Introduction

Hepatitis C virus (HCV) infection affects approximately 170 million individuals worldwide (Madhava et al. 2002). Iron parameters, especially ferritin, are frequently increased in patients with chronic hepatitis C suggesting an association between chronic hepatitis C and iron overload. However, it is yet unclear whether elevation of iron indices is an epiphenomenon of HCV-induced liver injury, liver inflammation, and virus replication, or whether elevation of iron parameters and HFE mutations may play a role in the evolution of HCV induced liver disease (Ishizu et al. 2012). Hereditary hemochromatosis (HH) is an autosomal recessive genetic disorder. HH is characterized by increased iron absorption from the small intestine and an increased iron deposition in parenchymal organs leading to multiorgan failure (Niederau et al. 1985).

A candidate gene of HH, termed HFE, has been identified on chromosome 6, coding for a protein homologous to MHC class I molecule. Two major mutations of the HFE gene, an exchange of cysteine to tyrosine at amino acid 282 (C282Y) and histidine to aspartic acid at amino acid 63 (H63D), are thought to be responsible for about 90 % of cases with HH (Erhardt et al. 2003).

The aims of this research are to determine the prevalence of C282Y and H63 HFE mutations among chronic HCV patients and to determine whether elevation of iron indices is related to HFE gene mutations in patients with chronic hepatitis C.

Subject and methods

This study is a cross-sectional study. It was carried out at the Faculty of Medicine, Cairo University. The study was approved by the Cairo University Clinical Research Ethics Committee, and informed consents were obtained from all participants according to the Helsinki guidelines.

The study population was 80 chronic HCV patients divided into two groups: group I, 40 patients with serum iron overload diagnosed by elevated transferrin saturation >50 % and elevated serum ferritin (Mainous et al. 2011), and group II, 40 patients without iron overload.

All included patients were with compensated chronic liver disease and documented as HCV-positive RNA and negative for HBsAg.

All patients were subjected to complete history taking and full clinical examination that was done by the hepatology unit specialists. All parameters of the liver function tests (total bilirubin, direct bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), total protein, albumin, alkaline phosphatase) and iron profile (serum iron, total iron binding capacity (TIBC), ferritin and serum transferrin saturation) were done on Cobas Integra 400 auto analyzer.

HFE mutation studied by PCR-RFLP

Genomic DNA was extracted using QIA amp DNA Mini and QIA amp DNA Blood Mini Kits (catalog number 51104) provided by QUIAGEN (Qiagen Strasse 1–40724 Hilden, Germany) as described by the manufacturer.

Human genomic DNA samples were subjected to PCR amplification by Taq PCR Master Mix kit 2X concentrated provided by Qiagen catalog no. 201445 containing ready to use PCR beads and using the hybrid PCR express thermo cycler (Perkin Elmer 9600).

The primers sequence was as follows: C282Y forward primer 5′-CAAGTGCCTCCTTTGGTGAAGGTGACACAT-3′, reverse primer 5′-CTCAGGCACTCCTCCTCTCAACC-3′; H63D forward primer 5′-ACATGGTTAAGGCCTGTTGC-3′, reverse primer 5′-CCTGCTGTGGTTGTGATTTTCC-3′. Expected amplified fragment 390 and 208 bp, respectively Forward and reverse primers were supplied by Midland Certified Reagent Company, 3112 West Cuthbert Avenue Midland, TX. 79701–5511 PCR. Amplification mix was 25 μL master mix, 2 μL of each primer (10 pmol/μL), 16 μL deionized water, and 5 μL genomic DNA, cycling program 2 min at 94 °C, 35 cycles as follows, 1 min at 94 °C, 1 min at 58 °C, 1 min at 72 °C and finally 72 °C for 10 min.

The amplified fragments were digested by Rsa1 and Bcl1 restriction enzyme for C282Y and H63D gene mutation (Calado et al. 2000; Saiki et al. 1988). Detection of the digested products was carried out by agarose gel electrophoresis. The gel was visualized by ultraviolet trans-illuminator and documented by Polaroid camera photography. The amplified bands appear as a single band of 390 and 208 bp for C282Y and H63D, respectively. Amplification with the primers for codon 282 produced a PCR product of 390-bp fragment which is digested into 250 and 140 fragments in the wild allele, but the mutant allele will be digested into 250, 111, and 29 bp; however, the 29-bp band is always invisible being lost early in the run. On the other hand, amplification for codon 63 gave a 208-bp product which is digested into 138 and 70 bp fragments in the wild allele, while the mutant allele will resist digestion giving the same size band (208 bp) (Figs. 1 and 2).

Fig. 1
figure 1

Detection of HFE C282Y mutation by PCR-RFLP with C282Y primers and digested with RsaI. Lane L, DNA ladder; lanes 1–9, normal wild C282 allele (250/140)

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Fig. 2
figure 2

Detection of HFE H63D mutation by PCR-RFLP with H63D primers and digested with Bcl1. Lane L, DNA ladder; lanes 3, 5, 6, 8, and 9 show normal wild H63D allele digested bands (138/70); lanes 1, 2, 4, and 7, heterozygote mutant allele showing normal digested bands as well as the undigested amplified fragment (208)

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Statistical methods

Data were statistically described in terms of mean ± standard deviation (±SD), frequencies (number of cases), and percentages when appropriate. All statistical calculations were done using computer programs Microsoft Excel 2003 (Microsoft Corporation, NY, USA) and Statistical Package for the Social Science (SPSS Inc., Chicago, IL, USA) version 15 for Microsoft Windows.

Student’s t test was done to compare means, Pearson’s bivariate correlation test was done to study correlation between different scale variables, and chi-squared test was done to test the relationships between different ordinal variables.

The results were then tabulated and figured using MS Office module and SPSS statistical package.

Results

Eighty chronic liver disease patients with HCV infection were enrolled in this study. They were classified into two equal groups; one group with iron overload and the other group with normal iron profile. They were matched for age and sex. Mean age was 50.3 ± 6.1 and 48.8 ± 5.5 in the iron overload and non-iron overload groups, respectively. There were 31 males and 9 females in the iron overload group and 26 males and 14 females in the non-iron overload group. In the iron profile data, serum iron was 232.6 ± 31.9 and 101.8 ± 19.3 μg/dL, TIBC was 346.6 ± 44.6 and 336.2 ± 46.1 μg/dL, ferritin was 375.8 ± 64.7 and 147.8 ± 66.8 μg/dL, and transferrin saturation was 67.6 ± 8.8 and 30.4 ± 5.0 % in the iron overload and non-iron overload groups, respectively. A significant difference was present regarding serum iron, ferritin, and transferrin saturation (P < 0.01).

Analysis of different biochemical parameters in the two study groups. There was a significant difference between iron overload and non-iron overload groups regarding AST, ALT, and total and direct bilirubin. The mean AST was 86.7 ± 20.7 U/L in the iron overload group and 70.5 ± 11.0 U/L in the non-iron overload group (P < 0.05). The mean ALT was 76.9 ± 15.2 and 62.6 ± 10.7 U/L in the iron overload and non-iron overload groups, respectively (P < 0.05). Regarding total and direct bilirubin, there was also a significant difference between the studied group where total bilirubin was 1.7 ± 0.9 and 1.2 ± 0.4 mg/dL and direct bilirubin was 0.8 ± 0.9 and 0.3 ± 0.2 mg/dL in the iron overload and non-iron overload groups, respectively (P < 0.05).

Regarding the HFE gene mutation testing done for study patients, the C282Y mutation was not found in any of the 80 patients, while the H63D mutation (heterozygous only) was found in 15 out of 80 patients (18.7 %). Eight patients (20 %) were found out of the 40 iron overload group patents and 7 patients (17.5 %) were in the non-iron overload group. Statistically, there was no significant relationship between the two studied groups regarding the presence of H63D mutation (P > 0.05) Table 1.

Table 1 Relationship between the carriage of C282Y and H63D mutations and iron overload
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Demographic and laboratory data in the study population was classified according to the presence of H63D mutation. The mutant group (n = 15) was comparable to the wild group (n = 65) regarding age, gender, iron profile data, and liver function tests data except alkaline phosphatase which showed a significant difference where it was 102.4 ± 30.3 in the mutant group and 120.7 ± 27.1 in the wild group (P < 0.05) (Tables 2 and 3).

Table 2 Demographic and laboratory data of studied patients divided according to gene type (mutant vs. wild)
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Table 3 Iron profile data among studied patients divided according to H63 gene mutation
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Bivariate correlation analysis was done for biochemical parameters in the entire study sample correlation between transferrin saturation and some of the laboratory data where there was a positive correlation with AST, ALT, total bilirubin, and direct bilirubin (P < 0.01). There was correlation between serum ferritin and liver function tests where positive correlation was found with AST, ALT, total bilirubin (P < 0.01) and direct bilirubin (P < 0.05) (Table 4).

Table 4 Correlation between transferrin saturation (%) and different laboratory measures in all studied patients
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Discussion

HFE mutations are the major gene variations in HH which is a common autosomal recessive disorder associated with iron overload in Caucasians. Therefore, there has been much interest in the roles of HFE mutations in patients with HCV infection. Several studies have been performed in order to assess the correlations among HFE mutations, hepatic iron overload, and disease progression in CHC. However, the effects of HFE mutations on hepatic iron concentration and disease severity remain controversial (Ishizu et al. 2012).

Although there is evidence that a mild to severe iron overload is common among patients with HCV chronic liver disease (Corengia et al. 2005), the significance of these observations in relation to the pathogenesis of liver injury and relevance to treatment remains uncertain (Bassett 2007).

The research methodology conducted in previous studies to track the relationship between HFE mutation and iron overload in HCV patients searched for mutations in HCV patients then studied their iron profile. In the current study, the methodology was different where we screened chronic HCV patients for the presence of iron overload and discriminated those having fasting transferrin saturation >50 % and elevated serum ferritin and picked them as our main study group. In addition, a sex and age-matched group of HCV patients with no iron overload was added to the study, and all patients were examined for the presence of HFE mutations. When we come to compare the results of the current study to the worldwide studies regarding HFE mutations in different communities, there were considerable variations according to different ethnic groups and races.

Absent C282Y mutations in the current study was in accordance with Roth et al. (1997) who showed absence of C282Y mutation in Algeria, Ethiopia, and Senegal and attributed this to the Celtic origin of the disease. Similar findings were found in Korean people in a study carried out by Lee et al. (2010).

On the other hand, Smith et al. (1998) found that the prevalence of C282Y heterozygous mutation among their HCV-positive English patients was 10 of 137 (7.3 %), and no homozygous ones were detected. Similarly, 13.7 % of their healthy controls were heterozygous and 1 % was homozygous. Still in Europe, in an open population screening study in Italy, Floreani et al. (2007) found that 2 % of their population had C282Y mutations; all of them were heterozygous. The same result of 2 % prevalence was obtained in Northern Italy by Valenti et al. (2007).

In another racial descent, a Brazilian study by Martinelli et al. (2000) found 4.4 % of their population with C282Y mutation, while Dhillon et al. (2007) found no C282Y mutations in their Indian study.

This may be explained by the geographic distribution of C282Y which was found to be absent in African and Asian populations and is restricted to north European population (Acton et al. 2006).

Regarding the prevalence of H63D mutation, there was no big difference between the current study and worldwide studies. While the prevalence in the current study was 18.7 %, Martinelli et al. (2000) from Brazil found that 23.7 % of patients and controls had H63D mutation. Also, in accordance with our results, the prevalence of H63D mutation in Valenti et al. (2007) in Italy was 25 %. A less prevalence was found in Asia where Dhillon et al. (2007) in Northern India found a prevalence of 13.98 %, and Lee et al. (2010) found a prevalence of 9.8 % in Korea. In Japan, the prevalence of H63D mutation was found to be 5.6 % (Ishizu et al. 2012). Regarding iron studies, results of the current study revealed no significant difference between chronic HCV patients with iron overload and those with normal iron profile regarding any of the HFE mutations where our positive mutation H63D heterozygosity was found in 20 % of the iron overload group and 17.5 % of the non-iron overload group (P > 0.05). Also, when dividing our patients into two groups according to H63D mutation, namely wild group (n = 65) and mutant group (n = 15), statistical comparison of serum iron indices between those two groups showed non-significant difference (P > 0.05).

Won et al. (2009), Elbahaie et al. (2008), Floreani et al. (2007), and Valenti et al. (2007) showed results that were in accordance with the results of the current study where they stated that the comparison between HCV patients carrying HFE wild type and those carrying H63D heterozygotes did not show a significant difference in the degree of intrahepatic iron deposition and serum levels of ferritin or TS. Dhillon et al. (2007) reported that H63D mutation is not associated with iron overload even in the homozygous state. In an open-population screening in Central Italy, Floreani et al. stated that there was no higher incidence of H63D mutation in people with iron overload than those with normal iron profile.

Another work that completely denies relationship between HFE mutation and iron overload in HCV patients is the work of Thorburn et al. (2002) who concluded that patients with chronic HCV infection frequently have elevated serum iron markers, although elevated liver iron concentration are uncommon. Elevated serum iron studies and liver iron concentrations occur in patients with more severe liver disease. Carriage of HFE mutations, although frequently observed in these HCV-infected patients, does not have a role in the accumulation of iron or the progression of liver disease in HCV infection. They stated that carriage of HFE mutations was not associated with any clinical, biochemical, virological, or pathological features, including accumulation of liver iron.

Experimental and clinical studies suggest that excess iron might exacerbate liver injury in patients with chronic hepatitis C, increasing the risk of hepatic fibrosis and cirrhosis (Meli et al. 2013; Bomfim et al. 2013; Bougle and Brouard 2013) favoring the development of hepatocellular carcinoma (Chapoutot et al. 2000) and preventing a sustained virologic response to antiviral therapy (Piperno et al. 1996; Fargion et al. 1997), although other studies denied such relationships (Hézode et al. 1999; Thorburn et al. 2002).

In conclusion, the current work emphasizes that the C282Y mutation is absent in our community, while presence of H63D mutation does not differ greatly from other Caucasian races especially in Europe. The current study did not detect any effect of HFE mutation on increasing serum iron indices. The controversies about the relation between HFE mutations and iron overload in chronic HCV patients still needs more studies considering novel factors and mutations involved in such mechanism such as hepcidin, ferroportin, hemojuvelin, β-globin, and transferrin receptor-2 (TFR2) (Harigae 2013).