Cloning of the hereditary hemochromatosis gene: Implications for pathogenesis, diagnosis, and screening

Cloning of the hereditary hemochromatosis gene: Implications for pathogenesis, diagnosis, and screening ANDREAS HIMMELMANN and JORG FEHR ZORICH,SWITZERLAND Hereditary hemochromatosis (HH) is one of the most common genetic disorders affecting populations of European ancestry. It is characterized by an inappropriately high iron absorption that leads to iron overload preferentially in the parenchymal organs. Although the severity of the phenotype can be modified by a number of factors, it is clear that most homozygotes will eventually become symptomatic. Clinical manifestations are often nonspecific and easily overlooked. Untreated HH can be associated with substantial morbidity and mortality. Diagnosis in the precirrhotic stage, however, results in normal life expectancy. Early diagnosis and the initiation of phlebotomy therapy are therefore keys to preventing complications related to iron overload. The recent isolation of a strong candidate gene for hemochromatosis has the potential to allow for genetic diagnosis and screening in the near future. This exciting finding is likely not only to change clinical practice but also to yield new insights into the poorly understood pathophysiology of the disease. (J Lab Clin Med 1999;133:229-36)

Abbreviations: HFE = hemochromatosis gene; HH = hereditary hemochromatosis; HLA = human leukocyte antigen

H

ereditary hemochromatosis is now considered one of the most common autosomal recessive disorders in populations of caucasian origin. It occurs with a prevalence of about 3 to 5 per 1000 in different populations. Although the disease has been known since the nineteenth century, it is probably one of the most underdiagnosed conditions. This is in part because of the initial notion that it is a rare disease characterized by a complex of symptoms comprising a particular skin pigmentation, diabetes, and liver disease. For several reasons this concept has changed enor-

From the HematologySection, Department of Internal Medicine, University Hospital of Zfirich. Submitted for publication July 16, 1998;revision submitted October 23, 1998;acceptedOctober29, 1998. Reprint requests: J6rg Fehr, MD, Division of Hematology,A Hof143, Department of Internal Medicine, UniversityHospital Zarich, RSxnistrasse 100, 8091 Ztirich, Switzerland. Copyright© 1999by Mosby,Inc. 0022-2143/99 $8.00 + 0 5/1/95877

mously over the last several years. First, as a result of earlier diagnosis, it has become apparent that HH has a much broader range of phenotypic expression that includes nonspecific symptoms such as lethargy and arthralgia or other organ manifestations such as gonadal or cardiac dysfunction. Family screening of probands also has provided evidence of a much higher prevalence of the disease than initially expected. Second, it has been clearly established that the organ manifestations of the disease can be prevented by regular phlebotomy treatment if the disease is identified at an early stage. Therefore early diagnosis has become a major goal in the management of this common disorder. There have also been major advances in the understanding of the genetic basis of HH that culminated in the isolation of a strong hemochromatosis candidate gene in 1996. The identification of this gene, HFE, will have major implications for diagnosing and screening the disease. The possibility of genetic testing will undoubtedly simplify these processes, but physicians will be confronted with a number of new questions. When is genetic testing

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indicated? Is a liver biopsy necessary in genetically identified homozygous patients? The isolation of a hemochromatosis gene will also shed new light on the pathophysiology of the disease, which has so far been poorly understood. It is likely that new mechanisms of iron regulation will be described because the HFE product has no resemblance to any of the proteins known to regulate iron transport and storage. This article will focus on the widening clinical spectrum and on the diagnosis and screening of HH, with special emphasis on the new genetic findings and the role of genetic testing. GENETIC ASPECTS

The genetic basis of HH was clearly established only 20 years ago by the demonstration of a close linkage to the HLA class I locus. 1 This important observation not only localized a putative hemochromatosis gene on the short arm of chromosome 6 but also allowed the determination of the mode of inheritance as autosomalrecessive. Furthermore, the identification of relatives of an index case as either homozygous, heterozygous, or normal became possible by HLA typing. Despite its known chromosomal localization, the hemochromatosis gene remained elusive until very recently. In 1996 a strong candidate gene was isolated by positional cloning. 2 This method has been successfully used in identifying the genetic basis of a great number of human monogenetic disorders and holds promise to play an important role in defining the genetic components of the more common polygenetic disorders. 3 The consequences of this important discovery for our understanding of the pathophysiology of HH will first be discussed. Cloning of a candidate gene for HH. Using a positional cloning approach, Feder et al 2 identified a strong can-

didate gene for HH. The gene is located more than 3 megabases telomeric from the HLA class I locus, explaining in part why the gene remained elusive for such a long time. The gene was initially called H L A - H but has now been renamed H F E . 4 It is inherent to the positional cloning approach that the function of the isolated gene is often unknown. Based on its amino acid sequence, HFE is an HLA class I-like molecule, although it is predicted not to function in antigen presentation. Two missense mutations of HFE were found in patients with HH, 2 one that substitutes tyrosine for cysteine at amino acid position 282 (C282Y) and another one that substitutes aspartic acid for histidine at position 63 (H63D). In a different numbering system that begins with residue one of the mature protein, these mutations are designated C260Y and H41D, respectively. 5 Already a large number of studies have examined the frequency of these mutations in patients with HH

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and have strongly suggested that HFE is responsible for HH. The frequency of the C282Y mutation has varied from 100% in an Australian series 6 to 93% in studies from England and FranceT, 8 to 65% in a study from Italy. 9 Several studies from the United States have found frequencies around 80%. 2,1°J1 The different results in these studies are in part explained by the different patient populations examined. In the Australian series, well-defined pedigrees were analyzed that evidently favors finding the same mutation in all patients that are related. On the other hand, in most other studies independent patients were genotyped for the mutations. But there also seems to be a true difference in the frequency of the mutations among different European populations. Higher prevalence rates exist in certain parts of France and England, 12 while lower rates were found in southern Europe, where the frequency of heterozygous carriers is also lower. 9 These initial results were confirmed in a study of the global prevalence of the C282Y and H63D mutations, which showed a higher prevalence among populations of northern European origin. 13 We are presently conducting a study on the frequency of HFE mutations in patients with proven HH from the northeastern part of Switzerland. Preliminary results indicate a high frequency of the mutation in the range of 80% to 85%. These data and the functional effect of the C282Y mutation on the interaction of HFE with other proteins (see below) clearly indicate that this mutation plays a major role in the pathogenesis of HH. On the other hand, the role of the H63D mutation is not yet clear. The observation that this mutation appears with similar frequency in patients and control subjects and that homozygotes for this mutation are very rare among patients have led to the hypothesis that it is a nonfunctional polymorphism. 9 However, if one determines the frequency of the H63D mutation in HH patients heterozygous for the C282Y mutation, there is a large, statistically significant excess of this mutation in this group as compared with control subjects.14,15 This observation is strong evidence that the H63D mutation is related to the disease and that compound heterozygotes are at an increased risk to develop symptomatic iron overload. The penetrance of this genotype is significantly lower than that of the homozygous C282Y genotype. A relative penetrance of 0.53% to 1.5% as compared with the C282Y homozygotes has been calculated for the compound heterozygous genotype. 15 There are presently not enough data to determine with certainty whether the homozygous H63D genotype is associated with iron overload. Biologic effects of HFE mutations. The function of the HFE product in regulating iron metabolism is not yet defined, although it is becoming clear that HFE plays

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a role in a major iron absorption pathway. A disulfite bridge is required in class I molecules for an interaction with the accessory molecule ~2-microglobulin to occur. The C282Y mutation prevents interaction with ~2-microglobulin and as a result the correct surface expression of the HFE product. This has already been shown directly in transfection experiments using normal and mutated cDNAs that carried the C282Y mutation. 16 No effect on the interaction of HFE and 132microglobulin was seen for the H63D mutation. In addition, it was recently shown that the HFE product forms stable complexes with the transferrin receptor 17. This interaction decreases the affinity of the transferrin receptor for its ligand. Again, the C282Y mutation almost completely abolishes this interaction, which probably leads to the accumulation of iron in cells that normally use HFE to regulate iron uptake. The crystal structure of HFE and a more detailed analysis of its interaction with the transferrin receptor further support this model. 5 So far the most direct evidence for a role of HFE in iron metabolism comes from gene targeting experiments. HFE-deficient mice show clear disturbances in iron homeostasis that result in a greatly increased hepatic iron concentration, thus resembling HH. 18 During the first 10 weeks of observation, no increased iron concentration was found in other organs in these mice. There is also increasing evidence that the regulation of the immune system and iron metabolism are connected. For example, a recently cloned iron transporter, 19 the murine homolog of which is mutated in microcytic mice, 20 is related to the NRAMP1 (natural resistance-associated macrophage protein) gene and has been designated NRAMP2. Once the precise function of HFE has been elucidated, it will be possible to test whether the H63D mutation also has a functional effect. Factors affecting phenotypic expression. The finding that the same genotype (C282Y mutation) is found in patients exhibiting very different phenotypes of HH was surprising. This observation supports the long-held view that modifying factors, either genetic or environmental, influence the phenotypic expression of HH. Among environmental factors, physiologic and pathologic blood loss, as well as excessive alcohol consumption, need to be considered. As an antosomal-recessive disorder, HH occurs with equal frequency in both sexes. Yet it is more frequently diagnosed in men and appears to be underexpressed in women. 21 This has been explained by the physiologic blood loss through menstruation and the increased iron requirements during pregnancy. It has also been speculated that regular blood donation can influence the course of the disease. 22 The role of excessive alcohol consumption has been controversial for many years. It now seems estab-

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lished that alcohol in itself does not result in significant iron overload but can contribute independently to organ damage and therefore increase the severity of the phenotype. In this context the liver is particularly affected. The prevalence of cirrhosis is higher in patients with HH who consume more than 80 mg of ethanol per day, and consequently their long-term survival is decreased. 23 Also, liver disease with severely compromised liver function is rare in HH and, if present, usually indicates concomitant alcohol abuse. Patients should therefore be advised to drink alcohol only moderately, but no other specific dietary restrictions seem to be indicated. 24 So far no genetic factors other than mutations in HFE have been identified that affect the phenotypic expression of HH. However, the fact that 15% to 20% of patients with HH do not have mutations in this gene indicates that mutations in other genes causing iron overload are likely to exist. DIAGNOSIS

The diagnosis of HH is made in a number of different clinical circumstances. Patients can be found to have signs and symptoms suggestive of the disease. In these patients some degree of organ damage caused by iron overload is usually present. Increasingly, however, the diagnosis is made during family studies of affected individuals or incidentally, either on the basis of abnormal iron parameters or by finding minor liver dysfunction. Finally, patients can be identified by screening programs in asymptomatic individuals. These patients are often detected in the pre-cirrhotic stage of the disease. Early diagnosis and the application of genetic markers have profoundly changed our concept of the symptomatology of HH. It has become apparent that HH can present with a number of often nonspecific symptoms and that the classic triad of liver disease, skin pigmentation, and diabetes mellitus is only at one end of a wide phenotypic spectrum. Early recognition of these symptoms as a manifestation of HH is of paramount importance to prevent the often irreversible organ damage, in particular liver cirrhosis. In the following section some of the less-well-known symptoms of HH and the available methods to confirm the diagnosis, including the role of the genetic testing, will be discussed. Widening clinical spectrum of HH. Three large recent studies have shown that weakness and lethargy are among the most common initial symptoms of HH and can be present as the sole complaints.212526 We have seen several patients over the last 2 years who suffered from unexplained weakness and lethargy. The only abnormal laboratory result was a moderately increased serum ferritin level. A further diagnostic work-up, either by liver biopsy or by testing for the C282Y muta-

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tion of HFE, lead to the diagnosis of HH. Removal of the excess iron by regular phlebotomy treatment improved their well being. This observation is in accord with the literature, which describes an improvement in lethargy in the majority of patients after treatment. 26,27 In our view this important finding makes the exclusion of early idiopathic hemochromatosis mandatory in patients who are considered to suffer from chronic fatigue syndrome. Another nonspecific symptom of HH is abdominal pain. The frequency as a presenting symptom has varied from 10% to 20% and up to 50% in different series, and the cause has frequently remained unclear.Z5, 26 Other presenting symptoms that are particularly common in younger homozygotes include arthralgia and sexual dysfunction. Arthropathy usually affects the small joints of the hand, but larger joints can also be involved. It is an important determinant of the quality of life in patients with HH 28 and often progresses even after initiation of treatment. Diminished sexual function can present as loss of libido, impotence, amenorrhea, and testicular atrophy. 25 In the majority of cases the search for hemochromatosis is prompted by abnormal laboratory results--either increased liver enzymes or abnormal iron parameters. Hepatomegaly, arthralgia, or abnormal skin pigmentation are less common initial clinical signs. 25 HH should also be included in the differential diagnosis of unclear liver disease, diabetes, and cardiomyopathy or cardiac arrhythmias. Despite an increased awareness of the frequency of HH and its variable clinical manifestations, the delay between the development of symptoms and the diagnosis is still very long. In a recent study it was found to be 4 years in women and 7 years in men. 25 Laboratory diagnosis of HH. Serum transferrin saturation, serum ferritin level, liver biopsy, and genetie analysis--either by HLA typing or mutation analysis of H F E - - a r e the most-valuable tests for the diagnosis of HH. For the interpretation of these tests and their use in screening programs, it is important to consider the pathophysiology of the disease. H H is characterized by an inappropriately high absorption of iron from the small intestine. This leads to an increase in circulating iron that is bound to serum transferrin and thus leads to an increase in transferrin saturation. After a variable time, usually in patients over 20 years of age, iron overload develops preferentially in the parenchymal organs, with relative sparing of the reticuloendothelial system. Therefore, transferrin saturation will be the first test to show abnormal results in patients with HH, and it is usually recommended as an initial screening test. 29,30 There are, however, several problems with this test. First, the determination of serum iron is affected by several biologic and analytic factors and must be repeated to confirm abnormal results. Second, the re-evaluation

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of serum transferrin saturation in patients with HH when using the presence of the homozygous C282Y mutation as a gold standard identified only 90% of homozygotes. In the same study, serum ferritin had a higher sensitivity of 96%. ~ Thus almost 10% of homozygotes would be missed if the transferrin saturation were used alone as an initial screening test. Third, transferrin saturation will be abnormal in individuals in whom significant iron overload has not yet developed and in some of those in whom it never will develo p - f o r example, heterozygotes. In one study, 12% of heterozygous relatives of probands had an increased transferrin saturation. 3a Other methods (eg, serum ferritin or a liver biopsy) are required to document iron overload. The serum ferritin levels correlate well with hepatic iron stores, and in homozygotes they increase with age. 33 In untreated patients, high levels in the range of several thousand micrograms per liter are observed. However, much lower levels that are only slightly abnormal can also be found. Indeed, genotyping has shown that the homozygous C282Y mutation of HFE can be found in patients who have only a slightly elevated serum transferrin level and relatively low iron body stores. 34 Therefore the possibility of hemochromatosis must also be considered in patients that have only a moderately elevated serum ferritin level. The current recommendation is that serum ferritin values above 400 gg/L in men and 200 gg/L in women require a further work-up. 24 When using serum ferritin assays with a different normal range, we would recommend further investigation in patients who have a serum ferritin level of more than 150% of the upper limit. Although the serum ferritin is a valuable test, problems of specificity exist, and the results must be interpreted within the clinical context. Serum ferritin levels can be inappropriately increased and might not reflect the actual iron body stores under a number of conditions, among them infection, malignancies, hemolysis, and liver diseaseY It needs to be kept in mind that serum ferritin is an acute phase reactant. Simultaneous determination of the C-reactive protein is therefore always done in our clinic to exclude inflammatory or infectious causes of an elevated serum ferritin level. This policy helps in directing further investigations in these patients. Chronic alcohol abuse with liver damage can by itself cause elevated serum ferritin levels, but these are usually slightly increased and accompanied by relatively higher levels of liver enzymes. In patients with HH, alanine aminotransferase and aspartate aminotransferase are usually less than two times above the normal range, even in patients with cirrhosis, 35 because there is a relatively small degree of liver necrosis associated with hemochromatosis. Both serum ferritin (which is preferred by us as a primary

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Screening

General Population? Family

C282Y +/-

C282Y +/+

C282Y -/-

\ Ferdtinl"

Ferritin--~

Ferritinl"

Liverbiopsy

Phlebotomy

Ferdtin---~

H63D? Liverbiopsy?

Monitor (-1 y.)

Phlebotomy

No follow up

l

Followup (~5 y.)

Fig 1. Possible role of genetic testing for HFE mutations in the screening for HH. Plus sign indicates a mutated allele; minus sign indicates a normal allele, y, Years.

screening test) and transferrin saturation are not infallible in distinguishing between homozygous and heterozygous relatives of index patients, 36 but this distinction will now be simplified by genetic testing. Although an otherwise unexplained increase in the serum ferritin concentration is an important lead to the diagnosis of HH, it requires further confirmation. To date, liver biopsy with a histologic determination of the degree and the distribution of hepatic iron--performed with a biochemical/biophysical quantification of the hepatic iron stores--is still considered the gold standard for confirming the diagnosis. Liver biopsy is also important to determine the absence or presence of cirrhosis, the most important prognostic factor of the disease. 26 In the case of cirrhosis, the likelihood of the development of a hepatoma is increased substantially, and the efforts to screen for this complication need to be intensified. On the Perl's iron stain, the liver biopsy of a patient with hemochromatosis typically shows grade 2 through grade 4 stainable parenchymal iron, predominantly in the periportal hepatocytes. Iron staining in the Kupffer cells is usually absent unless iron overload is severe. 3° Measurement of liver iron content by biochemical or biophysical methods is another important aspect of the liver biopsy. A liver iron concentration higher than 80 gmol/g dry weight or a hepatic iron index (hepatic iron in gmol/g dry weight/age in years) above 2 is considered reliable for distinguishing between hemochro-

matosis and other causes of increased liver iron--for example, alcoholism or viral hepatitis.37, 38 However, these criteria need to be re-evaluated now that a precise diagnosis can be made by genotyping. For example, a recent study determining the hepatic iron index in homozygous relatives of index cases (identified by HLA typing) found an index above 2 in only 93% of cases. 39 Another study has shown that in patients homozygous for the C282Y mutation, a hepatic iron index below 2 can be observed. 34 Therefore, it is recommended today that the diagnosis of HH should be based on genetic findings in conjunction with a liver biopsy. Role of genetic testing for the diagnosis of HH. An

important question arises with the observation that the vast majority of cases of HH are caused by a single mutation in a single gene. Can genetic testing replace liver biopsy as a confirmatory test for the diagnosis of HH? As genetic testing becomes more widely used, it is likely that the role of the liver biopsy will shift from a diagnostic to a staging procedure. Whether liver biopsy is used during the diagnostic process depends on the severity of symptoms, the laboratory results, and the ethnic background of the patient. Liver biopsy should still be performed in a patient with suspected HH who has no mutation of HFE, since the diagnosis is not ruled out. Depending on the ethnic background, 15% to 20% of patients with HH may not harbor a mutation of the

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gene. If a homozygous C282Y mutation of HFE is found, the liver biopsy is probably not needed to confirm the diagnosis and to start phlebotomy treatment. However, liver biopsy is still important to detect the presence or absence of cirrhosis, which heralds an increased risk of developing hepatoma. This is important, since phlebotomy treatment does not reduce the risk of hepatoma if a patient already has cirrhosis. 26 Therefore, at the present time we also offer a liver biopsy in patients homozygous for the C282Y mutation, particularly if they are young and have laboratory evidence of liver disease or a high serum ferritin level. Genetic testing for the C282Y mutation will also be very useful in distinguishing HH from other conditions associated with iron overload that sometimes cause diagnostic problems, such as alcoholic liver disease, viral hepatitis, or the heterozygous state for HH. SCREENING Screening of relatives of an index patient, The avail-

ability of a relatively simple genetic test will have its most immediate impact on family screening. In each newly diagnosed case of HH, screening of all firstdegree relatives is mandatory. This was usually done by HLA typing to identify putative homozygotes, heterozygotes, and normals by analysis of how many haplotypes were shared with the affected sibling. Most homozygous relatives are siblings, but vertical transmission can also occur as a result of homozygous/heterozygous matings. In one recent study, 11 of 255 children (4%) of index cases were found to be homozygous. 40 This rate is about 10 times higher than would be expected in the general population, and therefore screening of children of homozygous patients may be indicated. H ~ A typing can result in the misclassification of homozygotes as heterozygotes because of rare recombination events or in the case of homozygous/heterozygous matings. Now family screening can be done more precisely and probably more cost-effectively by testing for the C282Y mutation if this is present in the index patient. This test can easily distinguish between homozygous, heterozygous, and unaffected relatives, independent of age. Although genetic testing is important for family screening, it does not allow one to assess whether iron overload is present. Therefore measurement of serum ferritin levels should also be included in family screening, because clinical management is largely determined by the result of this test (Fig 1). Homozygous relatives of an index patient with normal serum ferritin levels should be monitored at yearly intervals, because significant iron overload is very likely to develop in these individuals in the future. Once the serum ferritin level becomes abnormal, phlebotomy treatment is initiated to keep the serum ferritin level in the low

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normal range. In homozygous relatives that have a significantly increased serum ferritin level on the initial examination, liver biopsy should be offered to asses the presence or absence of cirrhosis. In these patients treatment is started immediately. In heterozygous relatives, the serum ferritin level should also be determined. In most cases serum ferritin levels will be normal or only slightly increased. 41 There is, however, the possibility that hemochromatosis can also develop in these individuals. For instance, they might be compound heterozygotes for the C282Y and H63D mutations who are at an increased risk of developing toxic iron levels. 14 Furthermore, they might harbor alterations in other, yetunknown genes that could cooperate with the heterozygous C282Y mutation in causing iron overload. Therefore a liver biopsy and treatment should also be considered in heterozygous relatives of an index patient who have high serum ferritin levels. Heterozygous relatives with normal ferritin levels can be monitored at longer intervals (5 years). Population screening. As mentioned above, HH is one of the most common genetic disorders, with a prevalence of 3 to 5 per 1000. In contrast to many other genetic diseases, a simple and effective therapy is readily available and life expectancy is normal if the disease is detected in the pre-cirrhotic state. For these reasons widespread population screening for HH seems an attractive option and has been advocated based on several pilot studies, but so far only the screening of relatives of an index patient is widely accepted. 29 These studies were hampered by the fact that the distinction between homozygotes and heterozygotes is often difficult to make when using biochemical assays. Iron studies can be abnormal in both groups, and there can be substantial overlap. Furthermore, because of the age dependence of the iron studies, there is a chance that young homozygotes are missed in these screens. New interest in population screening will certainly come from the possibility of genetic screening for HH that can avoid the shortcomings of biochemical testing. A number of questions must be addressed before widespread genetic testing can be recommended. First, the cost-effectiveness of a genetic screening program depends on the prevalence and penetrance of a mutation. Therefore more data on the frequency of the C282Y mutation in different populations and on genotype/phenotype correlations are needed. Second, as in the case of other genetic screening programs, insurance discrimination based on test results is a concern. 42 It must be made certain that individuals homozygous for the C282Y mutation detected in the pre-cirrhotic state receive health and life insurance at standard rates, since treatment results in normal life expectancy. Third, screening for asymptomatic homozygotes will identify a large number of potential blood donors. A consensus has

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to be reached whether the blood removed in phlebotomy programs can be used for blood donation. As a result of these concerns, an expert panel from the Centers for Disease Control and the National Institute of Human G e n o m e Research has recently concluded that widespread genetic screening for HH cannot be recommended at the present time. 43 Screening in patients with manifestations of H H - - f o r example, diabetes m e l l i t u s - could be a valuable alternative to widespread population screening. 44 THERAPY

The simple and safe therapy for H H remains the same. Since the introduction o f p h l e b o t o m y treatment for HH, it has been r e c o m m e n d e d that an induction p h l e b o t o m y to the point o f mild iron depletion be performed, f o l l o w e d by life-long m a i n t e n a n c e therapy. After serum ferritin measurements have become available, this parameter has been most widely used to guide therapy, but no target ferritin levels have been defined in clinical studies. B a s e d on the observation that the transferrin saturation and the iron serum concentration normalize only after the serum ferritin level has fallen into the low normal range, one could make a theoretical argument to aim for low normal serum ferritin levels. If one assumes that the free iron is doing the damage, it seems prudent to continue therapy until a frankly normal iron status is achieved. Therefore, in the absence of well-defined target ferritin levels, we perform weekly removal of 500 ml of b l o o d until the serum ferritin level is in the lower n o r m a l range. Once this has occurred, maintenance therapy is begun that consists of phlebotomies approximately every 3 to 6 months. This therapy and the appropriate controls need to be continued for life. They should include regular screening for the d e v e l o p m e n t o f hepatoma, particularly if the liver b i o p s y has shown cirrhosis. A s i d e from the very u n c o m m o n c i r c u m s t a n c e in which h e m o c h r o m a t o s i s and a limited bone marrow reserve coexist (eg, in patients after bone marrow transplantation or under m y e l o t o x i c therapy), there is in our view no role for iron-chelating agents such as d e f e r o x a m i n e or L1 (deferiprone) in the therapy o f HH. These agents are much m o r e expensive than p h l e b o t o m y and have a number o f substantial side effects. Furthermore, there can be complications in relation to their intravenous or subcutaneous application. OUTLOOK

The isolation of a h e m o c h r o m a t o s i s candidate gene has been an important advance in understanding and managing HH. One major challenge will be to identify the genetic basis o f iron o v e r l o a d in families who do not have a mutation o f HFE. Observations in these

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patients indicate that there could be another gene on the short arm o f c h r o m o s o m e 6 that might be associated with HH. 9,45 Another interesting question that needs to be investigated is whether h e t e r o z y g o s i t y for the C282Y mutation might influence the course of other diseases. 46 It has been speculated that mild iron overload might negatively affect viral hepatitis and its response to interferon therapy. 47 Also, there have been claims that increased iron stores increase the risk for coronary heart disease.48, 49 So far an association has been d e s c r i b e d b e t w e e n p o r p h y r i a cutanea tarda and H F E mutation C282Y. 50 These results were confirmed in several subsequent studies, 51 but the association was not found in a study from Italy that showed a surprising association with the H63D mutation. 45 This unexpected observation highlights the fact that despite major advances in our understanding of the genetic basis and the p a t h o p h y s i o l o g y of HH, m a n y questions remain. Over the next few years, however, we can expect new insights into iron m e t a b o l i s m as well as new information about the capability of current and new laboratory tests to detect and screen for this common disease. REFERENCES

l. Simon M, Bourel M, Fauchet R, Genetet B. Association of HLA A 3 and HLA B 14 antigens with idiopathic hemochromatosis. Gut 1976;17:332-4, 2. Feder JN, Gnirke A, Thomas W, et al. A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet 1996;13:399-408. 3. Collins FS. Positional cloning moves from perditional to traditional. Nat Genet 1995;9:347-50. 4. Bodmer JG, Parham R Albert ED, Marsh SG. Putting a hold on "HLA-H". The WHO Nomenclature Committee for Factors of the HLA System. Nat Genet 1997;15:2345. 5. Lebron JA, Bennet MJ, Vaughn DE, et al. Crystal structure of the hemochromatosis protein HFE and characterization of its interaction with transferrin receptor. Cell 1998;93:111-23. 6. Jazwinska EC, Cullen LM, Busfield F, et al. Haemochromatosis and HLA-H. Nat Genet 1996; 14:249-51. 7. The UK Haemochromatosis Consortium. A simple genetic test identifies 90% of UK patients with haemochromatosis. Gut 1997;41:841-4. 8. Jouanolle AM, Fergelot R Gandon G, Yaouanq J, Le Gall JY, David V. A candidate gene for hemochromatosis: frequency of the C282Y and H63D mutations. Hum Genet 1997;100:5-6. 9. Carella M, D'Ambrosio L, Totaro A, et al. Mutation analysis of the HLA-H gene in Italian hemochromatosis patients. Am J Hum Genet 1997;60:828-32. 10. Beutler E, Gelbart T, West C, et al. Mutation analysis in hereditary hemochromatosis. Blood Cells Mol Dis 1996;22:187-94. 11. Barton JC, Shih WW, Sawada Hirai R, et al. Genetic and clinical description of hemochromatosis probands and heterozygotes: evidence that multiple genes linked to the major histocompatibility complex are responsible for hemochromatosis. Blood Cells Mol Dis 1997;23:135-45.

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