Inherited iron loading: genetic testing in diagnosis and management

Blood Reviews (2005) 19, 69–88

www.elsevierhealth.com/journals/blre

REVIEW

Inherited iron loading: genetic testing in diagnosis and management Mark Worwood* Department of Haematology, University of Wales College of Medicine, Cardiff CF14 4XN, UK

KEYWORDS

Summary Elucidation of the molecular pathways of iron transport through cells and its control is leading to an understanding of genetic iron loading conditions. The general phenotype of haemochromatosis is iron accumulation in liver parenchymal cells, a raised serum transferrin saturation and ferritin concentration. Four types have been identified: type 1 is the common form and is an autosomal recessive disorder of low penetrance strongly associated with mutations in the HFE gene on chromosome 6(p21.3); type 2 (juvenile haemochromatosis) is autosomal recessive, of high penetrance with causative mutations identified in the HFE2 gene on chromosome 1 (q21) and the HAMP gene on chromosome 19 (q13); type 3 is also autosomal recessive with mutations in the TfR2 gene on chromosome 3 (7q22); type 4 is an autosomal dominant condition with heterozygous mutations in the ferroportin 1 gene. In type 4, iron accumulates in both parenchymal and reticuloendothelial cells and the transferrin saturation may be normal. There are also inherited neurodegenerative conditions associated with iron accumulation. The current research challenges include understanding the central role of the HAMP gene (hepcidin) in controlling iron absorption and the reasons for the variable penetrance in HFE type 1. c 2004 Elsevier Ltd. All rights reserved.

Iron overload; Haemochromatosis; Iron absorption; Ferritin; Transferrin; HFE; Hepcidin; Transferrin receptor; Ferroportin 1; Clinical penetrance; Neurodegeneration




Introduction Iron metabolism In the adult human body there is normally about 50 mg Fe/kg in men (3.5–4.0 g in total) and 40 mg/kg in young women. The lower amount of body iron in women reflects the lower levels of storage iron resulting from the increased blood losses due to menstruation and childbirth. The largest compo-

*

Tel.: +44-29-2074-2726; fax: +44-29-2074-4655. E-mail address: [email protected].




nent is iron in circulating haemoglobin. Most of the remainder is contained in the storage proteins, ferritin and haemosiderin (Table 1). Iron stores are found mainly in the reticulo-endothelial (RE) cells of the liver, spleen and bone marrow, which acquire iron by breaking down senescent red cells, and in liver, parenchymal cells which normally gain most of their iron from the plasma. The functional iron-containing proteins of the tissues, including cytochromes and iron–sulphur proteins responsible for respiration, contain only small amounts of iron, at a similar level to other trace metals. Transferrin is the iron transport protein of plasma and extravascular fluid and there is normally a few mg Fe in this compartment.

0268-960X/$ - see front matter c 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.blre.2004.03.003

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Table 1 Distribution of iron in the body (75 kg man). Protein

Location

Iron content (mg)

Haemoglobin Myoglobin Cytochromes a Fe–S Proteins Transferrin Ferritin and Haemosiderin

Red blood cells Muscle All tissues

3000 400 50

Plasma and extra-vascular fluid Liver, spleen and bone marrow

5 100–1000

Normal iron balance The total iron content of the newborn is about 80 mg/kg at full term. Neonatal iron reserves are utilized for growth and from 6 months to 2 years virtually no iron stores are present. Thereafter, iron stores gradually accumulate during childhood to around 5 mg/kg. In men, there is a further increase between 15 and 30 years to about 10–12 mg/kg (up to approximately 1 g), whereas iron stores remain lower in women (average 300 mg) until the menopause. In men, iron losses from the body are about 1 mg per day, mostly as iron lost in red cells into the gut. Losses vary little with the amount of iron in the body and iron status is maintained by variation in the amount absorbed not the amount lost. For an adult, healthy male depletion of body iron stores and development of iron deficiency anaemia, solely due to lack of dietary intake or malabsorption, would take several years. Requirements are higher in menstruating women and during periods of rapid growth in infancy and adolescence. Menstrual loss has a median value of 30 ml per month and greater losses are associated

with iron deficiency. Requirements are highest of all in pregnancy. For basic information on iron metabolism, including absorption, losses, tissue concentrations, see Bothwell et al.1

Iron overload Normally iron absorption is closely regulated but if there is excessive iron accumulation this eventually causes tissue damage. When there is excessive iron absorption from food, iron initially accumulates in the parenchymal cells of the liver, whereas iron administered in transfused red cells is first taken up in senescent red cells by macrophages. A classification of iron overload is shown in Table 2. Genetic haemochromatosis is defined here as iron accumulation in the body due to inheritance of changes in genes regulating iron absorption.

Iron absorption Iron absorption varies with the amount of iron in the diet, the bioavailability of that iron, and the

Table 2 Causes of iron overload. Severe iron overload ( >5 g excess) Excess iron absorption

Increased iron intake Repeated red cell transfusions

Genetic haemochromatosis Massive ineffective erythropoiesis (e.g., b-thalassaemia intermedia, sideroblastic anaemia Sub-Saharan dietary iron overload (in combination with a genetic determinant of increased absorption) Congenital anaemias (e.g., b-thalassaemia major) Acquired refractory anaemias (e.g., myelodysplasia and aplastic anaemia)

Modest iron overload ( <5 g excess) Chronic liver disease (e.g., alcoholic cirrhosis) Porphyria cutanea tarda (also associated with homozygosity for HFE C282Y) Rare genetic disorders of iron metabolism (e.g., atransferrinaemia, aceruloplasminaemia) Rare inherited neurodegenerative conditions

Inherited iron loading: genetic testing in diagnosis and management

71

Table 3 Factors influencing iron absorption. Increased % absorption

Decreased % absorption

Luminal factors Haem iron Animal foods Acid pH (gastric HCl) Low molecular weight Soluble chelates (vitamin C, sugars amino acids)

Alkaline pH (e.g., pancreatic secretions) Iron binders producing insoluble complexes (phytates, tannates in tea, bran)

Systemic factors Iron deficiency Increased erythropoiesis Ineffective erythropoiesis Pregnancy Hypoxia

body’s need for iron. A normal western diet provides approximately 15 mg iron daily, from which only about 1 mg (or 5–l0% of dietary iron) is transferred to the portal blood in a healthy adult male. Iron absorption is regulated at several different stages in this process.

Dietary and luminal factors Much of dietary iron is non-haem iron derived from cereals (often fortified with additional iron), with a lesser component of haem iron from meat and fish. Iron is released from protein complexes by acid and proteolytic enzymes in the stomach and small intestine, and haem is liberated from haemoglobin and myoglobin. The maximum uptake is from the duodenum and decreases further down the small intestine, probably because the increasingly alkaline environment leads to the formation of insoluble ferric hydroxide complexes. Luminal and systemic factors enhancing or inhibiting absorption are listed in Table 3. Even in iron deficiency, the maximum iron absorption from a mixed western diet is no more than 3–4 mg daily. This figure is much less with the predominantly vegetarian, cereal-based diets of most of the world’s population. Therapeutic ferrous iron salts are well absorbed on an empty stomach, but when taken with meals, absorption is reduced due to the same ligandbinding processes that affect dietary non-haem iron.

Mucosal factors: molecular aspects of iron absorption and its regulation Recently discovered membrane transport proteins, regulatory proteins and associated oxido-reductases involved in iron transport through the intestinal cell have been reviewed by Miret et al.2 and

Iron overload Decreased erythropoiesis Infammatory disorders

are listed in Table 4. The proposed process is illustrated in Fig. 1. Non-haem iron is released from food as Fe3þ and reduced by Dcytb to Fe2þ . This is transported across the brush-border membrane by DMT1. Iron enters a small but dynamic “labile pool” and some is incorporated into ferritin and lost when the cells are exfoliated. Iron destined for retention by the body is transported across the serosal membrane by ferroportin 1 before uptake by transferrin as Fe3þ . Oxidation of Fe2þþ may be a function of either the intracellular protein hephaestin or caeruloplasmin in the plasma. Haem iron is initially bound by haem receptors at the brush border membrane (these have not yet been characterised) and released intra-cellularly by haem oxygenase as Fe2þ before entering the labile iron pool and following a common pathway with iron of non-haem origin.

Regulation of iron absorption Iron absorption may be regulated both during mucosal uptake and transfer to the blood. It has been assumed that as epithelial cells develop in the crypts of Lieberkuhn their iron status reflects that of the plasma (via the transferrin saturation) and this programmes the cells to absorb iron appropriately as they differentiate along the villous. There are, however, situations where this does not apply. In genetic haemochromatosis transferrin saturation is high, yet iron absorption is increased. One hypothesis3 concerning the transfer stage suggests that each of the iron-donating tissues (macrophages, liver and gut) supplies iron to plasma transferrin in proportion to the amount of available iron in those tissues, the iron being transported to satisfy the needs of the main receptor tissue, the erythroid marrow. The amount of iron supplied by

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Table 4 Iron transport proteins, oxido-reductases, storage proteins and regulators. Gene (protein)

Chromosome location Tissue expression

Structure

Function

Regulation

Mutations and disease

Dcytb, duodenal Cytochrome b1 DMT1 (SLC11A2) Ferroportin 1, SLC11A3 HAMP (Hepcidin)

–

Enterocyte +

TMP, 6 TMD

Ferric reductase

Fe (hepcidin)

–

12q13 2q32

TMP 568 aa TMP 571 aa 9 TMD

Fe uptake Fe export

Fe (30 IRE) Fe (50 IRE)

19q13

Widespread Liver,spleen, enterocyte Plasma (liver)

20–25 aa peptide

Fe (HFE)

Hephaestin

Xq11-q12

Enterocyte

HFE

6p21.3

widespread

TMP 1TMD Cu with homology to caeruloplasmin HLA class I heavy chain

Regulator of iron transport Fe2þ oxidase

Mk mouse Belgrade rat Human GH autosomal dom. Juvenile GH (digenic GH) Sla mouse

HFE2 (hemojuvelin)*

1q21

TFRC Transferrin receptor TFR2 Transferrin receptor 2

3q26.2-qter

Liver,heart and skeletal muscle Widespread – highest no. in erythroblasts Widespread

Transferrin

3

Ferritin Heavy Chain (FTH1)

11q13

FTL Ferritin Light Chain IRP 1

19q13.3-q13.4 9p22-p13

Widespread, cytosolic Widespread

IRP 2

15

Widespread

7q22

426 aa

TMP dimer of 90,000 kDa polypeptide 60% similarity in extracellular domain to TFRC Plasma, extravascular Single chain space polypeptide, glycoprotein Widespread, Subunit of ferritin cytosolic Subunit of ferritin Cytoplasmic, mol wt 98,000 with 4Fe–4S cluster

?

Human GH autosomal rec.

?

Juvenile GH autosomal rec (lethal in knockout mouse) Human GH autosomal rec.

Regulates Tf iron uptake and Hepcidin expression Modulates hepcidin expression? Binds transferrin

Fe (IRE)

Binds transferrin

No IRE

Iron transport

Iron stores

Atransferrinaemia

Iron storage (catalytic subunit for iron incorporation) Iron storage

Fe (IRE)

Autosomal dominant Fe overload (v rare)

Fe (IRE)

Hyperferritinaemia and cataract syndrome Not known

“Labile” iron pool Regulation of synthesis of FTH, FTL, TFRC, DMT1, ferroportin 1, ALAS2 As IRP1 “Labile” iron pool

Not known

GH, haemochromatosis; IRE, iron regulatory protein; rec, recessive; TMP, trans-membrane protein; TMD, trans-membrane domain; aa, amino acid. Data from OMIM http:// www.ncbi.nlm.nih.gov/omim/ and *118 .

M. Worwood

Cytoplasmic, mol wt 105,000. No 4Fe–4S cluster

–

Inherited iron loading: genetic testing in diagnosis and management Gut lumen

73

Haem receptor ? Haem Oxygenase

Haem 25% FP1 Non-haem Fe 0 - 20%

Labile iron pool Fe 2+

Fe2+

Cp

Fe3+

DCytb

Dietary

Hp Fe3+

factors

Fe2+

DMT1

Tf Ferritin

Mitochondria IRP

Figure 1 Molecular pathways of iron absorption. The diagram refers to iron absorption by the mature, villous epithelial cell (transfer from the gut lumen to transferrin). The numbers under “haem” and “non-haem iron” refer to the % dose transferred across the epithelial cell. Cp, caeruloplasmin; FP1, ferroportin 1; Hp, hephaestin; IRP, iron regulatory protein; Tf, transferrin. The transfer pathway appears to be regulated directly by the peptide hepcidin, synthesised in the liver. For further details see text and Table 4.

the intestinal cells would be dependent on the output from other donor tissues, being increased where there is tissue iron deficiency. Furthermore, a rise in plasma iron turnover due to increased erythron demands for iron, would increase output from all the donor tissues, including the gut, and so increase iron absorption. There now appears to be a more direct form of regulation via Hepcidin, the product of the HAMP gene (Table 4). This is a small peptide (20–25 amino acids) with several isoforms released from a large pre-propeptide of 84 amino-acids. It is predominantly expressed in the liver and is downregulated when iron stores are reduced and up-regulated when iron stores are increased or by inflammation.4 Evidence from transgenic mouse models indicates that hepcidin is the predominant negative regulator of iron absorption in the small intestine, iron transport across the placenta, and iron release from macrophages.5;6 A deficient hepcidin response to iron loading may contribute to iron overload in HFE linked haemochromatosis.7 In

several families with severe juvenile haemochromatosis, homozygous, nonsense mutations in the HAMP gene have been reported confirming the key role of the gene in the control of iron absorption (see later). In the anaemia of inflammation, hepcidin production is increased up to a 100-fold4 and this may account for the defining feature of this condition, sequestration of iron in macrophages. Much work has been focused on a possible role of HFE in the direct regulation of iron uptake into the developing enterocyte from transferrin but HFE may have a more indirect role in the control of iron absorption through the regulation of hepcidin synthesis (see later).

Classification of genetic haemochromatosis In Table 5 several types of genetic haemochromatosis are listed according to the causative gene.

Table 5 Classification of genetic haemochromatosis. Type

Gene

Inheritance and phenotype

Severity

Incidence

1

HFE Digenic (HFE and HAMP) HFE2 HAMP TFR2 Ferroportin 1

AR parenchymal Fe overload AR parenchymal Fe overload

Highly variable Variable Severe

AR parenchymal Fe overload AD reticulo-endothelial Fe

Severe Variable

Common Rare Rare Rare Rare Rare

2 (juvenile) 3 4

AR, autosomal recessive; AD, autosomal dominant. For gene symbols, see Table 4.

74 Types 1, 2 and 3 show autosomal recessive inheritance. Only type 4 haemochromatosis is inherited as a dominant condition. In type 4, mutations in the ferroportin1 gene are associated with iron accumulation in macrophages with a raised serum ferritin concentration. The transferrin saturation may be normal.

Type 1 haemochromatosis (HFE linked) This is one of the most common,autosomal recessive conditions found in populations of northern European origin. Homozygosity for the C282Y mutation of the HFE gene8 is strongly associated with genetic haemochromatosis, with about 90% of patients with genetic iron overload in the UK having this genotype.9 In the UK about 1 in 7 people are carriers of the C282Y and about 1 in 150 are homozygous for this mutation.10 In homozygotes, there is a gradual accumulation of iron, which may lead to tissue damage presenting as cirrhosis of the liver, diabetes, hypogonadism or arthritis, and a slate-grey skin pigmentation.11 Hepatocellular carcinoma develops in 25% of established cases with cirrhosis. Most patients present between the ages of 40 and 60 years but the clinical penetrance is low (see later). Full phenotypic expression of the disorder is dependent upon other factors, including dietary iron intake, blood loss and (mostly unknown) genetic factors modifying the genotype. Menstrual losses account for a lower frequency of iron overload and a generally delayed onset in women although women may present before the age of 30 years.

Nature of the defect An association between HLA-A and-B antigens and haemochromatosis was first described by Simon et al in 1975.12 The haemochromatosis gene was shown to be located on the short arm of chromosome 6 very close to the HLA A locus and association with HLA A3, and to a lesser extent B7, suggested a founder mutation in a chromosome carrying the A3, B7 haplotype.13 Subsequent linkage analyses using multiple genetic markers have supported this suggestion of a highly conserved, founder haplotype,14 and positional cloning led to the identification of a novel MHC class I-like gene, HFE, 5 Mb telomeric to the HLA A locus.8 In over 80% of patients with haemochromatosis initially studied there was homozygosity for a missense mutation in this gene. This mutation, G to A at nucleotide 845 in exon 4, results in a cysteine to

M. Worwood tyrosine substitution at amino acid 282 (C282Y). The authors speculated that this change would abolish a disulphide bridge, thus preventing binding to b2 -microglobulin. This is a separate protein that interacts with HLA class 1 proteins (including HFE) in the a3 homologous region. A second mutation, exon 2, C to G at nucleotide 187, results in a histidine to aspartic acid substitution at amino acid 63 (H63D).8 This is carried by about 25% of the general population.10 About 2% of the UK population are compound heterozygotes for the two mutations, as are about 4% of patients with GH in the UK.9 The HFE gene was therefore a strong candidate for being the haemochromatosis gene and this was confirmed by the demonstration that knockout mice for b2 -microglobulin15 and HFE16;17 accumulate iron. After the demonstration that in some cell lines the normal HFE protein bound to the transferrin receptor and reduced iron uptake from transferrin18–21 it was speculated that this would be the mechanism leading to increased iron absorption. However attempts to explain how increased iron uptake from transferrin, as a result of the failure of expression of the mutated HFE protein, caused increased iron absorption were not convincing.22 Interest is now focused on the role of hepcidin as a negative regulator of iron absorption. Lack of hepcidin may act to up-regulate the mucosal DcytB protein which reduces ferric to ferrous iron at the brush border membrane, thus increasing iron uptake and absorption.24 However, this finding in mice has not been confirmed in man.25 Hepcidin synthesis requires expression of HFE.7;23 HFE knockout mice have low levels of hepcidin7 and over-expression of the HAMP gene inhibited iron accumulation.6 Thus HFE may act by regulating expression of hepcidin. Hepcidin also controls iron release from macrophages.26 Changes in hepcidin levels in the plasma can increase both iron absorption and iron release from macrophages, explaining the findings in the early stages of haemochromatosis – increased iron absorption and a paucity of iron in macrophages. Although these are preliminary findings, the therapeutic possibilities of hepcidin in reducing iron absorption or mitigating the anaemia of chronic disease have already been noted.27

HFE mutation frequencies world-wide Genotypes have been reported for about 50,000 subjects world-wide.31 The C282Y mutation is confined to populations of European origin and within Europe is most frequent in the north. The

Inherited iron loading: genetic testing in diagnosis and management

75

Figure 2 HFE C282Y allele frequencies in Europe. Frequency (%) of chromosomes carrying the C282Y mutation in regions of Europe. Data from Merryweather-Clarke et al.31 except where indicated. Only studies of over 400 subjects are included. * Indicates that figures for the country are combined data reported by Merryweather-Clarke et al.31 Dublin,164 South Wales,10 Norwich,165 Scotland.166

highest frequencies for the allele are found in Ireland, Scotland, Wales, Brittany and parts of Scandinavia (Fig. 2) and the lowest frequencies in Southern Italy and Greece. Initial calculations of the age of the C282Y mutation suggest that there was a unique mutation which occurred from 69 to 250 generations ago on a chromosome carrying HLA-A3 and HLA-B7.28–30 Although the frequency map suggests a ‘Celtic’ origin, the widespread occurrence of the mutation in Northern Europe implies that the C282Y mutation occurred earlier rather than later in the range of 69–250 generations. The H63D mutation is found throughout the world but is most common in Europe where allele frequencies vary from 10% to 20% with a mean of 15%.31 The H63D mutation may have occurred independently in several places32 and is probably much older because it is geographically more widespread and does not occur on such an extended haplotype. The only other mutation found throughout Europe is S65C which has a frequency of 0.1–2%.33–41 At present there is no discernable pattern in the frequency of the S65C mutation in Europe but in most population surveys sample sizes

have been small. Other mutations and polymorphisms are listed in Table 6. The mutations linked to iron loading are rare. Polymorphisms listed may be valuable in establishing haplotypes. One has been implicated in possible mistyping of the C282Y mutation – IVS4(+48) G ! A.42;43 This did not appear to be a significant problem for 11 testing laboratories in Europe.44

HFE mutations and iron status It was suggested by Motulsky that the haemochromatosis gene may have increased in frequency because of a selective advantage for heterozygotes – protection against iron deficiency anaemia.45 Homozygotes would be unlikely to suffer the effects of iron overload before having children. Among family members of genetic haemochromatosis patients, about 25% of heterozygotes, identified by HLA typing, had either a raised TS or a raised sFn.46–48 Nevertheless co-existing disease may be responsible for the raised serum ferritin concentrations in many heterozygous family

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Table 6 Gene

Variants associated with iron loading

HFE (HLA-H)

Cys282Tyr His63Asp Ser65Cys Ile105Thr Gly93Arg Glu168Ter Trp169Ter Arg330Met Pro160DCys

IVS5 + 1 G ! A IVS3 + 1 G ! T a

G ! A at nucleotide 845 C ! G at nucleotide 187 A ! T at nucleotide 193 T ! C at nucleotide 314 G ! C at nucleotide 277 G ! T at nucleotide 502 G ! A at nucleotide 506 G ! T at nucleotide 989 Single cytosine deletion introducing a premature stop codon 50 amino acids downstream G ! A causing a splice site mutation whereby exon 4 is spliced to exon 6 G ! T causing a splice site mutation whereby exon 2 is spliced to exon 4

Frequencya

Reference

0.08 0.15 0.01 – – – –

8

149 150

151

152

–

153

–

154

–

155

In Northern European Populations. For information on other polymorphisms and SNPs within the HFE gene, see32;72;156–159 .

members.83 In population surveys, slightly but significantly higher values for serum iron and TS have been found in heterozygotes for either C282Y10;49–51 or H63D10;49;51 compared with subjects lacking these mutations. The differences in ferritin levels were smaller and not significant except that Jackson et al.10 found higher levels of sFn in men heterozygous for C282Y. In compound heterozygotes and subjects homozygous for H63D there are greater differences.10;51;52 In heterozygotes for C282Y50;51 and H63D51 Hb levels were slightly higher than in subjects lacking mutations. Beutler et al.51 noted a lower prevalence of anaemia among women carrying either mutation, but the differences were small and only significant if all subjects carrying mutations were compared with those lacking mutations. Jackson et al.10 did not find any evidence that the percentage of blood donors failing the screening test for anaemia differed among the genotype groups. Changes in transferrin saturation with genotype are shown in Fig. 3. The majority of 70 60

men over 30 years who are homozygous for C282Y will have a raised TS10;53 but only a minority of women. Serum ferritin concentrations in haemochromatosis may not accurately reflect tissue iron concentrations during the early stages of iron accumulation, particularly in heterozygotes. Serum ferritin concentrations are related to the levels of ferritin iron in macrophages and in haemochromatosis the iron initially accumulates in hepatic parenchymal cells. Edwards et al.54 noted that liver iron concentrations in heterozygote family members were above the reference range although serum ferritin concentrations were not elevated. Thus iron accumulation may be underestimated in heterozygotes. There is little information about S65C. Arya et al.34 did not find that heterozygosity for S65C was associated with elevated transferrin saturation in blood donors. In combination with C282Y or H63D, S65C may be associated with mild iron accumulation.33;55;56 Holmstrom et al.,35 in contrast,

Males Females

50 Transferrin Saturation (%)

40 30 20 10 0 HHCC HDCC HHCY DDCC HDCY HHYY

Figure 3 Transferrin saturation in 10,325 blood donors according to HFE genotype. Mean values in all variant genotype groups are significantly higher than the mean value for “wild-type” donors (p < 0:0001 for both men and women). Men (solid bars), women (open bars). HHCC, wild type; HDCC, H63D heterozygote; HHCY, C282Y heterozygote, etc.

Inherited iron loading: genetic testing in diagnosis and management found that 50% of healthy subjects carrying S65C had an elevated TS or serum ferritin, but compound heterozygosity for S65C and either C282Y or H63D did not significantly increase the risk of iron overload.

What other factors modify iron accumulation in homozygotes for C282Y? Homozygosity for HFE C282Y causes enhanced iron absorption and the degree of iron overload ensuing is modified by diet and blood loss. Alcohol is a significant risk factor for the development of cirrhosis in patients homozygous for C282Y.57 The significance of other genetic modifiers remains uncertain. Van Vlierberghe et al.58 described a high frequency of the haptoglobin 2–2 phenotype in haemochromatosis but this finding has not been confirmed.59;60

Diagnosis (see guidelines)61 Clinical The variety of clinical presentations and their lack of specificity for haemochromatosis means that a high degree of clinical suspicion is needed. Fatigue, diabetes mellitus, gonadal failure and arthritis may be present for several years before the diagnosis is made.62 Iron status In asymptomatic subjects, iron accumulation is indicated by a raised transferrin saturation (>55% for men and 50% for women). Most men and about 50% of women who are homozygous for HFE C282Y will have a raised transferrin saturation. As iron accumulates the serum ferritin concentration rises and values of >200 lg/l (women) and 300 lg/l (men) suggest iron overload. In patients with infection, inflammation, malignancy or undergoing surgery it should be remembered that the transferrin saturation may be depressed and the serum ferritin concentration elevated.63 In most cases genotyping will confirm the diagnosis of genetic haemochromatosis. In patients homozygous for C282Y with normal serum transaminase activity, serum ferritin concentration <1000 lg/l and without hepatomegaly there is no need for a liver biopsy in order to make a diagnosis of genetic haemochromatosis.64 In symptomatic patients, the serum transferrin saturation is usually greater than 80%, and the serum ferritin concentration greater than 1000 lg/l.

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A liver biopsy is essential to assess tissue damage in patients with evidence of liver disease or a serum ferritin concentration > 1000 lg/l. In patients with an unexplained, raised transferrin saturation and serum ferritin who are not homozygous for C282Y a liver biopsy may be required to confirm iron overload. Values in excess of 80 lmol/g dry weight (4.5 mg Fe/g dry weight) indicate iron overload. In the differential diagnosis of hereditary haemochromatosis, as there is a progressive increase in iron with age, it is useful to express the result as the ‘hepatic iron index’ (lmol iron/g dry weight  age in years).65 An elevated index (>2.0) generally separates patients with homozygous hereditary haemochromatosis from heterozygotes or those with relatively minor iron accumulation due to liver disease.66 Since alcohol is one of the factors that may enhance the phenotypic expression of hereditary haemochromatosis, this diagnostic distinction is important. Non-invasive methods to detect and quantify iron overload The SQUID-biosusceptometry technique is sensitive, accurate and reproducible. It depends on the paramagnetic properties of haemosiderin and ferritin. Unlike MRI it does not distinguish parenchymal from reticuloendothelial iron, but the result closely correlates with chemical estimation of liver iron except when fibrosis is present. Machines are expensive to build and run and currently there are only four worldwide. Results in a large series of patients with iron overload, including 164 patients homozygous for C282Y, have been described.67 Magnetic resonance imaging (MRI) techniques are being used increasingly as indirect measures of liver and cardiac iron. They have the advantage of being more widely available than SQUID, which is only suitable for liver iron. There are several MRI techniques that rely on a shortening of relaxation time, and thus reduction in signal intensity, with iron overload. At present MRI provides a means of investigating the 3-dimensional distribution of excess iron in the body, but provides only a semiquantitative assessment of storage iron.68;69

Genetic testing Numerous methods for detecting the C282Y and HFE variants have been described, encompassing almost all methods of mutation detection.70 Those used for diagnostic purposes in the UK have generally proved to be accurate (UKNEQAS, Histocompatibility and Immunogenetics, contact [email protected]). Doubts remain

78 about the application of patents relating to the HFE genotyping.71 If a non-HFE related condition is suspected, a more general approach to mutation detection is required as no common, causative mutations have yet been described for HFE2, HAMP, TfR2 and ferroportin 1. One such strategy is denaturing HPLC and sequencing.72

Treatment Removal of excess iron by regular phlebotomy greatly reduces the mortality from cardiac and hepatic failure, although hepatocellular carcinoma accounts for a substantial proportion of deaths in those who already have liver cirrhosis. Early diagnosis is therefore a priority, since patients identified and treated before the onset of cirrhosis of the liver have a normal life expectancy.73 Removal of excess iron is achieved by weekly venesection (450 ml blood, c 200 mg Fe) but the rate may need to be reduced if the haemoglobin concentration is not maintained above 12 g/dl. The amount of iron removed before anaemia develops is calculated after allowing for the 3 mg iron absorbed from the diet daily. The amount of storage iron measured by quantitative phlebotomy in normal adults has been shown to be about 750 mg in males and 250 mg in females.74 Treatment is monitored by measuring serum ferritin concentration (which may fluctuate initially) and the initial, weekly phlebotomy will need to be continued for at least 6 months to remove a storage iron load which is usually greater than 5 g with established symptomatic disease, but may be in excess of 20 g. Iron chelation, with desferrioxamine given as a continuous intravenous infusion, may have a limited role in the short-term management of patients with life-threatening cardiac failure. As the iron is exhausted, the serum iron and haemoglobin concentrations fall, and the frequency of phlebotomy should be reduced to two to four units each year, to continue indefinitely. The aim is to maintain a normal transferrin saturation (<50%) and a serum ferritin in the low normal range (<50 lg/l). Fatigue and transaminase elevation usually reverse on venesection. In some patients diabetes mellitus, hypogonadism and arthralgia improve, but cirrhosis and arthritis are not reversible.

Clinical penetrance Before the discovery of the HFE gene it was assumed that the natural history of the disorder meant that every person homozygous for haemochromatosis would eventually accumulate suffi-

M. Worwood cient iron to cause tissue damage and the resulting morbidity.75 However, a recent study in which subjects homozygous for HFE C282Y have been compared with “wild-type” subjects at a healthappraisal clinic has shown that the frequency of lethargy, arthralgia and diabetes was the same in both groups.53 There was a small but significant increase in the percentage of subjects with either raised serum transaminase activity or fibrosis/cirrhosis in the C282Y homozygous group. A large population based survey of haemochromatosis in Norway revealed iron accumulation but little morbidity amongst those homozygous for C282Y.76 Genetic testing of 10,500 blood donors from South Wales identified 72 who were homozygous for C282Y. Most of the men and 45% of the women had a raised transferrin saturation. None of the 63 C282Y homozygous donors interviewed showed physical signs of iron overload or were aware of relatives with haemochromatosis.10 Furthermore, a survey of clinical haemochromatosis in South Wales revealed that only 1.2% of adults homozygous for C282Y had received a diagnosis of haemochromatosis (2.8% of men over 45y).77 Despite much debate about ascertainment bias in family and population surveys78–82 it is becoming clear that most men homozygous for C282Y will have a raised transferrin saturation before the age of 30, a proportion will have an elevated serum ferritin concentration, but only a minority will eventually develop fibrosis and cirrhosis of the liver. Only about 50% of homozygous women have a raised transferrin saturation and progression through iron accumulation and tissue damage is usually, but not always, slower.

Family testing The availability of a genetic test which identifies about 90% of patients with haemochromatosis in patients of Northern European origin facilitates early identification of those at risk from iron overload, rather than late diagnosis in those who already have tissue damage. Within a family haemochromatosis may be prevented by genetic testing and following those at risk by measuring transferrin saturation and serum ferritin at regular intervals. Testing of relatives should be accompanied by appropriate genetic counselling. It is important to ensure that individuals being tested have received full information on the test and its consequences as well as on the disorder, and that they have given consent. All first degree relatives over the age of consent should be offered testing but genetic testing is not advised for healthy children under the age of consent. Transferrin

Inherited iron loading: genetic testing in diagnosis and management saturation and serum ferritin concentration should be measured along with HFE genotyping. Genetic testing may identify other family members homozygous for HFE C282Y. If the serum ferritin concentration is normal and there is no evidence of liver disease then transferrin saturation and serum ferritin should be measured at yearly intervals and treatment instituted if necessary. Compound heterozygotes are at much lesser risk of iron overload10 but should also be tested by measuring transferrin saturation and serum ferritin – perhaps at three yearly intervals. For heterozygotes iron status should be determined and, if normal, reassessed after 5 years in case other iron loading genes are present. About 25% of heterozygotes show minor abnormalities of iron metabolism, e.g., a raised transferrin saturation or serum ferritin concentration. However a raised serum ferritin concentration is often secondary to chronic disease.83 Iron accumulation similar to that in C282Y homozygotes is rarely seen.54 If the proband has haemochromatosis of type 2–4 then appropriate mutations should be tested for in other family members. If no causative genetic change has been identified, confirmation of iron overload may require liver biopsy.

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Shaheen et al.93 reported an association between HFE gene mutations and the risk of colon cancer. The effect was only demonstrable after adjusting for other risk factors and was only significant for H63D. In view of the limited phenotypic effect of H63D it is difficult to assess the significance of this finding in terms of iron metabolism. An association has been reported between HFE C282Y and childhood acute lymphoblastic anaemia in males.94 This was found in two groups of patients from Cardiff and Glasgow and confirmed the previous epidemiological finding that male heterozygotes for haemochromatosis have an increased risk of haemopoietic malignancies.95 The prevalence of HFE genotypes has been studied in patients with various haematological malignancies96 but it was not possible to confirm either the association with acute lymphoblastic anaemia in male children or the more general risk. An association between Factor V Leiden and HFE C282Y has been reported.97 Although this was refuted in a larger study98 MacLean et al.99 et al noticed that HFE C282Y frequency was increased among patients who had both FVL and a family history of thrombosis compared with those with FVL and no family history. This finding still awaits confirmation.

HFE mutations and morbidity Associations with other conditions There are long-established biochemical hypotheses linking increasing tissue iron concentrations with increasing risk of neoplastic,84 atherosclerotic,85;86 infectious86 and inflammatory conditions.87 Iron accelerates free radical generation which leads to inflammation, mutagenesis and atherosclerosis as well as bacterial growth. Recently there has also been much interest in the role of iron accumulation in the brain in the ageing process and in neurodegenerative disorders.88 Thus genotypes which increase transport and storage iron levels may be associated with increased risk for many common diseases. Furthermore, advanced haemochromatosis is characterised by diabetes, arthritis and liver disease, but there is little evidence that being a carrier of C282Y or H63D is a significant risk factor for these conditions.89 However, a significant risk for cardiovascular disease has been described for possession of HFE C282Y in association with smoking and hypertension.90 Homozygosity for C282Y is significantly more frequent in patients with cirrhosis and hepatoma than in the general population.91 In one study a significant increase in the frequency of homozygosity for C282Y was found for patients with late-onset, type I diabetes.92

It has long been thought that there may be a high frequency of heterozygosity for haemochromatosis in patients with porphyria cutanea tarda, sideroblastic anaemia, and hereditary spherocytosis with iron overload, amongst other conditions associated with some degree of iron overload. Until recently this was a matter for debate100 but the advent of genetic testing has resolved some of the arguments. Porphyria cutanea tarda Although patients with porphyria cutanea tarda demonstrate some iron accumulation and venesection (to cause iron depletion) results in clinical and biochemical remission, relatively few patients have a degree of iron overload that would be considered diagnostic for genetic haemochromatosis. In 1997 Roberts et al.101 showed that in 41 patients with sporadic porphyria cutanea tarda 45% carried at least one copy of the C282Y mutation of the HFE gene and over 20% were homozygous for this mutation. Patients homozygous for the C282Y mutation did not have iron overload consistent with genetic haemochromatosis, even though their average age of presentation with PCT was 67 years.

80 These findings have been confirmed in several countries.102–104 Brady et al.,105 extending the original study of Roberts et al.,101 concluded that in both sporadic and familial PCT, C282Y homozygosity is an important susceptibility factor but that heterozygosity for C282Y has much less effect on disease development. The relationship between haemochromatosis mutations and PCT is more subtle than just more rapid accumulation of iron due to the presence of an HFE C282Y mutation. In Italy the C282Y mutation is not common in the general population (frequency approx. 1%). Sampietro et al.106 found that patients with PCT did not have an increased frequency of this mutation. However they found a significant increase in the frequency of the H63D mutation. An increase has also been reported in patients from the USA.107 Sampietro et al proposed that the H63D mutation allows iron to inactivate porphobilinogen decarboxylase without general accumulation in the body. Haemochromatosis mutations appear to cause susceptibility to the development of sporadic porphyria cutanea tarda, but it is not necessary for major iron overload to develop for this to happen.

Inherited anaemias Because haemochromatosis mutations are common in some populations it seems likely that they will coexist with other conditions and in some cases exacerbate the clinical course. Of particular concern are those disorders associated with increased iron absorption due to an increased (often ineffective) production of red cells in the bone marrow. Such conditions include the b-thalassaemias, congenital/inherited sideroblastic anaemias and haemolytic anaemias. b-Thalassaemia intermedia is an anaemia of moderate to mild severity, caused usually by the homozygous or doubly heterozygous inheritance of b-thalassaemia mutations, or more rarely by the heterozygous inheritance of a ‘dominant’ b-thalassaemia. Iron absorption is increased, apparently exacerbated by splenectomy, and iron overload occurs in adult life. Iron overload in these patients does not require the co-inheritance of haemochromatosis genes but the co-inheritance of haemochromatosis heterozygosity or homozygosity may enhance the rate at which iron overload occurs.108 In two studies on patients with thalassaemia major from Italy, the frequency of the C282Y and H63D mutations was the same as that in normal controls (C282Y allele frequency only 1–2%).109;110

M. Worwood Borgna-Pignatti et al.109 found that heterozygosity for HFE mutations did not influence either the degree of iron overload or its consequences in regularly transfused and chelated patients with thalassaemia major. However, in Italian patients with genetic haemochromatosis, b-thalassaemia trait aggravated the iron loading in C282Y homozygotes.111 b-Thalassaemia carriers who were homozygous for HFE H63D were found to have higher serum ferritin levels that those with the wild-type genotype.112 Inherited/congenital sideroblastic anaemias are rare anaemias of varying severity. The X-linked form, responsive to pyridoxine, is usually an anaemia of moderate severity and is often associated with iron overload. Yaouanq et al.113 described a family with pyridoxine responsive sideroblastic anaemia in which one brother was a compound heterozygote for the haemochromatosis mutations. He had a significantly greater degree of iron overload than his older brother lacking both mutations. A subsequent study of 18 X-linked sideroblastic anaemia hemizygotes found a small but significantly higher frequency of C282Y amongst these patients, indicating a role for coinheritance of HFE alleles in the expression of this disorder.114 One proband with severe and early iron loading coinherited HH as a C282Y homozygote. The clinical and haematologic histories of two XLSA probands suggested that iron overload suppresses pyridoxine responsiveness. Notably, reversal of the iron overload in the proband homozygous for C282Y by phlebotomy resulted in higher haemoglobin concentrations during pyridoxine supplementation. The relationship between the HFE genotype and iron loading has been studied in 34 patients with pyruvate kinase deficiency from 29 Italian families.115 The allele frequency for C282Y was 1.8% and 16.1% for H63D. Iron overload occurred in patients with and without HFE mutations. In a family with inheritance of both hereditary spherocytosis and HFE C282Y there was severe iron loading in the proband who was homozygous for C282Y and also had spherocytosis.116

Population screening Widespread population screening on the basis of measures of iron status or by genetic testing has been proposed.75;117 However, genetic testing of the whole population would be premature, since the precise level of risk for a C282Y homozygote developing severe iron overload is not yet known. Once the factors associated with a high risk of developing significant iron overload and tissue

Inherited iron loading: genetic testing in diagnosis and management damage have been identified it may be appropriate to reconsider this question.

Type 2 haemochromatosis Juvenile haemochromatosis is a rare autosomal recessive disease, with clinical symptoms appearing in the second and third decades of life, characterised by cardiomyopathy and hypogonadism. The HFE2 locus has been mapped to chromosome 1q21 (Table 4.2). Iron absorption is greater than in Type 1 haemochromatosis. The severity of the clinical phenotype and the rapid rate of iron loading indicate that the responsible gene has a prominent role in the regulation of iron absorption. This HFE2 gene has recently been identified and its protein product named hemojuvelin.118 Mutations were identified in Greek, Canadian and French families with one,G320V, accounting for two-thirds of these mutations. Like HFE,1 HFE2 modulates hepcidin expression and serum hepcidin was low in homozygous affected individuals with HFE2 mutations. Mutations in the Hepcidin gene have been described in three families with a type of juvenile hemochromatosis not linked to chromosome 1. Affected subjects were homozygous for the mutation in each case.119;120

Type 3 haemochromatosis This is phenotypically similar to HFE-associated haemochromatosis but is due to mutations in the transferrin receptor 2 gene (Table 7).121–123 TFR2 (Table 4) shows moderate homology to TFR, may bind transferrin but is not iron-regulated in the same way as TFR. Its role in iron homeostasis is unclear.

Type 4 haemochromatosis Haemochromatosis type 4 differs from the other types in both inheritance and phenotype. It is inherited as an autosomal dominant trait and while patients have increased serum ferritin levels the transferrin saturation is not always elevated. Iron is increased in the Kupffer cells of the liver, as well as

81

in hepatocytes. These features suggest a different pathophysiology of the disease. Type 4 disease is due to heterozygous mutations in the newly identified iron exporter, ferroportin 1 (FPN1; MTP1; Ireg1; SLC11A3; SCL40A1),124–126 coded for by a gene on chromosome 2q32 (Table 4). Along with missense mutations (Fig. 4), a deletion of valine at amino acid 162 has been found in families from the UK, France, Italy and Australia, suggesting that this may be a relatively common cause of iron accumulation. The phenotype in one family with the Val162del was similar to that found in the anaemia of chronic disease.127 Weekly venesection rapidly caused anaemia and venesection with erythropoietin therapy was attempted. It is unlikely that a ferroportin 1 mutation will be suspected unless there is significant iron overload and a familial association. It is not yet clear whether the degree of tissue damage at a given serum ferritin level is similar to type 1 haemochromatosis or whether the predominantly reticuloendothelial distribution is less toxic. In people of African origin Gln248His is a common polymorphism that may be associated with a tendency to iron loading and mild anaemia.128;129

Neonatal haemochromatosis Neonatal hemochromatosis is a heterogeneous condition, which is recognized at birth but may occur in utero, characterized by heavy parenchymal iron deposition in several organs and irreversibile liver failure. Neonatal haemochromatosis may result from genetic or acquired factors. In the case of familial occurrence the most common inheritance seems to be autosomal recessive; however a mitochondrial inheritance has also been proposed. Mutations in the HFE gene have not been reported and chromosome 1q linkage has been excluded.131 The only therapeutic option for this severe disorder is liver transplantation.130

Increased iron intake African iron overload (Bantu siderosis) results from the combination of a dietary component (a

Table 7 Homozygous mutations in TFR2 associated with iron overload. Mutation

Type

Intra-gene location Country and pedigree information

Reference

E60X M172K Y250X AVAQ 594-97del Q690P

Frameshift Missense Nonsense Deletion Missense

Exon Exon Exon Exon Exon

123

2 4 6 16 17

Other heterozygous variants have been described.72;162;163

Southern Italy, 1 family with consanguinity Southern Italy, single patient Sicily, 2 families (1 with consanguinity) Northern Italy, 1 family Japan, 1 family Portuguese descent, 1 family

123 121 160;161 162

82

M. Worwood X5 Q182H

D157G N144T

Extracellular

V162del

N144H

A77D

Y

17

Y64N

85

12

33

8

5 6 7

4

YC

Y 57

15

57

1 2 3 57

G323V

13

9 48

N Q248H

Cytoplasm

G490D

Polymorphic in Africans

Figure 4 Mutations in the ferroportin 1 gene. Modified from Devalia et al.127 Original figure ª The American Society of Hematology. Y64N,167 A77D,168 N144T169 N144H,170 D157G, Q182H and G323V;171 V162del;127;172–174 Q248H;128;129 G490D.175

traditional beer that contains iron) and an unknown susceptibility gene.132 Iron deposition occurs both in hepatocytes and in RE cells. Serum ferritin is usually elevated, but transferrin saturation may be normal. It is a cause of hepatic fibrosis and cirrhosis in subSaharan Africa, and associations with diabetes mellitus, peritonitis, scurvy and osteoporosis have been described. The iron overload is associated with a high risk of death from hepato-carcinoma and poor outcome in tuberculosis.133 Mutations in the HFE gene have been excluded as the cause of iron overload (See also type 4 haemochromatosis, above).

Atransferrinaemia This rare recessive genetic disorder is associated with a severe hypochromic anaemia with iron overload.134 It can be treated by regular infusion of transferrin immediately preceded by phlebotomy to remove the excess iron.

Iron and neurodegeneration Aceruloplasminaemia A rare recessive disorder in which there is a deficiency of ferroxidase activity as a consequence of mutations in the ceruloplasmin gene on chromosome 3q. Clinically the condition presents in middle age with progressive degeneration of the retina and basal ganglia and with diabetes mellitus.135 Iron accumulates in the liver, pancreas and brain, with smaller amounts in the heart, kidneys, thyroid, spleen and retina. The serum iron is low, TIBC normal and ferritin normal or raised. Iron chelation with desferrioxamine may decrease brain iron

stores and halt the progression of the neurological degeneration.136 Hallervorden-Spatz syndrome Hallervorden-Spatz syndrome (HSS) is an autosomal recessive neurodegenerative disorder associated with iron accumulation in the brain. Clinical features include extrapyramidal dysfunction, onset in childhood, and a relentlessly progressive course. Histologic study reveals iron deposits in the basal ganglia. HSS is caused by a defect in a novel pantothenate kinase gene. This results in accumulation of cysteine. Iron binding by cysteine may account for the iron accumulation and oxidative stress which is a likely explanation for the pathophysiology of the disease.137

Neuroferritinopathy This is a recently described, dominantly inherited, late-onset basal ganglia disease, variably presenting with extrapyramidal features similar to those of Huntington disease (HD) or Parkinsonism. There is iron accumulation in the forebrain and cerebellum.138;139 The disorder was mapped 19q13.3, which contains the gene for ferritin light chain polypeptide (FTL). An adenine insertion at position 460–461 is predicted to alter carboxy-terminal residues of the gene product. Abnormal aggregates of ferritin and iron in the brain contrast with low serum ferritin levels. Friedrich’s ataxia In this neurodegenerative disease there is loss of sensory neurons in the spinal cord and dorsal root

Inherited iron loading: genetic testing in diagnosis and management ganglia associated with mitochondrial iron overload and a loss of activity of iron–sulphur clustercontaining enzymes. The majority of Friedrich’s ataxia (FRDA) cases result from the expansion of triple nucleotide repeats within an intron of the FRDA gene (human chromosome 9q13) leading to reduced expression of frataxin mRNA and protein.140 Point mutations have also been identified in a small number of cases. Frataxin is found in the mitochondrion. Yeast frataxin binds and stores iron in a way analogous to ferritin. The protein forms an oligomer in the presence of Fe2þ and O2 and as iron concentrations increase forms a 48-subunit multimer able to sequester about 2400 atoms of iron. Park et al.141 suggest that yeast frataxin can function either as an iron chaperone or as an iron store. In FRDA, there is increased oxidative stress and decreased activity of iron–sulphur proteins. Oxidative damage following iron accumulation is thought to precipitate the neuron loss in FRDA.142 Patients frequently die from cardiomyopathy. These diseases may serve as a model for complex neurodegenerative diseases, such as Parkinson disease, Alzheimer disease and Huntington disease, in which pathologic accumulation of iron in the brain is also observed.143 HFE mutations have been described as risk factors for Alzheimer disease144;145 or as a risk factor for early presentation.146 However a later study did not confirm either the increased frequency of H63D in Alzheimer Disease or a relationship with age of onset.147 No associations between HFE mutations and Parkinson disease have been reported. In a study of 438 Parkinson Disease patients and 485 controls the C282Y mutation appeared to offer some protection against the development of Parkinson disease.148

Practice points • In about 90% of cases of genetic iron overload in patients of Northern European origin there will be homozygosity for the C282Y mutation of the HFE gene. A small percentage will be compound heterozygotes (C282Y/H63D). • In cases where HFE mutations are not responsible for iron accumulation, mutations in a variety of iron transport genes may be involved: TFR2, HFE2 and HAMP (in juvenile haemochromatosis), and ferroportin-1 (particularly if there is reticuloendothelial iron overload and autosomal dominant inheritance).

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• In such cases a general mutation strategy is required as no common iron-loading mutations have yet been identified. • Phenotypic investigation of iron overload is essential. The clinical penetrance of the HFE C282Y mutation is low. The clinical manifestations seen in iron overload are also common in people without iron overload and homozygosity for this mutation is not necessarily an explanation for morbidity in a patient. • First degree relatives of patients should be offered counselling and both genetic and phenotypic testing.

Research agenda • Assessing the frequency and clinical significant of variants in the “new” haemochromatosis associated genes. • Understanding the role of hepcidin in controlling iron absorption and exploiting its therapeutic potential. • Identifying the significant genetic modifiers of clinical penetrance in type 1 haemochromatosis. • Making non-invasive quantitation of iron overload feasible and inexpensive in order to characterise and quantitate iron overload.

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