Section 4 -

Genetic Hemochromatosis Neil Theise, M.D. Romil Saxena, M.D.

Case 4A 42 year old man, hepatitis C positive; liver biopsy for grading and staging Diagnosis: Chronic hepatitis, moderately active with portal fibrosis (modified Ishak stage 1/4), compatible with hepatitis C. Hemosiderosis, grade 1/4 Comment: Patients with chronic hepatitis C infection often show evidence of iron overload in the form of elevations in serum ferritin and iron saturation. However, most of these patients do not have elevations of HIC; when an increase in HIC does occur, it is usually mild. The consequence of iron overload in chronic hepatitis C is two-fold. First of all, there may be more progression to fibrosis and cirrhosis. Secondly, these patients respond poorly to interferon therapy. Several studies (Smith, 1998; Bonkovsky, 2002; Erhardt, 2003) have shown an increased prevalence of the C282Y mutation in patients with chronic hepatitis C. These patients have higher serum iron saturations, more stainable iron and more advanced fibrosis at a younger age as compared to patients without the genetic mutation; this is especially true for those who are homozygous for C282Y. Whether the observations reflect the effect of iron on hepatic disease, or whether HFE mutations and iron overload in some way contribute to susceptibility for developing chronic hepatitis C after an acute infection is currently not known. Some studies have found a correlation between iron in the liver and degree of inflammation, as well as degree of fibrosis. The cellular distribution of iron correlates with response to therapy (Barton, 1995); the presence of stromal or endothelial iron seems to be especially important in this regard. Patients with hepatitis C who have high HIC respond poorly to standard short-acting interferon. These patients have been found to benefit from iron reduction therapy. Most show a drop in serum transminases; histological improvement, especially in the degree of inflammation, has been reported. Some studies have indicated an improvement in the fibrosis score in patients who underwent iron depletion. Current therapy for hepatitis C consists of a combination of long-acting pegylated interferon and ribavarin; results from randomized controlled trials on the benefits of iron depletion in this group are awaited. Case 4B: 44 year old man homozygous for genetic hemochromatosis Diagnosis: Hemosiderosis, grade 4/4, compatible with genetic hemochromatosis (homozygous by history) Comment: The identification of the HFE gene by positional cloning was the first step in the current explosion of knowledge of iron metabolism and its disorders. To date, multiple transport and regulatory proteins along with mutations in their genes have been identified, leading to a molecular classification of genetic or hereditary hemochromatosis (HH). The OMIM (Online Mendelian Inheritance in Man) database currently lists four types of HH: HFE associated hereditary hemochromatosis (type 1) C282Y homozygosity C282Y/H63D compound heterozygosity Non-HFE associated hereditary hemochromatosis Juvenile hemochromatosis (type 2) 2A hepcidin mutations, 2B hemojuvelin mutations Type 3 hemochromatosis (transferrin receptor 2 mutations) Type 4 hemochromatosis (ferroportin mutations, autosomal dominant hemochromatosis) The molecular genetics of hereditary hemochromatosis The gene called HFE encodes for a MHC class I-like molecule that is present ubiquitously on cell membranes, where it is expressed, like other MHC class I molecules, as a complex with b-2 microglobulin. HFE is expressed particularly strongly on hepatocytes and the cells in the deeper portions of duodenal crypts. Unlike MHC-class I molecules, the HFE protein lacks the peptide binding groove necessary for antigen presentation. The HFE/ b-2 microglobulin complex binds to transferrin receptor 1 (Tfr 1), a protein present ubiquitously on cell membranes that allows uptake of iron for cellular metabolism. The binding of HFE to Tfr results in decreased affinity of Tfr for diferric transferrin. The C282Y mutation disrupts the binding of HFE with b-2 microglobulin and decreases the surface expression of HFE. The H63D mutation does not affect the binding of HFE to b-2 microglobulin and does not decrease the surface expression of this complex. Although there is a strong relationship between HFE mutations and HH, the exact mechanism whereby mutations in this molecule cause iron overloading is not entirely clear. It is however becoming increasingly clear that the maintenance of normal iron homeostasis entails a complex interplay between numerous molecules and regulatory proteins, and mutations in at least 3 of these (hepcidin, hemojuvelin, Tfr 2) - in addition to HFE - are now known to cause iron overload similar to that seen with HFE mutations. Iron metabolism and homeostasis Ferric iron is first reduced in the alimentary tract by ferri-reductase or ascorbic acid to release ferrous iron. Transport of ferrous ions across the apical cell membrane is facilitated by a transporter called divalent metal transporter (DMT-1), also known as DCT1 (divalent cation transporter) or Nramp2 (Natural resistance associated macrophage protein 2). This molecule is highly expressed on enterocytes on the villus tips of the duodenum, the major site of iron absorption. After absorption, iron either egresses the cell through the basolateral membrane if required for body needs, or is stored within the cytoplasm as ferritin. The egress of iron out of the basolateral membrane of the cell is mediated by a membrane protein called ferroportin (also known as IREG 1 and MTP1). Another protein involved with egress of iron across the basolateral membrane is called hephaestin, a ceruloplasmin homolog. It is not certain if this protein acts in concert with, or independently from IREG1. In blood, iron circulates in blood tightly bound to transferrin. Each transferrin molecule binds 2 molecules of ferric iron, and normally 30% of the binding sites on transferrin are occupied. Transferrin binds to transferrin receptors which are present ubiquitously on cell membranes. Following interaction of Tf-Tfr, this complex is endocytosed within the cell. In the acidified endocytotic vesicle, iron is released and transported out of the vesicle into the cell cytoplasm or into endosomes by DMT1. Iron uptake and release from cells is tightly controlled and regulated by post-transcriptional control of expression of Tfr, DMT1, IREG1 and other genes. Hepcidin, a liver derived antimicrobial peptide is a repressor of iron absorption, and is increasingly thought to play a central role in the pathogenesis of most forms of HH. The gene encoding hepcidin is called HAMP and is located on 19q12. Hepcidin levels are greatly reduced in patients with type I HFE-associated HH as well as type 2A HH. Given the phenotypic similarity between type 2A and type 2B HH, it has been suggested that hemojuvelin may be a modulator of hepcidin. It has also been suggested that HFE and Tfr2 may be independent but complementary modulators of hepcidin, thus mutation in one gene may be compensated by the other in the presence of normal HAMP. Type I hereditary hemochromatosis Type I HH or HFE-associated HH is the commonest genetic disorder in Caucasians; it is inherited as an autosomal recessive disease, and is associated with missense point mutations in the HFE gene, which is located on chromosome 6. The commonest mutation is substitution of cysteine by tyrosine at site 282 (C282Y); the second commonest mutation is the substitution of aspartate for histidine at amino acid 63 (H63D). Both these mutations are common in people of Nordic and Celtic descent, and are uncommon in southern and Eastern European, Africa, Central and South America and Asia. At least one copy of the mutated C282Y gene is found in 10-20% of Caucasian subjects of North European descent, and the H63D mutation in 15-40%. The frequency of homozygosity for C282Y in people of North European descent is 0.5%; in other words every 1 person in 200 is homozygous for the C282Y mutation. In a large population based study of white residents of mostly Anglo-Celtic descent in Western Australia, 0.5% of the population was homozygous for the gene. Twelve percent of the population had the C282Y/ wild-type genotype and 2% were double heterozygotes (Olynk, 1999). Conversely, more than 90% of patients of North European descent with hereditary hemochromatosis are homozygous for the C282Y mutation. Screening for Hereditary Hemochromatosis (type I) An understanding of the genetic basis of HH and availability of molecular testing has provided the "gold standard" for diagnosis of the disease. However, the validity of universal routine screening at birth or early childhood is not established for a disease that is limited to a population group, and in which iron accumulation occurs progressively over time, and is modulated by host factors like consumption of alcohol. Thus, genetic testing is limited to subjects with abnormal biochemical tests found on screening, or as part of family screening. Screening for HH is based on biochemical tests, the most specific and sensitive of which is transferrin saturation; a serum transferrin saturation of 45% or higher identifies 98% of subjects with homozygous HH. The current policy is to perform serum transferrin and serum iron as screening tests in populations at risk, at an age when some degree of iron overload has developed but before serious organ damage has occurred. The role of the liver biopsy in hereditary hemochromatosis With the availability of molecular testing for HH, the role of liver biopsy in primary diagnosis is limited. However, till not very long ago, diagnosis was based largely on documentation of organ iron overload and HLA linkage analysis. The former included (1) grade 3 or 4 staining for iron on liver biopsy, (2) hepatic iron concentration (HIC) of >80 micromol (4500 micrograms)/g dry weight, (3) hepatic iron index (iron concentration/age) >1.9, or (4) removal of >5g of iron by phlebotomy without precipitating iron deficiency anemia. Currently, in the absence of non-invasive markers, the liver biopsy continues to provide valuable information on the degree of iron overload and the degree of fibrosis. A study based on multivariate analysis in C282Y homozygous patients found that there was no fibrosis in these patients if the serum ferritin was <1000 mg/L, there was no hepatomegaly and AST levels were normal; a liver biopsy is therefore not indicated in this group. Also, patients under 35 years of age detected through family screening never showed severe fibrosis; these persons had normal AST levels and serum ferritin <1000 mg/L. A separate study found that elderly patients with serum ferritin > 2000 mg/L may have high HIC and cirrhosis even in the face of normal liver tests; a liver biopsy is therefore indicated with serum ferritin levels >2000 mg/L (Fletcher, 2002). The relationship between iron overload and fibrosis is however not simple, and the absence of fibrosis in patients with severe iron overload suggests that iron by itself is not fibrogenic, and that complicating co-factors play an important role in fibrogenesis in the presence of iron overload. The role of co-factors in liver injury in hereditary hemochromatosis Alcohol consumption has emerged as an important co-factor in the progression of fibrosis in hereditary hemochromatosis; in one study, HH patients who consumed more than 60 grams of alcohol per day were 9 times more likely to develop cirrhosis even at modestly increased HIC than were HH patients who consumed less than 60 grams of alcohol per day. HIC was highest in those subjects with a heavy alcohol intake. Additionally, such patients developed cirrhosis at a younger age than HH patients who did not consume excess alcohol (Fletcher, 2002). In a study of C282Y homozygous relatives of probands with HFE-associated hemochromatosis, 36% of persons who consumed >60gms of alcohol had cirrhosis or advanced fibrosis (Powell, 2002). It is thus clear that alcohol accelerates the course of HH and its phenotypic expression. Alcohol consumption accentuates iron overload, and the two probably have an additive effect causing oxidative stress, hepatic stellate cell activation and hepatic fibrogenesis. Genotype Phenotype correlation: Homozygosity for the HFE gene predisposes to iron accumulation and potential for organ damage. However, this phenotype is not invariable and approximately a quarter of subjects who are homozygous for C282Y will not develop iron overload (Olynyk, 1999). A percentage does not even develop abnormal serum tests. Based on the gene frequency of H63D and C282Y mutations, one would expect to see many more people, both compound heterozygotes, as well as double the number of the C282Y homozygotes, with significant iron accumulation. This is not the case suggesting that penetrance of the C282Y homozygous genotype is not complete. The disease therefore has 4 stages: (1) genetic predisposition (2) iron overload (approximately 2-5 grams) but no symptoms (3) iron overload with early symptoms (lethargy, arthralgias) and (4) iron overload with organ damage. Progression from one stage to the other is not invariable, and the interval between the stages is highly variable; iron accumulation and progression of fibrosis in the liver is dependent on multiple variables which are as important, if not more important, than the accumulation of iron itself. Unlike other diseases like cystic fibrosis, disease expression in HH depends on genetic and environmental factors such as age, gender, amount of iron in the diet and factors that augment iron absorption or iron loss, and consumption of alcohol. Among patients with iron overload compatible with HH, 85-90% have homozygosity for C282Y. Another 3-5% are double heterozygotes for C282Y and H63D, and these patients have greater iron accumulation than the C282Y/ wild-type patients, suggesting a role for H63D in iron accumulation. Other forms of hemochromatosis Juvenile hemochromatosis, type 2A is caused by mutations in the HJV gene located at 1q21 that encodes for the protein, hemojuvelin. Type 2B HH is caused by mutations in the HAMP gene located on chromosome 19q13.1 that encodes for hepcidin. Both are inherited as autosomal recessive diseases. The earliest detectable biochemical abnormality is increased transferrin saturation. Parenchymal iron accumulation occurs in endocrine glands, heart and liver. The disease presents in the second or third decade of life, and death is commonly due to heart failure. Very little is known about type 3 HH. The gene is located on chromosome 7q22 and encodes for transferrin receptor 2. Type 3 HH is phenotypically similar to type I HH. Type 4 HH is inherited as an autosomal dominant disease. The gene, SLC 40A1, is located on chromosome 2q32 and encodes for ferroportin, a molecule involved in the export of iron out of parenchymal and reticuloendothelial cells. In the absence of functional ferroportin, as with mutations, iron accumulates within the reticuloendothelial cells of the liver and spleen, and to a lesser extent in hepatocytes and enterocytes. This decreases the plasma iron pool, as well availability of iron for erythropoiesis. The transferrin saturation is therefore low or normal, and patients may have borderline anemia, especially women and persons undergoing phlebotomy. Serum ferritin rather than transferrin saturation is the preferred test for early diagnosis, and it can be elevated as early as the first decade of life. This type of HH has the lowest propensity for organ damage. Suggested Reading: Case 4A Bonkovsky HL, Lambrecht RW, Shan Y. Iron as a co-morbid factor in nonhemochromatotic liver disease. Alcohol 2003; 30: 137-144. Eisenbach C, Gehrke SG, Stremmel W. Iron, the HFE gene and hepatitis C. Clin Liver Dis 2004; 8: 775-785. Ioannou GN, Tung BY, Kowdley KV. Iron in hepatitis C: villain or innocent bystander? Semin Gastrointest Dis 2002; 13: 95-108. George DK, Powell LW, Losowsky MS. The hemochromatosis gene: a co-factor for chronic liver diseases? J Gastroenterol Hepatol 1999; 14: 745-749. Case 4B Adams PC. 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