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Hemochromatosis Gene Mutations: Prevalence and Effects on Pegylated-Interferon and Ribavirin Therapy Response in Chronic Hepatitis C in Sardinia
Original Article
JOURNAL OF CLINICAL AND EXPERIMENTAL HEPATOLOGY
Hemochromatosis Gene Mutations: Prevalence and Effects on Pegylated-Interferon and Ribavirin Therapy Response in Chronic Hepatitis C in Sardinia Margherita Sini, Orazio Sorbello, Alberto Civolani, Luigi Demelia
Background/Aims: Considerable evidence suggests that iron could be a comorbid factor for liver injury in chronic hepatitis C (CHC). Elevated iron indices are frequently described in CHC and may impact negatively on the course of liver disease and on the response to interferon alfa therapy. The aim of this study was to evaluate the frequency of hemochromatosis gene mutations in Sardinian CHC patients, the association with iron overload and the impact on response to therapy. Methods: Sixty-nine CHC patients were enrolled. Iron indices, hepatic and viral parameters were detected. C282Y, H63D and S65C mutations were identified through a PCR. Liver biopsy was performed for hepatic fibrosis evaluation. All patients were treated for 6 months (viral genotype 2/3) or 12 months (viral genotype 1/4) with pegylated-interferon 180 mcg once weekly and ribavirin 1000– 1200 mg/daily. Sustained virological response (SVR) was defined as undetectable HCV RNA 24 weeks after the end of treatment. Results: HFE gene mutation was detected in 29 patients (42%). The presence of HFE mutations was significantly associated with elevated transferrin saturation (P < 0.01). Hepatic fibrosis was more advanced in HFE mutation carriers (c2, P = 0.04). Among mutation carriers 27.5% achieved responses at the end of treatment compared with 60% of non-carriers (P = 0.005). Patients with HFE wildtype produced significant SVR compared with patients with HFE mutations (P = 0.03). Conclusions: The literature shows discordant results about the prevalence, hepatic distribution and possible therapeutic implications of iron overload in chronic hepatitis C. Our findings shows that HFE gene mutations could favor, synergically with CHC and other genetic or acquired factors, the development of liver damage and could influence the outcome of interferon treatment with higher rate of non-response. ( J CLIN EXP HEPATOL 2012;2:211–217)
I
ron is an essential micronutrient which plays a key role in a wide range of biochemical pathways that govern cellular metabolism, including those that are essential for cellular respiration as well as DNA, RNA and protein synthesis. Iron balance is regulated at the absorptive step, but the mechanism by which the mucosa accomplishes this has not been defined. There is no effective physiological mechanism for the excretion of excess body iron, hence increased absorption of iron would increase body iron stores, mainly in the liver.1 Iron has been shown to increase the formation of reactive oxygen intermediates that lead to lipid peroxidation and subsequent oxidative damage to
Keywords: HFE gene, iron overload, viral hepatitis Received: 15.5.2012; Accepted: 9.6.2012; Available online: 27.8.2012 Address for correspondence: Orazio Sorbello, Department of Gastroenterology, Azienda Ospedaliero-Universitaria di Cagliari, SS 554 bivio per Sestu, 09130 Cagliari, Italy. Tel./fax: +39 070 51096100 E-mail: fi[email protected] Abbreviations: ALT: alanine aminotransferase; AST: aspartate aminotransferase; AP: alkaline phosphatase; CHC: Chronic hepatitis C; ETR: End of treatment response; GGT: g-glutamyl transpeptidase; HFE: Human hemochromatosis protein; HCV: Hepatitis virus C infection determination; HH: Hereditary Hemochromatosis; SVR: Sustained virologic response; TSI: Transferin saturation index; ULN: Upper normal limit; WT: wildtype http://dx.doi.org/10.1016/j.jceh.2012.06.004 © 2012, INASL
proteins and nucleic acids.2 Iron-induced oxidant stress is involved in this process as the primary cause of parenchymal cell necrosis or as activator of cells that are effectors or key mediators of hepatic fibrogenesis. The fibrogenic potential of iron in the liver is even more important when iron acts simultaneously with other hepatotoxic factors.3 The intestinal iron absorption appears to be disturbed in Hereditary Hemochromatosis (HH). The homozygous state in which both alleles of chromosome 6 possess the C282Y mutation or the compound heterozygous state with C282Y on one chromosome and H63D on the other, are the predominant genetic abnormalities associated with phenotypic HH.4 A third mutation, S65C, is considered to be a rather new polymorphism.5 “In Europe, the C282Y allele has a north to west frequency- decreasing gradient, with higher frequencies reported in Ireland (28.4%) and lower frequencies in Italy (3.2%). Conversely, the H63D allele has a higher frequency in southern Europe (Spain, 32.3%) and a lower frequency in the Celtic populations (5%).6 The Sardinian population is genetically differentiated from the other Caucasian populations. It represents a genetic isolate where the p.C282Y mutation is considered as rare or even absent. Candore et al studied the frequency of the HFE gene mutations in five Italian populations. In Italy, the allele frequency of the C282Y mutation decreases from
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Department of Gastroenterology, Azienda Ospedaliero-Universitaria di Cagliari, SS 554 bivio per Sestu, 09130 Cagliari, Italy
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northeast Italy (Friuli, 6%) to northwest Italy (Piemont, 4.8%) and to central Italy (Emilia-Romagna, 1.7%). However, this mutation is lacking in Sardinia. In contrast, no difference was observed in allele frequency of H63D in the five Italian regions (Friuli 12%—Sardinia 17.5%).7 Several studies assessed that no association exist between the HFE genetic variants and chronic liver disease. Overall, only a few studies have suggested an increased prevalence of HFE mutations in CHC patients,8,9 with respect to the general population; this observation was not confirmed in other studies.10 Laboratory abnormalities of iron metabolism have been detected in 15–20% of heterozygotes, but heterozygosity for hemochromatosis is rarely associated with liver damage due only to iron overload. Complications have been recognized only when other disorders, such as porphyria cutanea tarda, chronic anemia, alcoholism and hepatitis are also present.11 Over the last 20 years, considerable evidence suggested that a pathogenetic link exists between the iron content of the liver and viral hepatitis. Elevated iron indices are frequently described in CHC and may impact negatively on the course of liver disease and on the response to interferon alfa therapy.12 14 HFE gene mutations may play a role in the development of significant iron overload in patients with CHC and could represent a clinically relevant comorbid factor in patients with chronic hepatitis C.15–17 There are several host characteristics known to affect outcome of interferon treatment, including age, gender, immune surveillance system, nutritional state and iron status.18 The aim of our study was to evaluate the impact of HFE gene mutations on disease severity and response to interferon therapy in a cohort of Sardinian patients with Chronic Hepatitis C.
MATERIALS AND METHODS Patients Sixty-nine patients with chronic hepatitis C (53 male/16 female, mean age 51 2 years) were enrolled at the Division of Internal Medicine and Digestive Pathologies, University Hospital of Cagliari. The following specific inclusion criteria were fulfilled by all patients: age 18–65 years, elevated serum ALT levels above twice the normal range for at least 6 months before enrollment; positive test for anti-HCV antibodies; positive test for HCV RNA; histological diagnosis of chronic hepatitis with or without cirrhosis. The exclusion criteria included: decompensated liver disease, systemic diseases, cancer, hemolytic anemia, neutropenia <1000/mcl, thrombocytopenia <100 103/mcl, serological HBsAg positivity, HIV infection, drug addiction, alcohol abuse, hepatotoxic drugs usage, autoimmune hepatitis, pregnancy, psychiatric illness, renal impairment, Wilson's Disease, Hereditary Hemochromatosis and alpha1-antitrypsin deficiency. None of the patients had received previous interferon-alpha therapy. 212
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Liver Function Tests Liver function tests including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), g-glutamyl transpeptidase (GGT), pseudocholinesterase, bilirubin and albumin were detected.
HCV Determination Diagnosis of hepatitis C virus infection was based on a positive anti-HCV assay (ELISA III) and quantification of Hepatitis virus C infection determination (HCV) RNA by PCR (Cobas Amplicor; Roche, Basel, Switzerland), HCV genotypes were determined by INNO-LiPA HCV II assay (Bayer Diagnostics, Leverkusen, Germany) and classified according to Simmonds et al.19
Iron Parameters Quantitative determination of iron concentrations in serum was performed on automated clinical chemistry analyzers (Hitachi), using a colorimetric assay (Roche). Both ferritin and transferrin levels were measured by immunoturbidimetric assays using the Tina-quant reagents (Roche). Transferrin saturation index (TSI) was calculated as Fe/total Fe-binding capacity 100 (normal value 16–45%).
Histological Evaluation Liver biopsies were obtained employing the Menghini technique under ultrasound guidance in 69 patients. For histological examination, paraffin-embedded 4 mm sections were stained with hematoxylin and eosin, trichrome, and Perl's Prussian blue. Liver histology was evaluated in a blinded manner according to the Desmet classification.20
Genetic Analysis Genomic DNA was isolated from either EDTA anticoagulated whole blood. Detection of C282Y, S65C and H63D mutations in the Human hemochromatosis protein (HFE) gene were performed using PCR amplification.
Study Design and Protocol All patients were treated for 6 months (viral genotype 2/3) or 12 months (viral genotype 1/4) with pegylated-interferon (PEG-IFN) (Pegasys—Roche) 180 mcg once weekly, self-administered subcutaneously together with ribavirin (Rebetol—Schering-Plough) 1000–1200 mg/daily by body weight, orally in two divided doses. All patients were observed every 2 weeks for the first month and every 4 weeks thereafter during treatment. After the 24–48-week therapy period, patients were followed up at 4 week intervals for 6 months. For assessment of therapy compliance, adverse effects, response to the treatment and its relationship with HFE gene mutations, patients underwent laboratory measurements of liver function tests, full blood count, serum HCV RNA concentration, thyroid © 2012, INASL
JOURNAL OF CLINICAL AND EXPERIMENTAL HEPATOLOGY
Statistical Analysis Results are presented as mean SEM. Statistical analyses were performed using StatView 5.0 (SAS Institute). Descriptive statistics were calculated and test of normality were performed. For continuous variables, the values of which were found to be distributed normally, parametric statistical procedures were used, including paired t-test and unpaired t-test. For discontinuous variables, nonparametric procedures, including Wilcoxon's signed-rank test and Mann–Whitney U test, were carried out as appropriate. Comparisons between the frequencies of observation were performed using the c2 test. Significance criteria of P < 0.05 was used for all inferences.
Table 1 Base-line characteristics of 69 patients with HCV infection.a Range 69
Gender (M/F)
53/16
Age (years)
51 2
25–65
139 29
85–480
Alanine aminotransferase (UI/dl)
Viral genotype—n (%) 1–4
46 (67%)
2–3
23 (33%)
HFE genotype—n (%) Wildtype
40 (58%)
HFE mutation
29 (42%)
Serum iron (mg/dl)
127 8
15–312
Serum ferritin (ng/ml)
271 24
18–856
Transferrin saturation index (%)
46%
26–62
Normal range: Transferrin, 110–370 mg/dl. Plus–minus values are means SEM. a Upper normal limit (ULN) of laboratory data: ALT, 41 UI/l; Serum iron, 158 mg/dl; Serum ferritin, 250 ng/ml; Transferrin saturation, 45%.
Table 2 Base-line characteristics of 40 HFE wildtype patients and 29 patients with HFE mutation.a HFE wildtype
RESULTS HFE Genotypes The clinical characteristics of the 69 HCV-infected patients are reported in Table 1. The base-line characteristics of the 69 patients grouped by HFE genotype are shown in Table 2. At least one of the three HFE gene mutations was present in 42% of the patients. One patient was heterozygous for the C282Y mutation (1.4%), 22 were heterozygous for the H63D mutation (31.8%), three (4.3%) were heterozygous for the S65C mutation, two patients (2.8%) were homozygous for the H63D mutation and one patient (1.4%) was a compound heterozygote for the H63D and S65C mutation. No significant differences were observed for age, gender distribution, ALT, serum iron and ferritin levels and viral genotype between HFE mutant patients and wildtype homozygotes. Transferrin saturation index (TSI) showed a significant statistical difference between HFE mutant patients (50%)
N
HFE mutation
P-value
N
40
29
Gender (M/F)
32/8
25/4
ns
Age (years)
50 1
52 2
ns
ALT (UI/dl)
186 59
151 27
ns
AP (U/l)
183 13
209 22
ns
GGT (U/l)
71 8
108 20
<0.05
Bilirubin total (mg/dl)
0.6 0.06
0.8 0.1
ns
Serum iron (mg/dl)
118 9
149 13
ns
Serum ferritin (ng/ml)
280 34
Transferrin saturation(%) 43.4%
327 32
ns
50%
<0.01
4/29 (13.8%)
P = 0.04b
Histological fibrosis Stage I
10/40 (25%)
Stage II
15/40 (37.5%) 5/29 (17.2%)
Stage III
11/40 (27.5%) 10/29 (34.5%)
Stage IV
4/40 (10%)
10/29 (34.5%)
1–4
27 (67.5%)
19 (65.5%)
2–3
13 (32.5%)
10 (34.5%)
Genotype—n (%)
a
ns
Plus–minus values are means SEM. c test.
b 2
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function tests, autoantibody screening including antinuclear factors and anti-thyroid antibodies and serum iron parameters. A complete response at the end of treatment was defined as the normalization of serum ALT levels and negative serum HCV RNA. A sustained complete response was defined as maintenance of remission after cessation of treatment for 24 weeks or more. Patients were divided in two groups according to HFE gene mutations: Group A, which included subjects with HFE gene wildtype and Group B, subjects with HFE gene mutations. Biochemical parameters, iron indices, liver histology, End of Treatment Response (ETR) and Sustained Virologic Response (SVR) were compared between the two groups. Patients who discontinued treatment during follow-up were considered as virologic non-responders. All patients were informed of the purpose of the study and of known side-effects associated with both drugs, and written informed consent for liver biopsy and therapy was obtained.
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End of Treatment Response and Sustained Virological Response Evaluation of Antiviral Therapy
40
100 80 60
ETR
SVR
P=0.005
P=0.03
40 20 0
NR
R HFE mutation
NR
R WT
Figure 2 End of the Treatment Response (ETR) and Sustained Viral Response (SVR) were lower among patients with HFE gene mutations compared to those with HFE gene WT (wildtype) (c2, P = 0.005 and P = 0.03 respectively).
DISCUSSION Interest in the role of iron in CHC began when Di Bisceglie et al found that up to 36% of patients with chronic hepatitis C had elevated serum iron parameters.7 A substantial number of these patients also have increased iron deposition in the liver.21 Furthermore, HCVinfected patients with stainable iron in liver biopsies showed enhanced liver fibrosis compared with controls without detectable iron.22 The hypothesis that iron is a risk factor for liver injury in CHC patients was supported by a recent experimental report that excess dietary iron exacerbated liver injury in HCV-infected chimpanzees.23 The mechanism by which iron accumulates in liver infected with chronic HCV has not yet been established. Di Bisceglie et al reported that serum iron and ferritin levels were increased in patients with CHC because of their release from hepatocellular stores in association with cell necrosis.6 Alternatively, individuals with serum iron levels in the upper range of normal as a result of genetic polymorphisms or a high iron diet may be predisposed to develop more severe chronic HCV infections.6 It has been suggested that iron overload in the liver of patients with CHC is associated with higher ALT levels and decreased response to
35 30
120
25
100
Percentage of subjects
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At the end of treatment, both serum ALT levels normalized and HCV RNA became negative in 32 of 69 patients (46%). Seven of 69 patients (10%) did not complete the treatment; these patients stopped prematurely for side-effects associated with the use of PEG-IFN and ribavirin combination therapy (1 for neutropenia and thrombocytopenia, 3 for hypothyroidism and 3 for depression). The relationship between HFE gene mutations and response to antiviral therapy was investigated. At the end of treatment the viral and biochemical response rates were lower among the patients with HFE gene mutations 8/29 (27.5%) compared to those with HFE gene wildtype 30/40 (75%) (c2, P = 0.005) (Figure 2). Of the patients with genotype 1–4 infection, the end of treatment response rate was higher in group A (14/27; 51.8%) compared to group B (2/19; 10.5%) (c2, P = 0.003), while no significant difference was observed between patients with HFE gene mutation (5/10; 50%) and HFE gene wildtype with genotype 2–3 infection (11/13; 77%) (Figure 3). At the end of 24 weeks of post-treatment follow-up period, 32/69 (46.3%) patients produced SVR. The difference of SVR rate between 25/40 patients (62.5%) with HFE wildtype and 7/29 of HFE mutant (24.1%) was statistically significant (c2, P = 0.03) (Figure 3).
120
Percentage of subjects
and wildtype homozygotes (43%) (P < 0.01). The degree of hepatic fibrosis was evaluated by HFE genotype in 69 patients (Figure 1). Thirty-five of 69 (51%) patients had advanced fibrosis (Stage score III–IV); twenty of 29 (69%) patients had a HFE gene mutation and a histological stage III–IV, while only 15 of 40 (39%) HFE wildtype patients presented histological stage III–IV. There was a close association between advanced histological score and the presence of HFE gene mutation (c2, P = 0.04).
20 15 10 5 0 HFE WT Stage I
Stage II
HFE mutation Stage III
Stage IV
Figure 1 Relationship between stage of fibrosis and HFE gene status. A significant association exists between advanced histological score and the presence of HFE gene mutation (c2, P = 0.04). WT, Wildtype. 214
ns
P=0.003
80 60 40 20 0 1-4 NR
1-4 R HFE mutation
2-3 NR
2-3 R
WT
Figure 3 Relationship between HFE gene mutation and Sustained Viral Response (SVR) in 46 patients with viral genotype 1–4 and 23 patients with viral genotype 2–3. NR, NonResponders; R, Responders. © 2012, INASL
interferon.10 These observations indicate the important role of iron in the pathogenesis of CHC. In fact patients with combined hereditary hemochromatosis and chronic hepatitis C infection presented advanced fibrosis/cirrhosis at a younger age and at a lower hepatic iron concentration compared to HH patients. This supports the concept that the combination of iron overload and HCV has a potentiating effect on hepatic fibrogenesis.24 Iron can be a profibrogenic factor, acting as activator of both hepatic stellate cells and Kupffer cells and inducing liver inflammation or hepatocyte necrosis. In fact the presence of stainable iron on liver biopsy of patients with viral hepatitis correlates with a higher degree of necroinflammatory activity and a higher score for fibrosis compared to those with no stainable iron.25 The coexistence of hemochromatosis gene mutations represents a genetic risk factor affecting the severity of chronic hepatitis C and the response rate to interferon therapy.26 28 The results of our study indicate that the prevalence of the three known HFE gene mutations in Sardinia is in accordance with previous observations in other countries: (5–10% for the C282Y mutation and 6–30% for the H63D mutation),29,30 specifically 42% of our HCV-infected patients carried at least one of these mutations. Heterozygous C282Y status and homo- or heterozygosity for the H63D and S65C mutations do not usually induce hemochromatosis phenotype and are rarely associated with liver damage in healthy subjects.31 Nevertheless, laboratory abnormalities of iron metabolism (higher serum iron concentration, transferrin—saturation values and serum ferritin concentrations) have been detected in 15–20% of heterozygotes.6–26 Complications have been recognized only when other disorders, such as alcoholism and viral hepatitis are present.6–26 HFE gene mutations may contribute to iron storage and could represent a clinically relevant comorbid factor in patients with chronic hepatitis C. Sebastiani et al summarize the current status of the literature regarding the prevalence, hepatic distribution of iron overload in liver disease.32 In this review the role of HFE mutations as a risk factor for iron overload in CHC has been studied in different populations, with discordant results. Our results evidenced a correlation between HFE gene mutation and iron overload. TSI is the only serum iron parameter that showed a statistically significant difference between HFE gene wildtype and mutation, while serum iron and serum ferritin were higher in HFE mutation but did not obtain a statistically difference. We can assess that several aspecific factors seem to favor marked variations of serum iron and serum ferritin and we considered TSI as the most specific and sensitive parameter in identifying iron overload. The present study provides evidence supporting the view that the HFE gene mutations are associated with significant abnormalities of iron metabolism and suggests that patients with CHC accumulate iron as the result of interplay between genetic and acquired factors. In fact
transferrin saturation index (TSI) showed a significant statistical difference between HFE mutant patients (50%) and wildtype homozygotes (43.4%) (P < 0.01). Instead, no significant difference was observed for other indirect markers of iron stores such as serum iron and serum ferritin. These parameters lack specificity, particularly in the face of chronic inflammatory conditions or alcohol-induced liver disease.27 Previous reports suggested that TSI measurement appears to be the most sensitive method of detecting iron overload states and showed that TSI values exceeding 45% correctly identified 97.9% of homozygotes for Hereditary Hemochromatosis, with no false positives among the normal population, and 22.2% of the heterozygote population. Detecting the threshold for TSI to 45% could also identify other groups with relatively minor degrees of secondary iron overload, such as chronic hepatitis C.33,34 Recent evidence suggests that HFE gene mutations and a consequent mild iron overload may worsen the course of chronic hepatitis C and increase the progression of fibrosis.22–35 The results of our study showed that a significant association exists between advanced histological score and the presence of HFE gene mutation (c2, P = 0.04). The fact that patients heterozygote for hereditary hemochromatosis and infected with hepatitis C virus report greater fibrosis than those with homozygous wildtype provides additional evidence that iron modulates fibrosis in chronic hepatitis C. Despite these observations, the association between serum iron values, hepatic iron stores and hepatic necroinflammatory activity or fibrosis remains controversial. HFE gene mutations may have a potentiating effect on histological severity, acting synergically with CHC in the development of liver damage. Iron overload seems to impair antigen-specific immune responses by decreasing the generation of T cells and by impairment of natural killer and T helper cell function. Piperno et al 3 suggested that iron overload in patients with hemochromatosis may contribute to the persistence of HCV infection: iron overload may in theory promote viral replication. Moreover, iron overload has been incriminated as one of the essential factors that hamper response to interferon-alpha in CHC.3–36 The amount of hepatic iron has been identified as one of these factors that adversely affect the likelihood of response to interferon-alpha; those patients with higher hepatic iron content are less likely to respond to interferon therapy.37 Retrospective evidence that the amount of hepatic iron may modulate the response of the hepatitis C virus to interferon therapy was provided by Van Thiel et al13 who reported that hepatic iron content of interferon nonresponders was found to be almost twice that of responders. The influence of host and viral factors on the natural course of CHC and efficacy of PEG-IFN and Ribavirin therapy has been intensively studied. The presence of HFE gene mutations may be an additional factor to be considered among those implicated in the determination of a lower rate of sustained virological
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response to PEG-IFN plus ribavirin in chronic hepatitis C patients. In the present study, ETR rates were lower among patients with HFE gene mutations compared to those with HFE gene wildtype (P = 0.005). In patients with 1–4 HCV genotype infection, the ETR rate was higher in group A (51.9%) as compared with group B (P = 0.003). No significant response difference was observed between patients with HFE gene mutation and HFE gene wildtype in genotype 2–3. The small number of non-1–4 genotype patients could have accounted for the lack of statistical significance between the two groups. A sustained virological response was seen in 18.8%. The patients with HFE wildtype produced significant SVR compared with patients with HFE mutations (P = 0.03). Despite the number of patients in our study being too limited for meaningful statistical analysis, several comments can be made concerning the end of treatment and sustained responders. We have observed that PEG-IFN/ribavirin therapy is less effective in patients with 1–4 HCV genotype and HFE gene mutation. Moreover, in our patients with genotype 1–4 infection, TSI was significantly higher than those with genotype 2–3 infection and viral genotype distribution did not differ significantly between HFE mutant patients and wildtype homozygotes. Therefore, HFE gene mutations may act synergically with CHC in the development of liver damage, predicting a higher rate of non-response to PEG-IFN–ribavirin therapy. We suggest that screening for HFE mutations and iron parameters should be considered in patients with CHC. Therapies that specifically reduce iron levels should be designed to lessen the severity of HCV infection in patients with HFE gene mutation and to enhance the outcome of antiviral therapies.
CONFLICTS OF INTEREST There are no commercial associations (e.g. consultancies, stock ownership, equity interests, patent licensing arrangements, etc) with Margherita Sini, Orazio Sorbello, Alberto Civolani and Luigi Demelia that pose a conflict of interest in connection with the submitted article entitled “Hemochromatosis gene mutations: prevalence and effects on pegylated-interferon and Ribavirin therapy response in chronic hepatitis C in Sardinia”.
ACKNOWLEDGMENTS We acknowledge Dr. Barry Mark Wheaton for his expertise and help in the execution of this paper with gratitude. REFERENCES 1. Finch C. Regulators of iron balance in humans. Blood. 1994;84: 1697–1702. 2. Britton RS. Metal-induced hepatotoxicity. Semin Liver Dis. 1996 Feb;16(1):3–12.
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3. Piperno A, Fargion S, D'Alba, et al. Liver damage in Italian patients with hereditary hemochromatosis is highly influenced by hepatitis B and C virus infection. J Hepatol. 1992;16(3):364–368. 4. Riedel HD, Stremmel W. The haemochromatosis gene. J Hepatol. 1997;26:941–944. 5. Holmstrom P, Marmur J, Eggertsen G, et al. Mild iron overload in patients carrying the HFE S65C gene mutation: a retrospective study in patients with suspected iron overload and healthy controls. Gut. 2002;51(5):723–730. 6. Merryweather-Clarke AT, Pointon JJ, Jouanolle AM, et al. Geography of HFE C282Y and H63D mutations. Genet Test. 2000;4(2):183–198. 7. Candore G, Mantovani V, Balistreri CR, et al. Frequency of the HFE gene mutations in five Italian populations. Blood Cells Mol Dis. 2002 Nov–Dec;29(3):267–273. 8. Smith BC, Gorve J, Guzail MA, et al. Heterozygosity for hereditary hemochromatosis is associated with more fibrosis in chronic hepatitis C. Hepatology. 1998;27:1695–1699. 9. Kazemi-Shirazi L, Datz C, Maier-Dobersberger T, et al. The relation of iron status and hemochromatosis gene mutations in patients with chronic hepatitis C. Gastroenterology. 1999;116:127–134. 10. Martinelli AL, Franco RF, Villanova MG, et al. Are haemochromatosis mutations related to the severity of liver disease in hepatitis C virus infection? Acta Haematol. 2000;102:152–156. 11. Bulaj Zaneta J, Griffen Linda M, Jorde Lynn B, Edwards Corwin Q, Kushner James. Clinical and biochemical abnormalities in people heterozygous for hemochromatosis. N Engl J Med. 1996;335: 1799–1805. 12. Di Bisceglie AM, Axiotis CA, Hoofnagle JH, Bacon BR. Measurements of iron status in patients with chronic hepatitis. Gastroenterology. 1992;102:2108–2113. 13. Van thiel DH, Friedlander L, Fagiuoli S, et al. Response to interferon alfa therapy is influenced by the iron content of the liver. J Hepatol. 1994;20:410–415. 14. Farinati F, Carin R, De Maria N, et al. Iron storage, lipid peroxidation and glutathione turnover in chronic anti-HCV positive hepatitis. J Hepatol. 1995;22:449–456. 15. Tung BY, Emond MJ, Bronner MP, Raaka SD, Cotler SJ, Kowdley KV. Hepatitis C, iron status and disease severity: relationship with HFE mutations. Gastroenterology. 2003;124:318–326. 16. Geier A, Reugels M, Weischirken R, et al. Common heterozygous hemochromatosis gene mutations are risk factors for inflammation and fibrosis in chronic hepatitis C. Liver Int. 2004;24(4):285–294. 17. Hohler T, Leininger S, Kohler HH, Schirmacher P, Galle PR. Heterozygosity for hemochromatosis gene in liver diseases: prevalence and effects on liver histology. Liver. 2000;20(6):482–486. 18. Olynyc JK, Reddy KR, Di Bisceglie AM, et al. Hepatic iron concentration as a predictor of response to interferon alpha therapy in chronic hepatitis C. Gastroenterology. 1995;108:1104–1109. 19. Simmonds P, Alberti A, Alter HJ, et al. A proposed system for the nomenclature of hepatitis C viral genotypes (letter). Hepatology. 1994;19:1321–1324. 20. Desmet VJ, Gerber MA, Hoofnagle JH, Manns M, Scheuer PJ. Classification of chronic hepatitis: diagnosis, grading and staging. Hepatology. 1994;19:1513–1520. 21. Sartori M, Andorno S, La Terra G, et al. Evaluation of iron status in patients with chronic hepatitis C. Ital J Gastroenterol Hepatol. 1998;30:396–401. 22. Casaril M, Stanzial AM, Tognella P, et al. Role of iron load on fibrogenesis in chronic hepatitis C. Hepatogastroenterology. 2000;47: 220–225. 23. Bassett SE, Di Bisceglie AM, Bacon BR, et al. Effects of iron loading on pathogenicity in hepatitis C virus-infected chimpanzees. Hepatology. 1999;29:1884–1892. 24. Diwaakaran HH, Befeler AS, Britton RS, Brunt EM, Bacon BR. Accelerated hepatic fibrosis in patients with combined hereditary
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30.
hemochromatosis and chronic hepatitis C infection. J Hepatol. 2002;36:687–691. Pietrangelo A. Iron, oxidative stress and liver fibrogenesis. J Hepatol. 1998;28:8–13. Erhardt A, Maschner-Olberg A, Mellenthin C, et al. HFE mutations and chronic hepatitis C: H63D and C282Y heterozygosity are independent factors for liver fibrosis and cirrhosis. J Hepatol. 2003;38(3):335–342. Piperno A, Vergani A, Maltosio I, et al. Hepatic iron overload in patients with chronic viral hepatitis: role of HFE gene mutations. Hepatology. 1998;28. $9:1105–1109. Coelho-Borges S, Cheinquer H, Cheinquer N, Krug L, Ashton-Prolla P. HFE mutations prevent sustained virological response to interferon plus ribavirin in chronic hepatitis C patients with serum markers of iron overload. Am J Gastroenterol. 2002;97:1570–1572. Beutler Ernest, Felitti Vincent J, Koziol James A, Ho Ngoc J, Gelbart Terri. Penetrance of 845G*A (C282Y) HFE hereditary haemochromatosis mutation in the USA. Lancet. 2002;359: 211–218. Piperno A, Sampietro M, Pietrangelo A, et al. Heterogeneity of hemochromatosis in Italy. Gastroenterology. 1998;114(5):996–1002.
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Hemochromatosis Gene Mutations: Prevalence and Effects on Pegylated-Interferon and Ribavirin Therapy Response in Chronic Hepatitis C in Sardinia Margherita Sini, Orazio Sorbello, Alberto Civolani, Luigi Demelia
Background/Aims: Considerable evidence suggests that iron could be a comorbid factor for liver injury in chronic hepatitis C (CHC). Elevated iron indices are frequently described in CHC and may impact negatively on the course of liver disease and on the response to interferon alfa therapy. The aim of this study was to evaluate the frequency of hemochromatosis gene mutations in Sardinian CHC patients, the association with iron overload and the impact on response to therapy. Methods: Sixty-nine CHC patients were enrolled. Iron indices, hepatic and viral parameters were detected. C282Y, H63D and S65C mutations were identified through a PCR. Liver biopsy was performed for hepatic fibrosis evaluation. All patients were treated for 6 months (viral genotype 2/3) or 12 months (viral genotype 1/4) with pegylated-interferon 180 mcg once weekly and ribavirin 1000– 1200 mg/daily. Sustained virological response (SVR) was defined as undetectable HCV RNA 24 weeks after the end of treatment. Results: HFE gene mutation was detected in 29 patients (42%). The presence of HFE mutations was significantly associated with elevated transferrin saturation (P < 0.01). Hepatic fibrosis was more advanced in HFE mutation carriers (c2, P = 0.04). Among mutation carriers 27.5% achieved responses at the end of treatment compared with 60% of non-carriers (P = 0.005). Patients with HFE wildtype produced significant SVR compared with patients with HFE mutations (P = 0.03). Conclusions: The literature shows discordant results about the prevalence, hepatic distribution and possible therapeutic implications of iron overload in chronic hepatitis C. Our findings shows that HFE gene mutations could favor, synergically with CHC and other genetic or acquired factors, the development of liver damage and could influence the outcome of interferon treatment with higher rate of non-response. ( J CLIN EXP HEPATOL 2012;2:211–217)
I
ron is an essential micronutrient which plays a key role in a wide range of biochemical pathways that govern cellular metabolism, including those that are essential for cellular respiration as well as DNA, RNA and protein synthesis. Iron balance is regulated at the absorptive step, but the mechanism by which the mucosa accomplishes this has not been defined. There is no effective physiological mechanism for the excretion of excess body iron, hence increased absorption of iron would increase body iron stores, mainly in the liver.1 Iron has been shown to increase the formation of reactive oxygen intermediates that lead to lipid peroxidation and subsequent oxidative damage to
Keywords: HFE gene, iron overload, viral hepatitis Received: 15.5.2012; Accepted: 9.6.2012; Available online: 27.8.2012 Address for correspondence: Orazio Sorbello, Department of Gastroenterology, Azienda Ospedaliero-Universitaria di Cagliari, SS 554 bivio per Sestu, 09130 Cagliari, Italy. Tel./fax: +39 070 51096100 E-mail: fi[email protected] Abbreviations: ALT: alanine aminotransferase; AST: aspartate aminotransferase; AP: alkaline phosphatase; CHC: Chronic hepatitis C; ETR: End of treatment response; GGT: g-glutamyl transpeptidase; HFE: Human hemochromatosis protein; HCV: Hepatitis virus C infection determination; HH: Hereditary Hemochromatosis; SVR: Sustained virologic response; TSI: Transferin saturation index; ULN: Upper normal limit; WT: wildtype http://dx.doi.org/10.1016/j.jceh.2012.06.004 © 2012, INASL
proteins and nucleic acids.2 Iron-induced oxidant stress is involved in this process as the primary cause of parenchymal cell necrosis or as activator of cells that are effectors or key mediators of hepatic fibrogenesis. The fibrogenic potential of iron in the liver is even more important when iron acts simultaneously with other hepatotoxic factors.3 The intestinal iron absorption appears to be disturbed in Hereditary Hemochromatosis (HH). The homozygous state in which both alleles of chromosome 6 possess the C282Y mutation or the compound heterozygous state with C282Y on one chromosome and H63D on the other, are the predominant genetic abnormalities associated with phenotypic HH.4 A third mutation, S65C, is considered to be a rather new polymorphism.5 “In Europe, the C282Y allele has a north to west frequency- decreasing gradient, with higher frequencies reported in Ireland (28.4%) and lower frequencies in Italy (3.2%). Conversely, the H63D allele has a higher frequency in southern Europe (Spain, 32.3%) and a lower frequency in the Celtic populations (5%).6 The Sardinian population is genetically differentiated from the other Caucasian populations. It represents a genetic isolate where the p.C282Y mutation is considered as rare or even absent. Candore et al studied the frequency of the HFE gene mutations in five Italian populations. In Italy, the allele frequency of the C282Y mutation decreases from
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Department of Gastroenterology, Azienda Ospedaliero-Universitaria di Cagliari, SS 554 bivio per Sestu, 09130 Cagliari, Italy
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northeast Italy (Friuli, 6%) to northwest Italy (Piemont, 4.8%) and to central Italy (Emilia-Romagna, 1.7%). However, this mutation is lacking in Sardinia. In contrast, no difference was observed in allele frequency of H63D in the five Italian regions (Friuli 12%—Sardinia 17.5%).7 Several studies assessed that no association exist between the HFE genetic variants and chronic liver disease. Overall, only a few studies have suggested an increased prevalence of HFE mutations in CHC patients,8,9 with respect to the general population; this observation was not confirmed in other studies.10 Laboratory abnormalities of iron metabolism have been detected in 15–20% of heterozygotes, but heterozygosity for hemochromatosis is rarely associated with liver damage due only to iron overload. Complications have been recognized only when other disorders, such as porphyria cutanea tarda, chronic anemia, alcoholism and hepatitis are also present.11 Over the last 20 years, considerable evidence suggested that a pathogenetic link exists between the iron content of the liver and viral hepatitis. Elevated iron indices are frequently described in CHC and may impact negatively on the course of liver disease and on the response to interferon alfa therapy.12 14 HFE gene mutations may play a role in the development of significant iron overload in patients with CHC and could represent a clinically relevant comorbid factor in patients with chronic hepatitis C.15–17 There are several host characteristics known to affect outcome of interferon treatment, including age, gender, immune surveillance system, nutritional state and iron status.18 The aim of our study was to evaluate the impact of HFE gene mutations on disease severity and response to interferon therapy in a cohort of Sardinian patients with Chronic Hepatitis C.
MATERIALS AND METHODS Patients Sixty-nine patients with chronic hepatitis C (53 male/16 female, mean age 51 2 years) were enrolled at the Division of Internal Medicine and Digestive Pathologies, University Hospital of Cagliari. The following specific inclusion criteria were fulfilled by all patients: age 18–65 years, elevated serum ALT levels above twice the normal range for at least 6 months before enrollment; positive test for anti-HCV antibodies; positive test for HCV RNA; histological diagnosis of chronic hepatitis with or without cirrhosis. The exclusion criteria included: decompensated liver disease, systemic diseases, cancer, hemolytic anemia, neutropenia <1000/mcl, thrombocytopenia <100 103/mcl, serological HBsAg positivity, HIV infection, drug addiction, alcohol abuse, hepatotoxic drugs usage, autoimmune hepatitis, pregnancy, psychiatric illness, renal impairment, Wilson's Disease, Hereditary Hemochromatosis and alpha1-antitrypsin deficiency. None of the patients had received previous interferon-alpha therapy. 212
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Liver Function Tests Liver function tests including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), g-glutamyl transpeptidase (GGT), pseudocholinesterase, bilirubin and albumin were detected.
HCV Determination Diagnosis of hepatitis C virus infection was based on a positive anti-HCV assay (ELISA III) and quantification of Hepatitis virus C infection determination (HCV) RNA by PCR (Cobas Amplicor; Roche, Basel, Switzerland), HCV genotypes were determined by INNO-LiPA HCV II assay (Bayer Diagnostics, Leverkusen, Germany) and classified according to Simmonds et al.19
Iron Parameters Quantitative determination of iron concentrations in serum was performed on automated clinical chemistry analyzers (Hitachi), using a colorimetric assay (Roche). Both ferritin and transferrin levels were measured by immunoturbidimetric assays using the Tina-quant reagents (Roche). Transferrin saturation index (TSI) was calculated as Fe/total Fe-binding capacity 100 (normal value 16–45%).
Histological Evaluation Liver biopsies were obtained employing the Menghini technique under ultrasound guidance in 69 patients. For histological examination, paraffin-embedded 4 mm sections were stained with hematoxylin and eosin, trichrome, and Perl's Prussian blue. Liver histology was evaluated in a blinded manner according to the Desmet classification.20
Genetic Analysis Genomic DNA was isolated from either EDTA anticoagulated whole blood. Detection of C282Y, S65C and H63D mutations in the Human hemochromatosis protein (HFE) gene were performed using PCR amplification.
Study Design and Protocol All patients were treated for 6 months (viral genotype 2/3) or 12 months (viral genotype 1/4) with pegylated-interferon (PEG-IFN) (Pegasys—Roche) 180 mcg once weekly, self-administered subcutaneously together with ribavirin (Rebetol—Schering-Plough) 1000–1200 mg/daily by body weight, orally in two divided doses. All patients were observed every 2 weeks for the first month and every 4 weeks thereafter during treatment. After the 24–48-week therapy period, patients were followed up at 4 week intervals for 6 months. For assessment of therapy compliance, adverse effects, response to the treatment and its relationship with HFE gene mutations, patients underwent laboratory measurements of liver function tests, full blood count, serum HCV RNA concentration, thyroid © 2012, INASL
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Statistical Analysis Results are presented as mean SEM. Statistical analyses were performed using StatView 5.0 (SAS Institute). Descriptive statistics were calculated and test of normality were performed. For continuous variables, the values of which were found to be distributed normally, parametric statistical procedures were used, including paired t-test and unpaired t-test. For discontinuous variables, nonparametric procedures, including Wilcoxon's signed-rank test and Mann–Whitney U test, were carried out as appropriate. Comparisons between the frequencies of observation were performed using the c2 test. Significance criteria of P < 0.05 was used for all inferences.
Table 1 Base-line characteristics of 69 patients with HCV infection.a Range 69
Gender (M/F)
53/16
Age (years)
51 2
25–65
139 29
85–480
Alanine aminotransferase (UI/dl)
Viral genotype—n (%) 1–4
46 (67%)
2–3
23 (33%)
HFE genotype—n (%) Wildtype
40 (58%)
HFE mutation
29 (42%)
Serum iron (mg/dl)
127 8
15–312
Serum ferritin (ng/ml)
271 24
18–856
Transferrin saturation index (%)
46%
26–62
Normal range: Transferrin, 110–370 mg/dl. Plus–minus values are means SEM. a Upper normal limit (ULN) of laboratory data: ALT, 41 UI/l; Serum iron, 158 mg/dl; Serum ferritin, 250 ng/ml; Transferrin saturation, 45%.
Table 2 Base-line characteristics of 40 HFE wildtype patients and 29 patients with HFE mutation.a HFE wildtype
RESULTS HFE Genotypes The clinical characteristics of the 69 HCV-infected patients are reported in Table 1. The base-line characteristics of the 69 patients grouped by HFE genotype are shown in Table 2. At least one of the three HFE gene mutations was present in 42% of the patients. One patient was heterozygous for the C282Y mutation (1.4%), 22 were heterozygous for the H63D mutation (31.8%), three (4.3%) were heterozygous for the S65C mutation, two patients (2.8%) were homozygous for the H63D mutation and one patient (1.4%) was a compound heterozygote for the H63D and S65C mutation. No significant differences were observed for age, gender distribution, ALT, serum iron and ferritin levels and viral genotype between HFE mutant patients and wildtype homozygotes. Transferrin saturation index (TSI) showed a significant statistical difference between HFE mutant patients (50%)
N
HFE mutation
P-value
N
40
29
Gender (M/F)
32/8
25/4
ns
Age (years)
50 1
52 2
ns
ALT (UI/dl)
186 59
151 27
ns
AP (U/l)
183 13
209 22
ns
GGT (U/l)
71 8
108 20
<0.05
Bilirubin total (mg/dl)
0.6 0.06
0.8 0.1
ns
Serum iron (mg/dl)
118 9
149 13
ns
Serum ferritin (ng/ml)
280 34
Transferrin saturation(%) 43.4%
327 32
ns
50%
<0.01
4/29 (13.8%)
P = 0.04b
Histological fibrosis Stage I
10/40 (25%)
Stage II
15/40 (37.5%) 5/29 (17.2%)
Stage III
11/40 (27.5%) 10/29 (34.5%)
Stage IV
4/40 (10%)
10/29 (34.5%)
1–4
27 (67.5%)
19 (65.5%)
2–3
13 (32.5%)
10 (34.5%)
Genotype—n (%)
a
ns
Plus–minus values are means SEM. c test.
b 2
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function tests, autoantibody screening including antinuclear factors and anti-thyroid antibodies and serum iron parameters. A complete response at the end of treatment was defined as the normalization of serum ALT levels and negative serum HCV RNA. A sustained complete response was defined as maintenance of remission after cessation of treatment for 24 weeks or more. Patients were divided in two groups according to HFE gene mutations: Group A, which included subjects with HFE gene wildtype and Group B, subjects with HFE gene mutations. Biochemical parameters, iron indices, liver histology, End of Treatment Response (ETR) and Sustained Virologic Response (SVR) were compared between the two groups. Patients who discontinued treatment during follow-up were considered as virologic non-responders. All patients were informed of the purpose of the study and of known side-effects associated with both drugs, and written informed consent for liver biopsy and therapy was obtained.
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End of Treatment Response and Sustained Virological Response Evaluation of Antiviral Therapy
40
100 80 60
ETR
SVR
P=0.005
P=0.03
40 20 0
NR
R HFE mutation
NR
R WT
Figure 2 End of the Treatment Response (ETR) and Sustained Viral Response (SVR) were lower among patients with HFE gene mutations compared to those with HFE gene WT (wildtype) (c2, P = 0.005 and P = 0.03 respectively).
DISCUSSION Interest in the role of iron in CHC began when Di Bisceglie et al found that up to 36% of patients with chronic hepatitis C had elevated serum iron parameters.7 A substantial number of these patients also have increased iron deposition in the liver.21 Furthermore, HCVinfected patients with stainable iron in liver biopsies showed enhanced liver fibrosis compared with controls without detectable iron.22 The hypothesis that iron is a risk factor for liver injury in CHC patients was supported by a recent experimental report that excess dietary iron exacerbated liver injury in HCV-infected chimpanzees.23 The mechanism by which iron accumulates in liver infected with chronic HCV has not yet been established. Di Bisceglie et al reported that serum iron and ferritin levels were increased in patients with CHC because of their release from hepatocellular stores in association with cell necrosis.6 Alternatively, individuals with serum iron levels in the upper range of normal as a result of genetic polymorphisms or a high iron diet may be predisposed to develop more severe chronic HCV infections.6 It has been suggested that iron overload in the liver of patients with CHC is associated with higher ALT levels and decreased response to
35 30
120
25
100
Percentage of subjects
Chronic Hepatitis C
At the end of treatment, both serum ALT levels normalized and HCV RNA became negative in 32 of 69 patients (46%). Seven of 69 patients (10%) did not complete the treatment; these patients stopped prematurely for side-effects associated with the use of PEG-IFN and ribavirin combination therapy (1 for neutropenia and thrombocytopenia, 3 for hypothyroidism and 3 for depression). The relationship between HFE gene mutations and response to antiviral therapy was investigated. At the end of treatment the viral and biochemical response rates were lower among the patients with HFE gene mutations 8/29 (27.5%) compared to those with HFE gene wildtype 30/40 (75%) (c2, P = 0.005) (Figure 2). Of the patients with genotype 1–4 infection, the end of treatment response rate was higher in group A (14/27; 51.8%) compared to group B (2/19; 10.5%) (c2, P = 0.003), while no significant difference was observed between patients with HFE gene mutation (5/10; 50%) and HFE gene wildtype with genotype 2–3 infection (11/13; 77%) (Figure 3). At the end of 24 weeks of post-treatment follow-up period, 32/69 (46.3%) patients produced SVR. The difference of SVR rate between 25/40 patients (62.5%) with HFE wildtype and 7/29 of HFE mutant (24.1%) was statistically significant (c2, P = 0.03) (Figure 3).
120
Percentage of subjects
and wildtype homozygotes (43%) (P < 0.01). The degree of hepatic fibrosis was evaluated by HFE genotype in 69 patients (Figure 1). Thirty-five of 69 (51%) patients had advanced fibrosis (Stage score III–IV); twenty of 29 (69%) patients had a HFE gene mutation and a histological stage III–IV, while only 15 of 40 (39%) HFE wildtype patients presented histological stage III–IV. There was a close association between advanced histological score and the presence of HFE gene mutation (c2, P = 0.04).
20 15 10 5 0 HFE WT Stage I
Stage II
HFE mutation Stage III
Stage IV
Figure 1 Relationship between stage of fibrosis and HFE gene status. A significant association exists between advanced histological score and the presence of HFE gene mutation (c2, P = 0.04). WT, Wildtype. 214
ns
P=0.003
80 60 40 20 0 1-4 NR
1-4 R HFE mutation
2-3 NR
2-3 R
WT
Figure 3 Relationship between HFE gene mutation and Sustained Viral Response (SVR) in 46 patients with viral genotype 1–4 and 23 patients with viral genotype 2–3. NR, NonResponders; R, Responders. © 2012, INASL
interferon.10 These observations indicate the important role of iron in the pathogenesis of CHC. In fact patients with combined hereditary hemochromatosis and chronic hepatitis C infection presented advanced fibrosis/cirrhosis at a younger age and at a lower hepatic iron concentration compared to HH patients. This supports the concept that the combination of iron overload and HCV has a potentiating effect on hepatic fibrogenesis.24 Iron can be a profibrogenic factor, acting as activator of both hepatic stellate cells and Kupffer cells and inducing liver inflammation or hepatocyte necrosis. In fact the presence of stainable iron on liver biopsy of patients with viral hepatitis correlates with a higher degree of necroinflammatory activity and a higher score for fibrosis compared to those with no stainable iron.25 The coexistence of hemochromatosis gene mutations represents a genetic risk factor affecting the severity of chronic hepatitis C and the response rate to interferon therapy.26 28 The results of our study indicate that the prevalence of the three known HFE gene mutations in Sardinia is in accordance with previous observations in other countries: (5–10% for the C282Y mutation and 6–30% for the H63D mutation),29,30 specifically 42% of our HCV-infected patients carried at least one of these mutations. Heterozygous C282Y status and homo- or heterozygosity for the H63D and S65C mutations do not usually induce hemochromatosis phenotype and are rarely associated with liver damage in healthy subjects.31 Nevertheless, laboratory abnormalities of iron metabolism (higher serum iron concentration, transferrin—saturation values and serum ferritin concentrations) have been detected in 15–20% of heterozygotes.6–26 Complications have been recognized only when other disorders, such as alcoholism and viral hepatitis are present.6–26 HFE gene mutations may contribute to iron storage and could represent a clinically relevant comorbid factor in patients with chronic hepatitis C. Sebastiani et al summarize the current status of the literature regarding the prevalence, hepatic distribution of iron overload in liver disease.32 In this review the role of HFE mutations as a risk factor for iron overload in CHC has been studied in different populations, with discordant results. Our results evidenced a correlation between HFE gene mutation and iron overload. TSI is the only serum iron parameter that showed a statistically significant difference between HFE gene wildtype and mutation, while serum iron and serum ferritin were higher in HFE mutation but did not obtain a statistically difference. We can assess that several aspecific factors seem to favor marked variations of serum iron and serum ferritin and we considered TSI as the most specific and sensitive parameter in identifying iron overload. The present study provides evidence supporting the view that the HFE gene mutations are associated with significant abnormalities of iron metabolism and suggests that patients with CHC accumulate iron as the result of interplay between genetic and acquired factors. In fact
transferrin saturation index (TSI) showed a significant statistical difference between HFE mutant patients (50%) and wildtype homozygotes (43.4%) (P < 0.01). Instead, no significant difference was observed for other indirect markers of iron stores such as serum iron and serum ferritin. These parameters lack specificity, particularly in the face of chronic inflammatory conditions or alcohol-induced liver disease.27 Previous reports suggested that TSI measurement appears to be the most sensitive method of detecting iron overload states and showed that TSI values exceeding 45% correctly identified 97.9% of homozygotes for Hereditary Hemochromatosis, with no false positives among the normal population, and 22.2% of the heterozygote population. Detecting the threshold for TSI to 45% could also identify other groups with relatively minor degrees of secondary iron overload, such as chronic hepatitis C.33,34 Recent evidence suggests that HFE gene mutations and a consequent mild iron overload may worsen the course of chronic hepatitis C and increase the progression of fibrosis.22–35 The results of our study showed that a significant association exists between advanced histological score and the presence of HFE gene mutation (c2, P = 0.04). The fact that patients heterozygote for hereditary hemochromatosis and infected with hepatitis C virus report greater fibrosis than those with homozygous wildtype provides additional evidence that iron modulates fibrosis in chronic hepatitis C. Despite these observations, the association between serum iron values, hepatic iron stores and hepatic necroinflammatory activity or fibrosis remains controversial. HFE gene mutations may have a potentiating effect on histological severity, acting synergically with CHC in the development of liver damage. Iron overload seems to impair antigen-specific immune responses by decreasing the generation of T cells and by impairment of natural killer and T helper cell function. Piperno et al 3 suggested that iron overload in patients with hemochromatosis may contribute to the persistence of HCV infection: iron overload may in theory promote viral replication. Moreover, iron overload has been incriminated as one of the essential factors that hamper response to interferon-alpha in CHC.3–36 The amount of hepatic iron has been identified as one of these factors that adversely affect the likelihood of response to interferon-alpha; those patients with higher hepatic iron content are less likely to respond to interferon therapy.37 Retrospective evidence that the amount of hepatic iron may modulate the response of the hepatitis C virus to interferon therapy was provided by Van Thiel et al13 who reported that hepatic iron content of interferon nonresponders was found to be almost twice that of responders. The influence of host and viral factors on the natural course of CHC and efficacy of PEG-IFN and Ribavirin therapy has been intensively studied. The presence of HFE gene mutations may be an additional factor to be considered among those implicated in the determination of a lower rate of sustained virological
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response to PEG-IFN plus ribavirin in chronic hepatitis C patients. In the present study, ETR rates were lower among patients with HFE gene mutations compared to those with HFE gene wildtype (P = 0.005). In patients with 1–4 HCV genotype infection, the ETR rate was higher in group A (51.9%) as compared with group B (P = 0.003). No significant response difference was observed between patients with HFE gene mutation and HFE gene wildtype in genotype 2–3. The small number of non-1–4 genotype patients could have accounted for the lack of statistical significance between the two groups. A sustained virological response was seen in 18.8%. The patients with HFE wildtype produced significant SVR compared with patients with HFE mutations (P = 0.03). Despite the number of patients in our study being too limited for meaningful statistical analysis, several comments can be made concerning the end of treatment and sustained responders. We have observed that PEG-IFN/ribavirin therapy is less effective in patients with 1–4 HCV genotype and HFE gene mutation. Moreover, in our patients with genotype 1–4 infection, TSI was significantly higher than those with genotype 2–3 infection and viral genotype distribution did not differ significantly between HFE mutant patients and wildtype homozygotes. Therefore, HFE gene mutations may act synergically with CHC in the development of liver damage, predicting a higher rate of non-response to PEG-IFN–ribavirin therapy. We suggest that screening for HFE mutations and iron parameters should be considered in patients with CHC. Therapies that specifically reduce iron levels should be designed to lessen the severity of HCV infection in patients with HFE gene mutation and to enhance the outcome of antiviral therapies.
CONFLICTS OF INTEREST There are no commercial associations (e.g. consultancies, stock ownership, equity interests, patent licensing arrangements, etc) with Margherita Sini, Orazio Sorbello, Alberto Civolani and Luigi Demelia that pose a conflict of interest in connection with the submitted article entitled “Hemochromatosis gene mutations: prevalence and effects on pegylated-interferon and Ribavirin therapy response in chronic hepatitis C in Sardinia”.
ACKNOWLEDGMENTS We acknowledge Dr. Barry Mark Wheaton for his expertise and help in the execution of this paper with gratitude. REFERENCES 1. Finch C. Regulators of iron balance in humans. Blood. 1994;84: 1697–1702. 2. Britton RS. Metal-induced hepatotoxicity. Semin Liver Dis. 1996 Feb;16(1):3–12.
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3. Piperno A, Fargion S, D'Alba, et al. Liver damage in Italian patients with hereditary hemochromatosis is highly influenced by hepatitis B and C virus infection. J Hepatol. 1992;16(3):364–368. 4. Riedel HD, Stremmel W. The haemochromatosis gene. J Hepatol. 1997;26:941–944. 5. Holmstrom P, Marmur J, Eggertsen G, et al. Mild iron overload in patients carrying the HFE S65C gene mutation: a retrospective study in patients with suspected iron overload and healthy controls. Gut. 2002;51(5):723–730. 6. Merryweather-Clarke AT, Pointon JJ, Jouanolle AM, et al. Geography of HFE C282Y and H63D mutations. Genet Test. 2000;4(2):183–198. 7. Candore G, Mantovani V, Balistreri CR, et al. Frequency of the HFE gene mutations in five Italian populations. Blood Cells Mol Dis. 2002 Nov–Dec;29(3):267–273. 8. Smith BC, Gorve J, Guzail MA, et al. Heterozygosity for hereditary hemochromatosis is associated with more fibrosis in chronic hepatitis C. Hepatology. 1998;27:1695–1699. 9. Kazemi-Shirazi L, Datz C, Maier-Dobersberger T, et al. The relation of iron status and hemochromatosis gene mutations in patients with chronic hepatitis C. Gastroenterology. 1999;116:127–134. 10. Martinelli AL, Franco RF, Villanova MG, et al. Are haemochromatosis mutations related to the severity of liver disease in hepatitis C virus infection? Acta Haematol. 2000;102:152–156. 11. Bulaj Zaneta J, Griffen Linda M, Jorde Lynn B, Edwards Corwin Q, Kushner James. Clinical and biochemical abnormalities in people heterozygous for hemochromatosis. N Engl J Med. 1996;335: 1799–1805. 12. Di Bisceglie AM, Axiotis CA, Hoofnagle JH, Bacon BR. Measurements of iron status in patients with chronic hepatitis. Gastroenterology. 1992;102:2108–2113. 13. Van thiel DH, Friedlander L, Fagiuoli S, et al. Response to interferon alfa therapy is influenced by the iron content of the liver. J Hepatol. 1994;20:410–415. 14. Farinati F, Carin R, De Maria N, et al. Iron storage, lipid peroxidation and glutathione turnover in chronic anti-HCV positive hepatitis. J Hepatol. 1995;22:449–456. 15. Tung BY, Emond MJ, Bronner MP, Raaka SD, Cotler SJ, Kowdley KV. Hepatitis C, iron status and disease severity: relationship with HFE mutations. Gastroenterology. 2003;124:318–326. 16. Geier A, Reugels M, Weischirken R, et al. Common heterozygous hemochromatosis gene mutations are risk factors for inflammation and fibrosis in chronic hepatitis C. Liver Int. 2004;24(4):285–294. 17. Hohler T, Leininger S, Kohler HH, Schirmacher P, Galle PR. Heterozygosity for hemochromatosis gene in liver diseases: prevalence and effects on liver histology. Liver. 2000;20(6):482–486. 18. Olynyc JK, Reddy KR, Di Bisceglie AM, et al. Hepatic iron concentration as a predictor of response to interferon alpha therapy in chronic hepatitis C. Gastroenterology. 1995;108:1104–1109. 19. Simmonds P, Alberti A, Alter HJ, et al. A proposed system for the nomenclature of hepatitis C viral genotypes (letter). Hepatology. 1994;19:1321–1324. 20. Desmet VJ, Gerber MA, Hoofnagle JH, Manns M, Scheuer PJ. Classification of chronic hepatitis: diagnosis, grading and staging. Hepatology. 1994;19:1513–1520. 21. Sartori M, Andorno S, La Terra G, et al. Evaluation of iron status in patients with chronic hepatitis C. Ital J Gastroenterol Hepatol. 1998;30:396–401. 22. Casaril M, Stanzial AM, Tognella P, et al. Role of iron load on fibrogenesis in chronic hepatitis C. Hepatogastroenterology. 2000;47: 220–225. 23. Bassett SE, Di Bisceglie AM, Bacon BR, et al. Effects of iron loading on pathogenicity in hepatitis C virus-infected chimpanzees. Hepatology. 1999;29:1884–1892. 24. Diwaakaran HH, Befeler AS, Britton RS, Brunt EM, Bacon BR. Accelerated hepatic fibrosis in patients with combined hereditary
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25. 26.
27.
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