( Fig. 4.10 ). Most demonstrate a milder degree of iron overload than those with HFE -hereditary HC, although there is progression to cirrhosis in some published cases.

Transferrin receptor 2 (TfR2) haemochromatosis. In this 21-year-old man with a homozygote Q317X TfR2 mutation, iron deposits are seen within zone 1 hepatocytes. (Perls stain.)

Treatment, as for classic hereditary HC, is by venesection, although there is often a persistence of Tf saturation after phlebotomy. The diagnosis is made by clinical presentation, serum iron indices and elimination of the HFE genotype. Although TfR2- hereditary HC is even rarer than juvenile HC, both stand to be of considerable importance in the understanding of the molecular pathogenesis of iron regulation.

HJV- and HAMP- associated haemochromatosis

HJV -associated HC and, more rarely, HAMP -associated HC account for almost all cases of ‘juvenile HC’. Juvenile HC otherwise differs considerably from HFE -hereditary HC with respect to age, an almost equal ratio between genders, greater frequency of cardiac and endocrine disturbances and lower frequency of diabetes and hepatic involvement. The patient usually presents in the second decade, typically with hypogonadism that manifests as primary infertility in the female. A dilated cardiomyopathy that often becomes refractory to treatment is a common complication, and the untreated patient usually dies of cardiac disease by the 30th year. The hepatic complications of iron overload in juvenile HC are not as common as in the case of hereditary HC, despite that iron indices are usually much higher in these younger patients. The hepatic pathology may be profound, however, with histologically diagnosed cirrhosis developing even at a young age in up to 40% of patients. However, the clinical diagnosis of juvenile HC is often coincidental, relating to investigation of endocrine or cardiac abnormalities, including cardiac shock. Glucose intolerance is manifest in almost two-thirds of patients, and the presentation may involve arthropathy or skin changes. Of note, iron indices are often considerably higher than in patients with hereditary HC.

The histological features of juvenile HC are similar to those of hereditary HC in that there is progressive iron loading of parenchymal cells with, at least initially, sparing of the reticuloendothelial system ( Fig. 4.11 ). Untreated patients, even if clinical picture is dominated by hypogonadotropic hypogonadism and cardiomyopathy, often present with advanced liver fibrosis.

Juvenile haemochromatosis in a 40-year-old woman with a homozygote G171R HJV mutation affected by severe cardiopathy and hypogonadism. Biopsy shows massive iron deposits in the hepatocytes. (Perls stain.)

As with hereditary HC, aggressive venesection remains the cornerstone of therapy for HJV – or HAMP -associated HC. Depending on the extent of progression of the disease, there may be a place for chelating therapy, with successful cardiac transplants performed in some patients.