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Update on Hereditary Hemochromatosis and the HFE Gene

      Hereditary hemochromatosis (HHC) is the most common inherited single gene disorder in people of northern European descent. Hereditary hemochromatosis is characterized by increased intestinal absorption of iron leading to its deposition into multiple organs. The classic description of HHC is bronze diabetes in a patient with cirrhosis. Hereditary hemochromatosis is increasingly being diagnosed at an earlier, less symptomatic stage. Diagnosis is based on an elevated fasting early morning transferrin saturation. Treatment is by phlebotomy, which, if initiated before the development of cirrhosis or diabetes, is associated with a normal life expectancy. Recently, a gene associated with HHC was discovered and named HFE. Two point mutations of this gene have been referred to as C282Y and H63D. Several US and European studies have found that 60% to 93% of patients with suspected HHC are homozygous for C282Y. Positive results of HFE gene testing may eliminate the need for a liver biopsy in selected cases. The greatest utility otHFE gene testing will likely be in screening family members of an identified proband and in helping to resolve ambiguous cases.

      Abbreviations:

      HHC (hereditary hemochromatosis), ORs (odds ratios), PCT (porphyria cutanea tarda)
      Hereditary hemochromatosis (HHC) is an autosomal recessive disorder associated with increased intestinal absorption of iron and deposition of excessive amounts of iron into the liver, pancreas, and other organs. Hereditary hemochromatosis is the most common genetic disorder in the US white population. Approximately 1 in every 250 to 300 persons is homozygous for the hemochromatosis mutation, and at least 1 in every 10 persons is a carrier for the mutation. Most physicians will encounter only a few cases of HHC in a lifetime of clinical practice, probably because they are not testing their patients for iron overload and because not all patients with HHC will have disease manifestations. Hereditary hemochromatosis should be distinguished from secondary iron overload, which includes entities such as anemia with ineffective erythropoiesis and iron overload due to chronic liver disease of various causes as well as factors such as multiple blood transfusions and parenteral or oral iron supplements.

      CLINICAL FEATURES

      The classic description of HHC was that of bronze diabetes or hyperpigmentation, diabetes mellitus, and cirrhosis of the liver. This description emphasized that, before the incorporation of serum iron studies on many chemistry panels, HHC was usually diagnosed only at an advanced stage. Clinical features include fatigue, hyperpigmentation, hepatomegaly, arthropathy, hypogonadism, hypothyroidism, elevated serum aminotransferase values, cirrhosis, diabetes, and cardiomyopathy. Because HHC can present with various disease manifestations, clinicians must have a high index of suspicion in patients with any of these disorders. Recent studies indicate that HHC is being diagnosed earlier, and as a result approximately 75% of patients are asymptomatic and have neither cirrhosis nor diabetes.
      • Bacon BR
      • Sadiq SA
      Hereditary hemochromatosis: presentation and diagnosis in the 1990s.

      DIAGNOSIS

      A diagnosis of HHC is suggested by an elevated transferrin saturation (100 x [serum iron concentration ÷ total iron binding capacity]) and ferritin level. There is diurnal variation in serum iron values, and measurements may be affected by the ingestion of food; therefore, an elevated transferrin saturation should be repeated as a fasting early morning determination. An elevated transferrin saturation is the earliest phenotypic abnormality in HHC. Debate exists about the appropriate cutoff for screening. A recent report from Australia used statistical modeling in populations with and without HHC and found that a transferrin saturation threshold of 45% identified 98% of homozygotes without misidentification of any healthy persons who were not heterozygous carriers.
      • McLaren CE
      • McLachlan GJ
      • Halliday JW
      • et al.
      Distribution of transferrin saturation in an Australian population: relevance to the early diagnosis of hemochromalosis.
      Although transferrin saturation is the best initial screening test, results may be normal early in the course of HHC. In addition, the serum ferritin level and transferrin saturation are often elevated in patients with acute or chronic viral hepatitis, alcoholic liver disease, and nonalcoholic steatohepatitis.
      Hemochromatosis is a disease for which screening may be appropriate. Hereditary hemochromatosis is much more common than many clinicians think; a simple, inexpensive, minimally invasive test is available to allow detection at an early stage, and treatment positively influences outcome. Several studies have demonstrated that screening the general population by determining the transferrin saturation would be cost-effective.
      • Balan V
      • Baldus W
      • Fairbanks V
      • Michels V
      • Burritt M
      • Klee G
      Screening for hemochromatosis: a cost effectiveness study based on 12,258 patients.
      • Phatak PD
      • Guzman G
      • Woll JE
      • Robeson A
      • Phelps CE
      Cost-effectiveness of screening for hereditary hemochromatosis.
      • Adams PC
      • Gregor JC
      • Kertesz AE
      • Valberg LS
      Screening blood donors for hereditary hemochromatosis: decision analysis model based on a 30-year database.
      Normalization of total body iron stores may diminish many of the clinical features of HHC, including diabetes and cardiomyopathy. A landmark study found that survival of HHC patients was the same as that of age- and sex-matched controls without HHC if treatment was initiated before the development of cirrhosis or diabetes.
      • Niederau C
      • Fischer R
      • Sonnenberg A
      • Stremmel W
      • Trampisch HJ
      • Strohmcyer G
      Survival and causes of death in cirrhotic and in noncirrhotic patients with primary hemochromatosis.
      Despite this evidence, none of the major medical organizations have endorsed systematic population screening for HHC.

      TREATMENT

      Treatment of patients with HHC and evidence of iron overload is by phlebotomy, removal of 500 mL of blood on a weekly basis until the hemoglobin concentration is less than the reference range. Once the hemoglobin concentration is lower than the reference range for 3 consecutive weeks without phlebotomy, the transferrin saturation and ferritin level are reassessed. Iron depletion is confirmed if the ferritin level is not greater than 50 μg/L with a subnormal transferrin saturation. Most patients with HHC will not have iron deficiency until at least 5 g of iron has been removed (500 mL of blood = 250 mg of iron), and many with advanced disease have total body iron stores greater than 20 g. Once iron depletion is accomplished, lifelong maintenance phlebotomy should be initiated. Most patients require 4 to 8 phlebotomies per year to keep the ferritin level lower than 50 Ug/L. Both the transferrin saturation and the ferritin level should be determined at least annually to allow adjustments in the frequency of maintenance phlebotomy.

      The HFE Gene

      Recently, a gene associated with HHC was discovered
      • Feder JN
      • Gnirke A
      • Thomas W
      • et al.
      A novel MHC class Iike gene is mutated in patients with hereditary haemochromatosis.
      and was initially named HLA-H; it has since been renamed HFE. This gene, located on the short arm of chromosome 6, contains 1029 base pairs that encode a 343 amino acid protein, similar in structure to major histocompatibility complex class I proteins.
      Two point mutations in the HFE gene were initially identified. The first is a mutation at nucleotide 845 in which adenine is substituted for guanine, resulting in a tyrosine substitution for cysteine at amino acid position 282 (C282Y). This mutation disrupts a disulfide bond at the binding site for β2-microglobulin. This may have clinical relevance because mice deficient in β2-microglobulin develop parenchymal iron overload in a pattern similar to that observed in HHC.
      • Rothenberg BE
      • Voland JR
      β2 knockout mice develop parenchymal iron overload: a putative role for class I genes of the major histocompatibility complex in iron metabolism.
      The second mutation is a guanine substitution for cytosine at nucleotide 187, resulting in an aspartate substitution for histidine at amino acid position 63 (H63D). This amino acid substitution is in the peptide-binding domain of the molecule. More recently, 2 other mutations were described, but these are rare and are not likely of major clinical importance.
      • Wallace DF
      • Dooley JS
      • Walker AP
      A novel mutation of HFE explains the classical phenotype of genetic hemochromatosis in a C282Y hetérozygote.
      • Mura C
      • Raguenes O
      • Ferec C
      HFE mutations analysis in 711 hemochromatosis probands: evidence for S65C implication in mild form of hemochromatosis.

      Clinical Importance of the HFE Gene

      In the initial study that reported the discovery of the HFE gene, the frequencies of the C282Y and H63D mutations were determined in 178 US patients with HHC and in 155 controls.
      • Feder JN
      • Gnirke A
      • Thomas W
      • et al.
      A novel MHC class Iike gene is mutated in patients with hereditary haemochromatosis.
      The C282Y homozygous state was observed in 83% of those with suspected HHC and was not present in any of the controls. Of 9 patients with HHC who were heterozygous for C282Y, 8 were also heterozygous for H63D (compound heterozygote), whereas there were no compound heterozygotes among the 10 C282Y heterozygotes from the control group. In addition, 12 (6.7%) of 178 patients with clinically diagnosed HHC had no HFE mutation.
      In several other studies in the United States and Europe,
      • Burke W
      • Thomson E
      • Khoury MJ
      • et al.
      Hereditary hemochromatosis: gene discovery and its implications for population-based screening.
      • Brandhagen DJ
      • Fairbanks VF
      • Baldus WP
      • Smith CI
      • Kruckeberg KE
      • Thibodeau SN
      Prevalence of HFE gene mutations in normal blood donors and in patients with iron overload [abstract].
      60% to 93% of patients with iron overload were homozygous for C282Y. Comparing these studies is difficult, however, because each used different criteria for the diagnosis of HHC. Of importance, almost all studies have described patients with clinically meaningful iron overload in the absence of any HFE gene mutations.
      The importance of HFE mutations other than the C282Y homozygous state is controversial. The risk of the development of iron overload for individual HFE mutations is unknown. To aid in quantifying this risk, we pooled data from our study and 8 other US and European studies to calculate the odds ratios (ORs) for the development of iron overload for each genotype compared with the homozygous wild type.
      • Brandhagen DJ
      • Fairbanks VF
      • Baldus WP
      • Smith CI
      • Kruckeberg KE
      • Thibodeau SN
      Prevalence of HFE gene mutations in normal blood donors and in patients with iron overload [abstract].
      Homozygosity for C282Y was associated with the greatest risk with an OR of 2300, followed by compound heterozygotes (OR = 49.4), H63D homozygotes (OR = 6.3), C282Y heterozygotes (OR = 3.1), and H63D heterozygotes (OR = 1.6). These findings are consistent with those from several other studies that have estimated low penetrance (about 1.5%) for both the compound hétérozygote and the homozygous H63D genotype. In addition, several studies have confirmed that H63D is more common in patients with iron overload compared with controls when C282Y-positive chromosomes are excluded from analysis.
      • Fairbanks VF
      • Brandhagen DJ
      • Thibodeau SN
      • Snow K
      • Wollan PC
      H63D is an haemochromatosis associated allele [letter].
      • Beutler E
      The significance of the 187G (H63D) mutation in hemochromatosis [letterl.
      • Risch N
      Haemochromatosis, HFE and genetic complexity [letter].
      The exclusion of C282Y chromosomes is important in this type of analysis because the C282Y and the H63D mutations do not occur on the same chromosome.

      Functional Importance of the HFE Protein

      Several recent studies have begun to explore the functional importance of the HFE protein. One study using anti-HFE antibodies showed that the HFE protein is normally present in most human cells.
      • Parkkila S
      • Waheed A
      • Britton RS
      • et al.
      Immunohistochemistry of HLA-H. the protein defective in patients with hereditary hemochromatosis, reveals unique pattern of expression in gastrointestinal tract.
      Interestingly, however, the HFE protein in the small bowel was localized in the crypts near the nuclei of epithelial cells, whereas it was expressed on the plasma membrane elsewhere throughout the alimentary tract. This unique localization of the HFE protein in the small bowel, where iron is absorbed, suggests a fundamental role for this protein in regulating iron absorption. Another study demonstrated that wild-type HFE and H63D protein bind to β2-microglobulin, but the C282Y protein does not.
      • Feder JN
      • Tsuchihashi Z
      • Icrinki A
      • et al.
      The hemochromatosis founder mutation in HLA-H disrupts β3-microglobulin interaction and cell surface expression.
      The observation that the C282Y protein does not bind to β2-microglobulin is relevant because of the studies that have shown that β2-microglobulin-deficient mice develop iron overload in a pattern similar to that seen in HHC.
      • Rothenberg BE
      • Voland JR
      β2 knockout mice develop parenchymal iron overload: a putative role for class I genes of the major histocompatibility complex in iron metabolism.
      Finally, the most convincing evidence of the functional importance of HFE proteins comes from a recent study that used an HFE gene “knockout” mouse model.
      • Zhou XY
      • Tomatsu S
      • Fleming RE
      • et al.
      HFE gene knockout produces mouse model of hereditary hemochromatosis.
      Mice deficient in HFE proteins developed iron overload in a pattern similar to that observed in HHC. Although the exact mechanism of the regulation of iron absorption by the HFE protein remains to be elucidated, these studies provide strong support for the functional importance of the HFE proteins.

      HFE Gene Mutations in Other Liver Diseases

      Results of serum iron studies are frequently abnormal in patients with viral hepatitis, alcoholic liver disease, and other chronic liver conditions. Because iron may cause free radical-induced tissue injury, excess iron may adversely influence disease outcome in these conditions. Therefore, several studies have examined the role of HFE gene mutations in other chronic liver diseases associated with iron overload. Several investigators have examined the importance of HFE gene mutations in porphyria cutanea tarda (PCT) and have reached different conclusions. A British study
      • Roberts AG
      • Whatley SD
      • Morgan RR
      • Worwood M
      • Elder GH
      Increased frequency of the haemochromatosis Cys282Tyr mutation in sporadic porphyria cutanea tarda.
      found an increased prevalence of C282Y in PCT patients, whereas an Italian study
      • Sampietro M
      • Pipemo A
      • Lupica L
      • et al.
      High prevalence of the His63Asp HFE mutation in Italian patients with porphyria cutanea tarda.
      found an association between PCT and H63D. A recent study of North American patients with PCT found that 73% had HFE mutations with an increased prevalence of both C282Y and H63D.
      • Bonkovsky HL
      • Poh-Fitzpatrick M
      • Pimstone N
      • et al.
      Porphyria cutanea tarda, hepatitis C, and HFE gene mutations in North America.
      Another study examined the clinical importance of HFE mutations in 51 patients with nonalcoholic steatohepatitis.
      • George DK
      • Goldwurm S
      • MacDonald GA
      • et al.
      Increased hepatic iron concentration in nonalcoholic steatohepatitis is associated with increased fibrosis.
      The C282Y mutation was significantly associated with the intensity of Perls stain, hepatic iron concentration, and transferrin saturation percentage. Linear regression analysis showed that increased hepatic iron closely correlated with severity of fibrosis. Two studies have confirmed that HFE gene mutations are not more common in patients with chronic viral hepatitis.
      • Smith BC
      • Grove J
      • Guzail MA
      • et al.
      Heterozygosity for hereditary hemochromatosis is associated with more fibrosis in chronic hepatitis C.
      • Piperrno A
      • Vergani A
      • Malosio I
      • et al.
      Hepatic iron overload in patients with chronic viral hepatitis: role of HFE gene mutations.
      In 1 of these studies, however, patients with chronic hepatitis C who were C282Y heterozygotes had more fibrosis and cirrhosis (40% vs 9%; P = .01) despite having only a mild increase in hepatic iron. Therefore, heterozygosity for C282Y or H63D may influence disease severity in chronic liver disease that is primarily due to other causes such as viral hepatitis or steatohepatitis.

      Use of HFE Gene Testing in Clinical Practice

      The role of HFE mutation analysis in the diagnosis of iron overload disorders is summarized in Figure 1. The gene test is most useful for screening adult family members of an identified proband and should replace more expensive and cumbersome HLA typing. In addition, HFE gene testing is useful in helping to resolve ambiguous cases. Whether it becomes appropriate for general population screening depends on its cost, although when automated, it can be done at no greater cost than the serum ferritin assay. Of importance, however, pronounced iron overload can occur without HFE mutations, and some patients with HFE mutations may not develop clinically important iron overload.
      Figure thumbnail gr1
      Fig. 1Screening algorithm for hereditary hemochromatosis (HHC). AST = aspartate aminotransferase; TS = transferrin saturation.
      HFE gene testing may eliminate the need for a liver biopsy in many cases. Traditionally, a liver biopsy was performed in patients with iron overload to confirm a diagnosis of HHC and to exclude cirrhosis. However, in patients with iron overload who are C282Y homozygotes, a liver biopsy may be unnecessary to confirm the diagnosis of HHC. A liver biopsy is still the “gold standard” for assessing the degree of fibrosis. Definitively excluding cirrhosis is important because hemochromatosis patients with cirrhosis have a greater than 200-fold increased risk of developing hepatocellular carcinoma. In such patients, screening with ultrasonography and α-fetoprotein tests every 6 months may be appropriate. There may be a subset of HHC patients whose risk of cirrhosis is minimal, and a liver biopsy would be unnecessary. A recent study confirmed that certain noninvasive predictors were accurate in excluding cirrhosis in C282Y homozygotes.
      • Guyader D
      • Jacquelinel C
      • Moirand R
      • et al.
      Noninvasive prediction of fibrosis in C282Y homozygous hemochromatosis.
      There were no cases of cirrhosis in 96 C282Y homozygotes with serum ferritin levels lower than 1000 μg/L, normal aspartate aminotransferase values, and absence of hepatomegaly. Although these clinical variables were able to predict the absence of cirrhosis accurately, they were not nearly as accurate in predicting the presence of cirrhosis.

      SUMMARY

      Hemochromatosis is the most common inherited disorder in people of northern European ancestry. Hereditary hemochromatosis may present with various disease manifestations, including cirrhosis and bronze diabetes. Recently, in more patients, the diagnosis has been made at an early, asymptomatic stage. Diagnosis is suggested by an elevated fasting first morning transferrin saturation confirmed on repeated determinations in association with an elevated ferritin level. Although liver biopsy has traditionally been necessary to confirm a diagnosis of HHC in patients with iron overload, HFE gene testing may be the most appropriate initial confirmatory test in some patients. The diagnosis of HHC is confirmed in patients with iron overload who are homozygous for C282Y and do not have an alternative explanation for their increased iron stores. The HFE gene test is also a useful adjunct for screening relatives of an identified proband and in helping to resolve ambiguous cases. Currently, performing HFE gene testing on the general population with no HHC risk factors does not seem appropriate or cost-effective. However, screening for HHC with transferrin saturation may be cost-effective. Treatment is weekly phlebotomy until iron stores are depleted, followed by lifelong maintenance phlebotomy approximately 4 to 8 times per year. Early diagnosis and treatment are crucial because many of the manifestations are reversible and survival is similar to that in persons without HHC if iron stores are normalized before the development of diabetes or cirrhosis.

      ACKNOWLEDGMENT

      We thank Linda D. Veer, Heidi M. Hettinger, and Gwen M. Boe for their help in the preparation of the submitted manuscript.

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