12.3.2 Haemochromatosis

Hereditary haemochromatosis is an inherited iron storage disorder in which altered iron metabolism leads to an increase in intestinal iron absorption. This results in a progressive accumulation of body iron stores particularly in the liver, heart, pancreas, and pituitary. The excess iron deposited in tissues may result in cirrhosis, diabetes, cardiac failure and arrhythmias, hypogonadism, arthritis, hepatocellular carcinoma, and a shortened life expectancy.

Defects in the HFE gene were identified as the most common cause of hereditary hemochromatosis in 1996. A homozygous G→A mutation resulting in a cysteine to tyrosine substitution at position 282 (termed C282Y homozygous) has been identified in 85–90% of patients with hereditary haemochromatosis in populations of northern European descent but is found in only 60% of cases from Mediterranean populations (e.g. southern Italy). Although most cases of haemochromatosis are due to C282Y homozygosity, there is now good evidence that not all those who are homozygous will progress through all stages of the disease. These stages comprise genetic predisposition without abnormality; iron overload (raised serum ferritin in the presence of a raised fasting transferrin saturation) without symptoms; iron overload with haemochromatosis-associated symptoms such as arthritis and fatigue; and iron overload with organ damage, particularly cirrhosis (Fig. 12.3.2.1).

Iron is biologically an important element since it is an essential metabolic requirement for electron transport, oxygen transport, and enzyme activity in living organisms. However, it also has the potential to be highly toxic when present in unsequestered forms because of its ability to initiate free radical reactions leading to lipid peroxidation of cell membranes, which results in cell and tissue damage. Therefore, an appropriate iron balance must be maintained for species survival. In haemochromatosis, however, the ability to down-regulate iron absorption is lost and iron is absorbed at a high rate irrespective of body iron stores. The excess iron eventually leads to clinical complications. Hepatic fibrosis and/or cirrhosis can occur, ultimately resulting in hepatocellular carcinoma. Removal of iron by venesection therapy before tissue damage has occurred prevents the development of most clinical complications.

The disease was first recognized in France in the late 1800s where the association of diabetes, liver cirrhosis, pancreatic fibrosis, and pigmentation was first described and referred to as ‘bronze diabetes’. von Recklinghausen (1889) believed the pigment originated from the blood and coined the term haemochromatosis (1), however, the publication of a monograph by the English physician Sheldon in 1935 suggested that the multiorgan involvement probably represented a single disease resulting from an inborn error of iron metabolism (2). There were others, however, who believed that the iron loading resulted from nutritional factors and alcohol. In 1976, Marcel Simon made the important observation that there was a close link between haemochromatosis and the human leucocyte antigen (HLA) class I loci on chromosome 6, the disease being inherited as an autosomal recessive trait (3). Localization of the genetic defect was finally determined some 20 years later when the C282Y mutation in the HFE gene that results in haemochromatosis was identified and found to be responsible for the majority of clinical cases with hereditary haemochromatosis (4).

Total body iron content in humans is 3–5 g. Mammals depend on nutrition for an adequate supply of iron, and since there is no effective mechanism for excretion of large amounts of iron, the amount that the body contains is tightly regulated by controlling the daily absorption of iron in the diet. In iron deficiency, iron absorption from the gastrointestinal tract is increased and release of iron from macrophages is facilitated. When the body’s iron burden is excessive, absorption is decreased and release of iron from macrophages is inhibited.

Dietary iron is absorbed mainly by villous enterocytes of the duodenum. Haem iron is more readily absorbed from the lumen than nonhaem iron, however, absorption of the latter may be enhanced by dietary factors. Nonhaem iron is reduced from ferric to ferrous ion by ferrireductases such as duodenal cytochrome B (DcytB) at the apical surface of the brush border (5). Proton pump inhibitor therapy lowers the concentration of vitamin C in gastric juice and the proportion of the vitamin in its active antioxidant form i.e. ascorbic acid. This has secondary effects on nonhaem iron absorption and has been anecdotally employed in the management of haemochromatosis.

Iron is absorbed via the epithelial cells of the intestine and transported to the iron-requiring cells of the body. In the past few years, the number of proteins implicated in the regulation of iron metabolism has increased greatly. The first mammalian proteins known to mediate transmembrane transport of ionic iron were Nramp2, the murine form, and DCT1, the rat isoform, which are expressed in the small intestine and up-regulated in iron deficiency (6). The human equivalent, divalent metal transporter 1 (DMT1) is highly expressed in the apical surface of the duodenum, and it transports ferrous ion across the brush border from the lumen into the enterocyte (7). Iron in the ferrous state is then transported across the basolateral membrane of the enterocyte by ferroportin (8). Ferrous iron is oxidized to ferric iron by hephaestin, a multicopper oxidase located in the basolateral membrane, and ferric iron binds to transferrin in the blood.

Since ionic iron is rapidly oxidized in an oxygen-rich environment, biological iron is either chelated or complexed to a protein. The two proteins that play a prime role in the transport and storage of iron are transferrin and ferritin. Both are capable of tightly binding iron. Following absorption, it is the function of transferrin to transport the largest fraction of iron to the erythroid marrow cells for synthesis of haem. Iron is carried to the liver and proliferating cells. The transferrin molecule has two binding sites and when fully saturated can carry two atoms of iron. Iron not immediately required is stored as ferritin, a large protein composed of 24 H and L subunits surrounding a hollow core that holds up to 4500 atoms of Fe³⁺ ions as ferrihydrite. Iron sequestered in this form is relatively nontoxic. When iron stores increase, large amounts of iron are deposited as haemosiderin, a complex consisting of iron and degraded proteins such as ferritin. Approximately one-third of body iron stores occur within ferritin molecules in the liver. This store is readily accessible to supply the body’s daily iron needs.

The level of ferritin present in the serum reflects body iron stores in the absence of inflammation, malignancy, or hepatocellular necrosis, all of which increase the serum concentrations of ferritin, which is also an acute phase reactant. Transferrin-bound iron is taken up by almost all cells through a transferrin receptor (TFR1)-mediated process. Diferric transferrin binds to TFR1 at the cell surface and is internalized into endosomes, where the iron is released from transferrin by endosomal acidification. This receptor-mediated endocytosis is a unique process in which transferrin and its receptor are reutilized repeatedly in iron delivery. Once inside the cell, iron is released from the complex when the pH in the endosome drops to 5.6. Transferrin remains complexed to the receptor which recycles to the cell surface (9). It is only at neutral pH that iron-free transferrin dissociates from its receptor and is available once again to chelate iron. Both transferrin and the transferrin receptor function normally in haemochromatosis, although transferrin is highly saturated with iron, a fact used in the diagnosis of the disease.

The uptake and storage of iron are normally controlled at the post-transcriptional level by iron regulatory proteins (IRPs) that recognize iron responsive elements (IREs) (10). Recently hepcidin, a 25 amino acid antimicrobial peptide produced by the liver has been identified as the master regulator of iron homoeostasis (11). Hepcidin transcription is up-regulated by inflammatory cytokines, iron and bone morphogenetic proteins and is down-regulated by iron deficiency, ineffective erythropoiesis, and hypoxia. The iron transporter ferroportin is the cognate receptor of hepcidin and is destroyed as a result of interaction with the peptide. Except for inherited defects of ferroportin and hepcidin itself, all forms of iron-storage disease appear to arise from hepcidin dysregulation.

Haemojuvelin is a membrane protein that is responsible for the iron overload condition known as juvenile haemochromatosis. Haemojuvelin, highly expressed in the liver, skeletal muscle and heart, seems to play a role in iron absorption and release from cells and has anti-inflammatory properties. Haemojuvelin is a bone morphogenetic protein (BMP) coreceptor and signals via the SMAD (human homologue of Drosophila mad-mother against decapentaplegic) pathway to regulate hepcidin expression. HJV acts as a BMP coreceptor. Moreover, HJV plays an essential role in the regulation of hepcidin expression, specifically in the iron-sensing pathway, although through unknown mechanisms. Dietary iron-sensing and inflammatory pathways converge in the regulation of the key regulator hepcidin, but how these two pathways intersect remains unclear.

Haemochromatosis usually presents in adults in the third or fourth decade of life. The disease is underdiagnosed and the clinical manifestations, notably fatigue, hepatic disease, diabetes, arthritis, skin pigmentation, cardiomegaly, and hypogonadism, are often nonspecific. Chronic fatigue is the commonest manifestation of the disease on presentation. This fatigue improves in approximately 60% of subjects following therapy (12).

The liver is the first organ affected by the increased iron deposits, and hepatomegaly is common in symptomatic patients with elevated iron stores. Biochemical and histological signs of liver disease are commonly found, although the liver may be enlarged in the absence of clinical symptoms or abnormal liver function tests. Histologically, the iron deposits in the liver consist of ferritin and haemosiderin. A classic pattern is seen in that the iron is deposited in the hepatocytes (parenchymal cells) initially in a periportal distribution (Fig. 12.3.2.3a). Within these hepatocytes the iron is found particularly in the lysosomes in the pericanalicular cytoplasm. Kupffer cell iron loading, if present, occurs only in the late stages of the disease. In other diseases where iron may be deposited such as alcoholic liver disease, Kupffer cell iron loading is a common feature with little iron in hepatocytes. In haemochromatosis, where heavy deposition of iron is present, periportal and lobular fibrosis occurs followed by cirrhosis. Hepatocellular carcinoma represents the most important and frequent cause of death in haemochromatosis patients and develops in approximately 18–30% of patients with cirrhosis (13). The relative risk of development of hepatocellular cancer in patients with iron overload who present with cirrhosis is 200-fold (14).

The excessive skin pigmentation of exposed areas that occurs in most symptomatic patients results from iron-induced increased melanin deposits in the dermis, which usually give rise to bronzing and a metallic grey hue seen in 25–50% of patients. Excessive pigmentation is less common if diagnosis is made early during the course of the disease.

Diabetes mellitus has long been recognized as being associated with haemochromatosis. Indeed, the earliest description of a patient with haemochromatosis was reported during a lecture on diabetes delivered by Trousseau in 1865. Subsequently the term ‘bronze diabetes’ was used to describe the association between pigmentation due to iron and associated diabetes. The prevalence of diabetes mellitus in haemochromatosis was initially reported to be in excess of 80% (12, 15) but recent population studies have brought this into question (16). A decrease in prevalence of diabetes among those with haemochromatosis appears to have occurred since the discovery of the gene in 1996 and might be related to earlier ascertainment of genetic risk and increased prevention of severe iron-overload related sequelae such as cirrhosis and associated diabetes.

The development of diabetes in haemochromatosis may result from direct damage to the pancreas by iron deposits, a family history of diabetes and/or the existence of cirrhosis which may impair glucose tolerance. Exocrine cells show the heaviest deposits of iron while within the islets, the iron is found in the pancreatic β cells (Fig. 12.3.2.3b). Chemical determination of iron concentration shows high level of iron accumulation in the pancreas, although slightly less than that which occurs in the liver, however, dense pancreatic fibrosis may occur in untreated individuals. Insulin resistance develops in cirrhotic cases, but has also been described in some noncirrhotic patients and has been attributed to hepatocyte iron deposits. Diabetes in haemochromatosis is encountered less often than in earlier times, partly due to the increasing awareness of the disease and early intervention by venesection therapy which results in less deposition of iron in the pancreas with fewer pathological consequences. Some studies have shown that patients treated by venesection require less insulin than those patients who do not undergo venesection. Others have suggested that iron-chelation therapy using desferrioxamine to remove excess iron results in improved glycaemic control of diabetes. However, venesection therapy is much more effective at removing large amounts of iron than is chelation (17).

Although a number of studies have shown a low prevalence of haemochromatosis in subjects attending diabetic clinics, screening of patients by the determination of transferrin saturation remains a simple and effective method of identifying the disease in diabetic clinics. Subsequent investigations of family members can be useful in identifying haemochromatosis. There has been some debate as to the cost-effectiveness of screening for haemochromatosis in diabetic clinics. Prior to the cloning of the haemochromatosis gene, a number of studies advocated screening while others found that screening was not cost-effective (18). However, in order to determine if there was a genetic link between diabetes and the haemochromatosis mutation, Frayling et al. determined the prevalence of the C282Y mutation in 238 unrelated patients with type 2 diabetes. They found that the mutation was not associated with type 2 diabetes per se (19). A population study of more than 31 000 subjects did not find a higher prevalence of diabetes among C282Y homozygotes compared with wild-type controls (16) although the prevalence of disease in all genotype groups was low and the study was relatively underpowered to address this issue effectively.

Nonetheless diabetes is found in association with haemochromatosis although it is not clear whether this is associated with iron overload per se or secondary to development of cirrhosis. Diabetes developed as a complication of cirrhosis is known as hepatogenous diabetes. Around 30–60% of cirrhotic patients have this metabolic disorder. Insulin resistance in muscular, hepatic, and adipose tissues as well as hyperinsulinaemia, seem to be pathophysiological bases for this form of diabetes. An impaired response of the islet beta cells of the pancreas and the hepatic insulin resistance are also believed to be contributing factors. Diabetes develops when defective oxidative and nonoxidative muscle glucose metabolism develops.

In haemochromatosis iron deposits can occur in the thyroid, parathyroid and anterior pituitary, and adrenal glands. As a consequence, loss of libido is common in haemochromatosis and approximately 24% of haemochromatotic men have testicular atrophy. These signs may precede other manifestations of the disease especially in young individuals and hypogonadal symptoms may occur before liver function is disturbed. Iron deposition in the pituitary affects the gonadotropin-producing cells, selectively impairing hormone secretions (luteinizing hormone and follicle-stimulating hormone (FSH)) and resulting in hypogonadism in about 20% of haemochromatosis patients (17). The hypogonadism has been shown to be due to pituitary complications of iron directly affecting pituitary function in contrast to alcoholic cirrhosis, where testicular failure is predominant.

Osteoporosis is reported to be a frequent complication of iron-loading conditions such as haemochromatosis thalassaemia, sicklaemia, and African siderosis, as well as on the cessation of menstruation. The metal suppresses osteoblast formation of bone and may also stimulate osteoclast resorption of bone. Iron also inhibits anterior pituitary synthesis of gonadotrophs. This, in turn, results in depressed formation of gonadal hormones. The tendency of iron-loaded persons to become osteoporotic may be enhanced by gonadal hormone deficiency. It has been postulated that iron-binding agents that could specifically withhold excess skeletal iron (and be excreted as the iron chelate) might have therapeutic utility. (20). In a study of 87 patients with hereditary hemochromatosis, osteoporosis was detected in 25%, and osteopenia in 41%. Osteoporosis was observed independently of the genetic background, and was associated with alkaline phosphatase (ALP), hypogonadism, body weight, and severity of iron overload (21).

Arthropathy is recognized in approximately 20% of patients with the disease and may be one of the earliest symptoms. The arthropathy, however, appears to be unrelated to either the extent or duration of iron overload and indeed may even occur following venesection therapy. The arthropathy may be a presenting feature in older male patients and the first symptoms are often pain and limited flexion of the metacarpophalangeal joints of the hands characteristically of the second and third joints. It is characterized by cystic and sclerotic changes, cartilage defects, and narrowing of the joint cavity. The exact mechanism of injury is unknown, although a similar pathology in other iron-loading disorders suggests that it may be a direct effect of iron-initiating free radical reactions.

Evidence of cardiac dysfunction has been reported in up to 30% of haemochromatosis patients. Presentation with heart failure is uncommon, however, it may be the presenting problem in some young patients (Fig. 12.3.2.3c) (13). Cardiac symptoms take the form of arrhythmias, or progressively severe heart failure caused by the deposition of iron in cardiac muscle. The level of iron, however, is much less than that which is deposited in the pancreas and liver and fibrosis rarely occurs. Tuomainen et al. (22), using the concentration ratio of TfR/ferritin as a measurement of body iron stores, showed an increased risk of acute myocardial infarction in men with increased iron stores. Venesection treatment often improves cardiac arrhythmias.

The search for the mutation that results in excessive iron deposits in tissues began following the recognition by Simon in the late 1970s that there was an association between HLA alleles A3 and B14 and haemochromatosis (3). This enabled the use of HLA typing to assist in diagnosis of the disease within the family of an affected subject. It was possible to predict homozygosity and heterozygosity among affected siblings and for quite some time was invaluable in allowing effective management of affected siblings. Haplotype analysis and genetic linkage analysis located the gene within one centimorgen of HLA-A on chromosome 6 at 6p21.3. The tremendous advances in molecular biology with the advent of the polymerase chain reaction and the development of microsatellite markers made it possible to narrow down the area of interest further. The microsatellite marker D6S105 showed a highly significant association with haemochromatosis and further genetic characterization of haemochromatosis gene region defined a predominant ancestral haplotype involving HLA-A3, D6S105-8 and D6S265-1, which segregated with the gene. This haplotype was later found in patients in the UK, Italy, France, and the USA, indicating a common origin for the gene in these populations. The identification of this ancestral haplotype, which has survived many generations suggests that this haplotype may convey some genetic advantage which may or may not be related to iron status. This topic has been reviewed by Worwood (23).

The gene was finally cloned by Feder et al. (4) using linkage disequilibrium and high resolution haplotype analysis. The gene, formerly called HLA-H, is now termed HFE. A mutation at nucleotide 845 of the open reading frame of a major histocompatibility complex (MHC) class I type gene resulted in a cysteine to tyrosine substitution at amino acid position 282 (the C282Y mutation). This mutation was detected in 85% of all haemochromatosis chromosomes and only in 3.2% of control chromosomes, giving a carrier frequency of 6.4%. Extensive sequencing of the gene also revealed a second missense mutation of histidine to aspartic acid (H63D), however, the role of this mutation in iron overload disease remains controversial. It has been suggested that subjects who are heterozygous for the both the C282Y and H63D mutations (that is, compound heterozygotes) have an increased risk of developing iron overload, however, the penetrance is very low and it has recently been shown that less than 1% of compound heterozygotes express the disease (24). A significant proportion of C282Y heterozygotes, however, do have some evidence of elevated iron indices even though they do not develop clinical disease (25).

Despite the lack of an obvious function related to the alteration of body iron balance, there is overwhelming evidence to implicate HFE as the gene affected in haemochromatosis.

The HFE gene encodes a 343 amino acid type I transmembrane glycoprotein, which shows remarkable similarities to MHC class I molecules. As with other MHC class I proteins, the heavy chain comprises three domains (α1, α2, α3) (Fig. 12.3.2.4) and associates with the class I light chain, β₂-microglobulin. Unlike most other MHC class I proteins however, the groove between the α1 and α2 domain is structured such that the protein does not bind peptides for presentation to the cell surface. The function of the HFE gene product therefore appears to be different from other MHC class I proteins. The crystal structure of the gene product has been resolved (26) and confirms the putative structure proposed by Feder et al. in that in addition to the three cytoplasmic domains, there is a single transmembrane domain and a short cytoplasmic tail.

The observation that β₂-microglobulin-deficient mice developed iron overload provided evidence that this molecule was an important part of the complex mechanisms that interact to regulate iron absorption. These mice were shown to have elevated plasma iron levels, transferrin saturations, and hepatic iron concentrations that mimic haemochromatosis (27). It is known that MHC class I type molecules must bind to β₂-microglobulin in order to function normally. The formation of a heterodimer between HFE and β₂-microglobulin has been shown to be essential for the correct intracellular trafficking and transport of the protein to the cell surface (28). The 845G→A mutation in the open reading frame of the haemochromatosis gene causes a cysteine to tyrosine substitution at a position in the molecule that has important structural consequences. The mutation disrupts a critical disulphide bond which results in a conformational change in the molecule which subsequently fails to undergo late Golgi processing and is rapidly degraded. The molecule therefore does not bind β₂-microglobulin and so is not carried to the cell surface (29).

Targeted disruption (‘knockout’) of the HFE gene in mice has supported previous evidence that HFE is indeed the gene responsible for maintaining iron homoeostasis. The loss of function in the mutated gene product results in body iron accumulation and a phenotype that mimics haemochromatosis with periportal iron accumulation in hepatocytes (30). Although the mutation in the gene has been identified as being the cause of haemochromatosis, the role of the gene product in normal iron metabolism and the mechanism by which the abnormal protein resulting from the C282Y mutation results in the disruption of the regulation of iron absorption is still unknown.

HFE mRNA in humans is broadly expressed in many tissues, with the highest levels in the liver and small intestine (4). HFE has been localized to the crypt region of the small intestine in normal humans, indicating a role in the regulation of intestinal iron absorption in humans (31).

Haemochromatosis is one of the most common disorders inherited as a recessive trait in Caucasian populations with an estimated disease frequency of 1:200 in such populations in Australia, Europe, and North America (32, 33). Since the discovery of the HFE gene and the mutation responsible for the disease, populations have been screened for the genetic defect. The largest of these studies is that of Merryweather-Clarke et al. (34). which examined population samples from a variety of ethnic backgrounds all over the world and more recently the HEIR study (16). The C282Y mutation was found to be most prevalent in Northern European populations with the highest frequency seen in Ireland (10%) and the lowest frequencies in African, Asian, and indigenous Australian populations. In contrast, the H63D mutation was present in almost all the populations studied. Worldwide frequencies were reported to be 1.9% for the C282Y mutation and 8.1% for H63D.

Haemochromatosis shows a wide variation in phenotypic expression as determined by the clinical measures of serum ferritin, transferrin saturation, hepatic iron concentration, hepatic iron index, and histochemical grading of parenchymal iron. There are a number of environmental, genetic, and nongenetic factors, for example, pathological and physiological blood loss and blood donation, which can influence the degree of iron loading in affected individuals. The disease is transmitted genetically as an autosomal recessive trait and therefore one would expect equal disease expression in men and women, however, this is not the case. Iron stores vary greatly according to age and gender. Women accumulate iron at a slower rate than men as determined by serum ferritin and transferrin saturation levels and hence men usually present with symptoms earlier in life than women. There is further evidence for this in that fact that iron indices are more likely to rise in female homozygotes after menopause (35). However, not all of the phenotypic variation observed can be accounted for by environmental differences. Both manifestations of haemochromatosis and biochemical markers of iron loading can be seen to be concordant within families rather than between families indicating that genetic factors primarily determine the extent of iron accumulation.

A number of investigators have reported correlations between phenotypic expression of the disease and genotype. These observations suggest that other genes may act as genetic modifiers of disease expression. Since the rate of clinical expression is similar in same-sex siblings with the same HLA haplotype, it seems that any potential modifying genes are possibly located close to HFE in the region spanning the ancestral haplotype. In addition, in Italy and Southern France a large number of patients do not carry the C282Y or H63D mutations.

Normal iron concentrations in the liver and pancreas are approximately 5–40 µmol/g dry weight. In established disease the hepatic and pancreatic iron levels can reach 50–100 times normal. The iron is deposited in a characteristic pattern which is diagnostically important. Haemosiderin deposits appear firstly in the hepatocytes surrounding the portal tract (the periportal region of acinar zone 1) and later in the lysosomes. Hepatic injury and fibrosis occurs in this area and fibrosis can occur early following the deposition of iron. If iron levels exceed 400 µmol/g dry weight, cirrhosis is often found to be present (36). Although the precise pathophysiological pathways initiating the fibrotic stimuli are still unclear, remarkable advances have been made in recent years. It is now well established that the hepatic stellate cells are primarily responsible for increased collagen production in the iron-laden liver. These cells, which are normally quiescent, become activated by the iron, possibly due to free radical mediated mechanisms involving products of lipid peroxidation, the collagen-producing genes are switched on and fibrosis ensues (37).

The aim of diagnosis is early detection and early therapy to prevent organ damage and initiation of fibrotic lesions. Diagnosis therefore involves a high degree of clinical suspicion combined with careful laboratory testing and histological examination indicative of excessive iron stores and tissue damage. The diagnosis should be considered in all patients who present with hepatomegaly, unexplained fatigue, diabetes, skin pigmentation, cardiomyopathy, arthropathy, and hypogonadism (Fig. 12.3.2.5). Factors to be considered include a careful history relating to parenteral iron administration, excessive menstrual blood loss, multiple pregnancies, and blood donations, which may account for low iron indices. In the event of a patient presenting to an endocrinology clinic with unexplained endocrinological insufficiencies, biochemical tests relating to liver function and iron indices may well identify the underlying cause of the endocrinological problem.

Diagnosis is now frequently made in individuals who remain asymptomatic. Patients usually develop elevated serum iron, serum transferrin saturation, and serum ferritin levels before marked symptoms occur. The accumulation of iron is a slow process, is often silent in the early stages, and only when iron stores reach toxic levels does tissue injury develop. Asymptomatic patients are usually diagnosed by biochemical screening tests following family investigations usually involving both biochemical and genetic testing.

Figure 12.3.2.5 shows proposed guidelines for the diagnosis and management of haemochromatosis. It is well accepted that the most reliable marker for identifying affected individuals is the presence of persistently elevated transferrin saturation (which is defined as serum iron divided by the total iron-binding capacity and which is expressed as a percentage) in the context of elevated serum ferritin. Transferrin is usually approximately 33% saturated with iron. A raised fasting transferrin saturation (greater than 45%), which remains elevated following fasting and which is accompanied by an elevated serum ferritin level, usually indicates increased body iron stores. Detection of the C282Y mutation in such cases helps to establish the diagnosis of haemochromatosis.

Since iron accumulation in haemochromatosis increases with age, liver biopsy followed by chemical determination of the hepatic iron concentration permitted the calculation of a hepatic iron index (hepatic iron concentration divided by age) (38). An index of 2.0 or greater was considered to be abnormal and, in the absence of secondary causes of iron overload such as iron-loading anaemias, usually represented homozygous subjects. The removal of 4–5 g of iron by serial venesection is also diagnostic and has been used in the past to confirm the presence of the disease (39). Following the localization of the disease gene, genetic testing is recommended in those subjects with persistently elevated iron indices. Some individuals who are heterozygotes (i.e. carriers of one copy of the C282Y mutation) or compound heterozygotes (i.e. carriers of one copy of the C282Y mutation and one copy of the H63D mutation) do have elevated iron indices although the degree of iron loading is not usually as great as that associated with cirrhosis (400 µmol/g). Screening is not generally recommended for children less than 18 years of age since the onset of disease does not occur until at least the third decade of life and unnecessary limitation of dietary iron risks iron deficiency at a time of rapid growth and increased iron requirements in healthy children. If one parent is homozygous for C282Y the general recommendation is to test the other parent to assess whether they carry one allele of the gene (40). If they do not then the parents may be reassured that offspring are at no risk of C282Y-associated haemochromatosis.

Once the diagnosis of genetic homozygosity in the light of elevated iron indices and C282Y genotyping is established, liver biopsy needs to be considered to determine whether fibrosis or cirrhosis is present. There is a 200-fold excess risk in these patients of developing hepatocellular carcinoma, and approximately 30% of patients who present with cirrhosis will die of hepatocellular carcinoma (12, 14). Recently, a French/Canadian study showed that haemochromatosis patients who did not have hepatomegaly and who had a serum ferritin level of less than 1000 μg/l with normal liver function tests were unlikely to have remarkable fibrosis and they concluded that liver biopsy was therefore not necessary in these individuals (41). Powell and colleagues have confirmed that cirrhosis is highly unlikely in asymptomatic homozygotes with SF<1000 μg/l (42).

If cirrhosis is established, it is recommended that patients at risk (particularly males over 55 years of age) participate in active surveillance programmes and are regularly screened using ultrasonography and A-fetoprotein measurements to detect cancer development early when treatment can be more effective. If cirrhosis is not present, venesection therapy is effective, often reverses fibrosis if present, and the 10-year survival rate is identical to the general population (Fig. 12.3.2.6a).

Increased body iron stores have been implicated in the pathogenesis of diseases other than haemochromatosis. Alcoholic liver disease where hepatic iron deposition is present has in the past caused some confusion in the diagnosis of haemochromatosis (43), and a number of reports have highlighted increased iron indices in hepatitis C patients. The molecular basis of iron involvement in these diseases is uncertain and is most likely due to factors not involving the C282Y mutation in the haemochromatosis gene.

Family screening based on an index case is now well established and usually first degree relatives are screened with fasting transferrin saturation and serum ferritin. Genotyping for the C282Y mutation has now been implemented and is widely used for cascade screening. The use of transferrin saturation as a screening tool has been evaluated in several studies (44). Although the test has advantages of being inexpensive and easy to perform, two determinations are necessary to confirm abnormal values and this form of cascade screening is unlikely to be cost-effective.

Haemochromatosis is an ideal condition for population-based genetic screening since the disease is common and venesection therapy provides a simple effective treatment that improves the life expectancy in precirrhotic individuals. Since the discovery of the gene, the question arises whether it would be preferable to screen populations based on phenotypic characteristics or on genotype. The presence of a single causative mutation in HFE allows relatively simple screening at the molecular level. The Centers for Disease Control and Prevention and the National Human Genome Research Institute in the USA issued a consensus statement recommending that genetic testing was not recommended at the present time in population-based screening for the disease, due to uncertainties regarding the penetrance of HFE mutations and optimal care of asymptomatic carriers of the mutation. The centres agreed that genetic testing was to be recommended for confirming diagnosis in people with elevated iron indices (45). This recommendation was based on the fact that although more than 90% of cases are due to C282Y homozygosity (46), there is now good evidence that not all those who are homozygous will progress through all stages of the disease. These stages comprise genetic predisposition without abnormality; iron overload (raised serum ferritin in the presence of a raised fasting transferrin saturation) without symptoms; iron overload with haemochromatosis-associated symptoms such as arthritis and fatigue; and iron overload with organ damage, particularly cirrhosis (47). Although most of those who are homozygous appear to develop raised serum ferritin and raised transferrin saturation by the fifth decade of life (25) until now there have been few reliable data on the number of homozygous individuals who develop disease as a result of iron overload.

Population estimates of the prevalence of nonspecific signs and symptoms of haemochromatosis (e.g. arthritis and fatigue) and disease due to documented iron overload (e.g. cirrhosis) in C282Y homozygous individuals have been hindered by either the failure to clinically assess individuals before knowledge of their genetic status or an inability to account for the long lead time of preclinical iron overload status. A cross-sectional population study of participants aged 20–80 years suggested that disease attributable to haemochromatosis occurs in fewer than 1% of those who are homozygous, regardless of sex (48). However, this study did not conduct clinical examinations or liver biopsies, and a quarter of the homozygous patients were excluded on the basis that they had been previously diagnosed. This exclusion would be expected to reduce the estimate of clinical penetrance of C282Y homozygosity. Furthermore, the study included homozygous patients of ages at which disease would not be expected to have developed. Until the time of writing, there had been only two longitudinal studies of hereditary haemochromatosis designed to accurately estimate the proportion of homozygous patients who will develop disease secondary to iron overload (49, 50). However, with a combined total of 23 patients, they were substantially underpowered to assess disease prevalence.

In the largest longitudinal prospective study to date, 203 homozygous individuals among a healthy population of 31 192 were followed up over 12 years (16). Data were collected by physicians who were blinded to genotype, and liver biopsies were performed as clinically indicated (serum ferritin >1000 μg/l, unexplained hepatomegaly or raised serum aminotransferase levels) (15). The study that homozygous individuals with a serum ferritin level higher than 1000 μg/l were at increased risk of haemochromatosis-associated signs and symptoms, when compared with either those who were homozygous with a serum ferritin level of 1000 μg/l or less, or individuals with other HFE genotypes. In particular, homozygous men with a serum ferritin level higher than 1000 μg/l reported greater fatigue, use of arthritis medication, and history of liver disease than men without the C282Y mutation.

The proportion of homozygous individuals with disease that was directly attributable to iron overload was assessed using the combined definition of documented iron overload (51) and one or more of the following: cirrhosis, liver fibrosis, hepatocellular carcinoma, raised aminotransferase concentration, physician-diagnosed symptomatic hereditary hemochromatosis, and arthropathy of the second and third metacarpophalangeal joints. Iron overload-related disease developed in 28% of homozygous men, but only 1% of homozygous women (16). These new findings suggest that targeted population screening may be more cost-effective than previously believed. Certainly the risks associated with screening are believed to be low since the condition is preventable through venesection, individuals do not experience unnecessary anxiety following screening (52), and in Australia at least, insurance industry agreements abrogate the risk of genetic discrimination (53).

The aim of treatment is to return the body iron stores to normal levels and this is achieved by venesection which allows the tissue iron to be mobilized to meet the demand for increased haem synthesis. Treatment in the majority of cases of haemochromatosis is simple, effective and safe. Treatment is usually initiated with weekly or fortnightly venesection therapy of 500 ml of whole blood which removes approximately 250 mg of iron from the body. Serum ferritin and haemoglobin levels are monitored at each venesection. Therapy is ceased when serum ferritin levels fall to approximately 50 µg/L or if the haemoglobin level declines and does not rise rapidly on cessation of venesection although this practice of aggressive venesection has recently come in to question for those who are asymptomatic at presentation since these previous guidelines were developed prior to the identification of the gene and the ability to detect asymptomatic iron overload. For those who are symptomatic at presentation, 2–3 monthly maintenance venesection is common although venesection two to three times per year is often all that is required for asymptomatic homozygotes. Iron chelation therapy using desferrioxamine infusion removes less iron than venesection. However, it may be useful in patients who present with cardiac problems and who may not tolerate venesection. Fatigue, liver function and diabetic control are often improved after treatment, however, gonadal failure and arthropathy usually do not improve significantly.

Ultimately, the prognosis appears to depend on the amount of iron present in the parenchymal cells and the coexistence of liver disease of other aetiology. The prognosis of untreated haemochromatosis is poor. Fibrosis followed by the development of cirrhosis is the most important adverse prognostic factor in the disease and the 5-year survival in patients with cirrhosis may be as low as 50%. However, if patients present with considerable iron loading and fibrosis, which has not yet developed to cirrhosis, with no remarkable tissue damage, life expectancy in venesected subjects does not differ from that of the general population (Fig. 12.4.2.6b) (12). Therefore, in patients in the early stages of disease progression and in relatives of patients who are closely monitored, the natural course of the disease can be prevented. Early diagnosis of the disease is therefore of the utmost importance so that venesection therapy can be immediately initiated to remove excess iron before tissue damage, cirrhosis, or the development of diabetes.

Cumulative survival in diabetic patients is reduced when compared with nondiabetic patients (Fig. 12.4.2.4c). Since the risk of developing hepatocellular carcinoma in cirrhotic patients is high, cirrhotic patients should routinely be screened with regular ultrasonography and serum A-fetoprotein levels. Small tumours may be locally resected, chemoembolized or treated by ethanol injection, whereas orthotopic liver transplantation remains an option for endstage liver disease.

With the discovery of the HFE gene, ascertainment of individuals at risk of developing haemochromatosis is more likely to occur through cascade genetic screening of HH-affected probands. There is evidence that asymptomatic homozygous individuals with serum ferritin <1000 μg/l are at low risk of cirrhosis (42) as well as other HH-associated disease (16). Furthermore it appears that the majority of C282Y homozygotes who are likely to develop serum ferritin levels sufficient to place them at risk of iron-overload-related disease will have done so by mean age 55 years (35) (Fig. 12.3.2.7).

The author acknowledges the contributions of Drs Linda Fletcher and June Halliday to this chapter in the previous edition.