Heterozygosity for Hereditary Hemochromatosis Is Associated With More Fibrosis in Chronic Hepatitis C

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1 Heterozygosity for Hereditary Hemochromatosis Is Associated With More Fibrosis in Chronic Hepatitis C BELINDA C. SMITH, 1 JANE GROVE, 1 MUNA A. GUZAIL, 1 CHRISTOPHER P. D AY, 1 ANN K. DALY, 2 ALASTAIR D. BURT, 1 AND MARGARET F. B ASSENDINE 1 Hepatic iron has been associated with more aggressive liver disease in chronic viral hepatitis. We evaluated whether the recently described C282Y mutation of the hemochromatosis gene, designated HFE (responsible for at least 83% of hereditary hemochromatosis), was associated with more advanced liver disease in chronic hepatitis C. One hundred thirty-seven patients with biopsy-proven chronic hepatitis C were studied and liver biopsies scored for necroinflammation (grade 0-18) and fibrosis (stage 0-6). Genomic DNA was amplified by polymerase chain reaction and the C282Y mutation identified by restriction with RsaI and electrophoretic separation of restriction fragments. Ten (7.3%) patients had the C282Y mutation. No C282Y homozygous patients were identified. Age, sex distribution, and estimated weekly alcohol consumption were not significantly different between those with and without the mutation. Serum ferritin was higher in the heterozygotes (mean, 339 g/l) compared with homozygous wild types (153 g/l; P.0005). In the majority of patients, liver iron was graded 0 out of 4, but hepatocyte iron staining was more commonly present in heterozygotes compared with homozygous normals (30% compared with 4% [P.02]). Liver disease was more advanced in those with the mutant allele (mean fibrosis stage: 3.6, compared with wild type: 1.5 [P.01]). Cirrhosis was found more often in those with the mutation (40%) compared with those without (8.7%) (P.01; odds ratio: 7.6 [ ]). There was no significant difference in inflammation scores between heterozygotes and wild type (mean, 5.4 compared with 4.1). Hepatitis C virus (HCV)-RNA titers were measured by branched DNA assay (HCV RNA 2.0-Chiron), and there was no difference between heterozygous and homozygous normal patients. Thus, despite relatively minor increases in iron stores, individuals who are heterozygous for hemochromatosis appear to develop more fibrosis in chronic hepatitis C. Venesection may be useful therapy in this subgroup. (HEPATOLOGY 1998;27: ) Abbreviations: HCV, hepatitis C virus; HH, hereditary hemochromatosis; HIC, hepatic iron concentration; HCC, hepatocellular carcinoma. From the 1 Centre for Liver Research and 2 Department of Pharmacological Sciences, University of Newcastle, Newcastle upon Tyne, England. Received August 27, 1997; accepted February 9, Dr. Smith is supported by the Sir Jules Thorne Charitable Trust. Dr. Grove is supported by the Medical Research Council (UK). Address reprint requests to: Prof. M. F. Bassendine, Professor of Hepatology, Centre for Liver Research, 4th floor William Leech Building, The Medical School, University of Newcastle, Newcastle upon Tyne, NE2 4HH, UK. Fax: Copyright 1998 by the American Association for the Study of Liver Diseases /98/ $3.00/0 Hepatitis C virus (HCV) infection is persistent in the majority of infected individuals, and carriers develop chronic liver disease with approximately 20% developing cirrhosis after 20 years. 1-3 It is not clear why some infected individuals develop progressive disease and others have a more indolent course. There is, however, data to support a pathogenic role for iron in HCV. Serum markers of iron overload are frequently elevated in HCV, more often in HCV than other causes of chronic liver disease excluding hereditary hemochromatosis (HH). 4-6 Elevated hepatic iron concentration (HIC) has been associated with a poor response to interferon alfa, 7,8 but with an improved response after phlebotomy. Phlebotomy alone has also been shown to result in significant reduction in transaminase levels in patients with chronic HCV. 9 In addition, iron has been shown to modulate the course of hepatitis B. 10 Subjects with elevated serum iron and ferritin are more likely to be hepatitis B surface antigen carriers, and to develop hepatocellular carcinoma (HCC). 7,11-13 There is an increased prevalence of both hepatitis B and C markers in patients with hepatic iron overload, such as those with porphyria cutanea tarda, HH, and thalassemia. 11,14 HH, an autosomal recessive condition, causes excessive intestinal absorption of dietary iron, the lifelong accumulation of which results in its deposition in organs including the liver. In 1996, a candidate gene for HH, HLA-H, now redefined as HFE, 15 was cloned by Feder et al. 16 Two mutations of HFE have been identified: cdna nucleotide 845G=A (C282Y) and 187 C=G (H63D). 16 Jouanolle et al. 17 found that the C282Y mutation was responsible for 91% of HH in a group of well-characterized patients in France. Similarly, 91% of United Kingdom patients with HH were homozygous for the C282Y mutation, 18 as were all 112 HH patients tested in Australia. 19 The C282Y mutation, which is found in 6% to 11% of the U.K. population, 18,20 is in complete linkage disequilibrium with the H63D mutation. The H63D mutation is found in 16% to 25% of the population, 21 but its role, if any in HH, is controversial. It seems to be associated with hemochromatosis only when inherited together with the C282Y mutation. The compound heterozygote state seems to have low penetrance. 22 The discovery of the HFE gene has enabled examination of the heterozygote state of hemochromatosis to determine whether there is any associated morbidity with this mutation. We evaluated 137 patients with biopsy-proven chronic hepatitis C to determine if there was any link between heterozygosity for HFE at position 282 (C282Y) and severity of liver disease. 1695

2 1696 SMITH ET AL. HEPATOLOGY June 1998 PATIENTS AND METHODS One hundred thirty-seven white patients with chronic HCV were evaluated. Patients with secondary iron overload were excluded. All were HCV antibody positive by second- or third-generation recombinant immunoblot assay (RIBA) with elevated transaminases and/or positive HCV RNA by reverse-transcription polymerase chain reaction. All patients had liver biopsies performed for diagnostic purposes and staging of their disease. Liver histology was scored using the modified histological activity index, 23 with necroinflammation (grade) scored 0-18, and fibrosis (stage) scored 0-6. Liver iron concentration was graded 0-4 using Perls stain, according to the method of Searle et al., 24 by two observers (M.A.G. and A.D.B.) unaware of HFE genotype. Data on estimated weekly alcohol consumption in units during the year before biopsy were recorded (1 unit equivalent to approximately 10 g alcohol). Serum ferritin was measured using standard methods. Ferritin was elevated above the upper limit of normal (300 µg/l) in 5 heterozygous patients and 7 homozygous normals, and in these cases transferrin saturation was determined. Serum was collected at the time of liver biopsy and HCV-RNA titer determined using the branched DNA assay (HCV RNA 2.0-Chiron; Chiron Corporation, Emeryville, CA) according to the manufacturer s instructions. After obtaining informed consent, blood (10 ml) was collected and genomic DNA obtained by standard phenol/chloroform extraction and amplified by polymerase chain reaction to give a 387 base pair (bp) product. 16 Polymerase chain reaction products were digested with RsaI to identify the C282Y mutation, which creates a new RsaI restriction site. Ten percent polyacrylamide gel electrophoresis was used to separate restriction fragments: wild type giving bands at 247 and 140 bp; mutant alleles showing bands at 247, 111, and 29 bp. Prevalence of the C282Y mutation was also evaluated in a local control group of 117 healthy adults. Differences between genotypes were analyzed using Fisher s Exact test and the Mann-Whitney test. Relative risks were calculated with the Woolf-Haldane modification. Correlation between serum ferritin, liver iron, and histology were assessed by linear regression analysis. RESULTS Ten of 137 (7.3%) patients were found to be heterozygous for the C282Y mutation. No patients were homozygous for this mutation. Similarly, 13.7% of controls were heterozygous for the mutation with 1 control homozygous. There was thus no significant difference in prevalence of the mutation between patients and controls (P.1). Among the patients with hepatitis C, the sex, age, and alcohol consumption were not significantly different between HFE heterozygotes and normals (Table 1). Both serum ferritin and iron saturation were significantly higher in patients with the C282Y mutation compared with wild-type individuals (Table 2). Stainable liver iron was absent in the majority of subjects with and without the mutation; however, it was present significantly TABLE 2. Serum and Hepatic Iron Indices in Patients With Chronic HCV Genotyped for HFE C282Y Mutation HFE Heterozygote (n 10) Homozygous Normal (n 127) Mean ferritin (µg/l) (range) 339 ( ) 153 (9-567).0005 Iron saturation (%) (range) (n 12)* 60.2 (48-74) 37.3 (22-48).0001 Iron stain positive (%) 3 (30) 5 (4).02 Iron stain graded 0-4, mean (range) 0.7 (0-3) 0.04 (0-1).17 *Measured only when ferritin was abnormal. more often in C282Y heterozygotes compared with wild-type patients (Table 2). Liver disease was more advanced in heterozygotes reflected by higher fibrosis scores (Fig. 1). In addition, 40% of those with the mutation had cirrhosis compared with 8.7% of wild types (P.01; odds ratio: 7.6 [ ]). Cirrhosis occurred at a younger age in heterozygous patients ( years) compared with homozygous normals ( years; P.03). There was no significant difference in necroinflammation scores between HFE heterozygotes and homozygous normals (Fig. 1). Similarly, there was no difference in HCV-RNA titers between the two groups (Table 1). Serum ferritin levels correlated with stainable iron on liver biopsies (r.22; P.002). In addition, there was a weak correlation between ferritin and fibrosis (r.207; P.04), but the correlation between ferritin and inflammation was not significant. DISCUSSION The prevalence of the C282Y mutation was similar in the patients with hepatitis C (7.3%) compared with both local controls (13.7%) and that reported for other U.K. populations (6% 18 and 11% 20 ). This implies that presence of the mutation does not predispose to establishment of chronic hepatitis C. The finding of no homozygotes among the patients is consistent with the reported prevalence of C282Y/C282Y homozygosity in controls of 1%. 18,20 The data show a significant correlation between liver fibrosis and presence of the C282Y mutation in patients with chronic HCV. There is at present no evidence that the C282Y mutation is harmful in any other way than through accumulation of excess body iron stores. However, preliminary functional analysis demonstrates that the C282Y mutation abrogates binding between 2 -microglobulin and the protein encoded by the major histocompatibility class 1 like HFE gene. 25 This prohibits proper intracellular processing and P TABLE 1. Characteristics of Patients with Chronic HCV Genotyped for HFE C282Y Mutation Heterozygous (n 10) Homozygous Wild Type (n 127) P Male Female Age (range) 39.1 (22-55) 40.8 (20-75).67 Alcohol (units/wk), mean (range) 24.5 (0-80) 18.1 (0-100).43 HCV-RNA titer (Meq/mL) mean (range) 5.59 ( ) 4.39 ( ).87 FIG. 1. Mean hepatic histology scores in patients with chronic HCV genotyped for the HFE C282Y mutation. *Error bars show maximum values seen. (O), heterozygous mutant; (S), wild type.

3 HEPATOLOGY Vol. 27, No. 6, 1998 SMITH ET AL cell-surface presentation of the protein. Presence of the C282Y mutation could potentially have effects in a variety of diseases; the mutation is found commonly in patients with porphyria cutanea tarda, 20 and there is a preliminary report of increased fibrosis in patients with nonalcoholic steatohepatitis who have the mutation. 26 Thus, the presence of the mutation could potentially alter the natural history of HCV either by an unknown mechanism resulting from the loss of HFE cell-surface expression or through alteration in liver iron stores. If fibrosis in HCV is accelerated by elevated liver iron in HFE C282Y heterozygotes, it must first be established that HIC is indeed increased in these patients. Serum markers of iron status (ferritin and transferrin saturation) are used to estimate body iron stores, but have limited utility in patients with inflammatory liver disease. Even in healthy subjects, there is wide variability in these markers, with the coefficient of variation for ferritin being 27% to 29%. 7 Nevertheless, mean serum ferritin and transferrin saturation have been shown to be higher in heterozygotes compared with normals 27 : mean ferritin higher by 0 to 71 µg/l, transferrin saturation higher by 5% to 9%. There is, however, considerable overlap between heterozygotes and both normals and HFE homozygotes. 27,28 The findings of this study are consistent with these results, with both ferritin and iron saturation significantly higher in heterozygotes (Table 2), but with overlap demonstrated between the groups. Histological evaluation of liver iron stores using Perls staining may be insufficiently sensitive to identify slight increases in liver iron in heterozygotes. Within a single histological grade, there may be a substantial range of HIC. A limitation of this study is that hepatic iron has been estimated using the modified Perls staining method rather than by quantitative methods. However, Ludwig et al. 29 reported that in cases of mild hepatic iron abnormalities, for example, where hemosiderosis is focal, iron quantitation may give false-negative results. This is particularly a problem when the sample for quantitation is small ( 5 mg wet weight) or in livers with extensive fibrosis. Table 2 demonstrates that significantly more HFE heterozygotes had positive hepatocyte staining for iron compared with homozygous normals. However, the majority of both heterozygotes and wild types had grade zero iron staining in hepatocytes. This finding is consistent with Bulaj s 27 report of HIC in 39 subjects heterozygous for HH (17 of whom had normal transferrin saturation and ferritin): the stainable iron ranged from grades 0 to 3, with a mean of 0.82 in those with normal serum markers, and 1.3 in those with elevated serum markers. HIC was 23 µmol/g dry weight and 32 µmol/g/dry weight, respectively. Thus, only a handful of heterozygotes have HIC above the normal value of 25 µmol/g dry weight. It has been suggested that there is a threshold concentration of iron above which hepatic fibrosis develops in HH, with fibrosis developing only with HIC above 400 µmol/g (22.3 mg/g) in the absence of alcoholism. 30,31 However, there is an imperfect correlation between HIC and fibrosis 31,32 ; other factors may be involved in the initiation of fibrosis. 33 HCV may act synergistically with iron in producing liver damage in which the HIC alone is insufficient to cause fibrosis. Thus, even slight increases in liver iron, as seen in heterozygotes, may be sufficient to contribute to fibrosis in HCV. In a study of patients with chronic viral hepatitis, DiBisceglie et al. 6 found that 46% had elevations in serum iron, ferritin, or transferrin saturation. However, marked elevations in hepatic iron were uncommon with only 4 of 80 patients (5%) having HIC above the upper limit of normal (25 µmol/g dry weight). In contrast, in this study, only 12 of 137 (9%) of patients had elevated serum ferritin, and only 2 patients had abnormal stainable liver iron ( 2). This may in part be a reflection of the relatively young age (mean, 40 years) and mild disease (mean fibrosis score, 1.6) of these patients. In addition, more patients with abnormal biochemical markers may have been detected if serum iron and transferrin saturation had been measured in all patients (rather than only those with elevated ferritin). However, because of the nonspecific effects of hepatic inflammation on ferritin, one would expect serum ferritin to be over-, rather than undersensitive, as a screening test for abnormal iron stores. Di Bisceglie et al. 6 found that only serum ferritin was significantly associated with HIC. Both serum ferritin levels 6 and HIC have previously been shown to correlate with liver fibrosis in chronic viral hepatitis. 34 This study found that the effect of the C282Y mutation on fibrosis occurred independent of changes on necroinflammation. This is consistent with studies in HH in which cirrhosis can develop without significant biochemical abnormalities 35 and in which fibrosis is seen histologically without associated cell necrosis or inflammatory infiltrate. 36 Iron may contribute to HCV-related liver disease by facilitating viral replication, possibly by provision of nutrients to the virus as has been suggested for hepatitis B virus 37 and other viruses and bacteria. 38,39 Iron is an essential cofactor for RNA replication. A number of factors are, however, against this theory. First, no increase in HCV-RNA titers were seen in patients heterozygous for HFE (Table 1). Second, iron depletion has not been shown to result in a fall in HCV-RNA titers. 40 Finally, if heterozygosity for HH was associated with increased HCV replication, an increase in necroinflammatory scores may be expected, and this was not observed. Another mechanism whereby iron may cause liver fibrosis is as a result of free radical production leading to cell damage. Iron is a potent catalyst of oxidative stress; it reacts with oxygen to generate hydroxyl free radicals and ferryl radicals. 11 Lipid peroxides have been shown to be important in the pathogenesis of HH. 41 Hepatic iron may cause fibrosis due to oxidant stress, by both lipid peroxidation or independent of lipid peroxidation via effects on intracellular signalling. 33 Serum and hepatic levels of thiobarbituric acid reacting substances, a marker of oxidative injury, are elevated in HCV infection. 11 Similarly malondialdehyde levels, a measure of lipid peroxidation and glutathione turnover, are both increased in HCV. 5 These putative markers of free radical mediated liver damage correlate with liver iron measurements. 5 Levels of antioxidants in liver and plasma are reduced in rats with experimental iron overload and in patients with HH. 42 In a preliminary study, the antioxidant N-acetylcysteine was found to improve transaminase abnormalities in chronic HCV, 43 providing support to the importance of oxidant stress in the pathogenesis of HCV. The size of this study prohibited the examination of the effect of HH heterozygosity on risk of HCC in HCV. As HCC risk is related to the presence of cirrhosis, it seems likely that heterozygotes will be more at risk for HCC than wild-type patients. Whether this risk is increased above the risk of

4 1698 SMITH ET AL. HEPATOLOGY June 1998 cirrhosis alone must be determined. In the United States First National Health and Nutrition Examination Survey, overall cancer risk correlated with transferrin saturation. 44 In HH, risk of HCC correlates with the amount and duration of iron overload, 45 consistent with the work in animal models 28 and tissue culture, 10 in which elevated hepatic iron is associated with carcinogenesis and removal of iron reduces HCC growth. The evidence that iron depletion improves liver function tests in HCV, even in subjects without iron overload, 9 supports the hypothesis that iron is in part responsible for liver injury. 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