Hepatitis C virus genotype 4 (HCV-4) is mainly found in Egypt, the country with the highest prevalence of HCV worldwide (15%), where HCV-4 represents 90% of all cases with HCV (1). For genotype 4–infected patients, the most effective therapy to eradicate the virus consists of a combination of pegylated interferon (PEG-IFN)-α and ribavirin (RBV). Unfortunately, the rate of sustained virological response (SVR) is approximately 50% in genotype 1– and genotype 4–infected patients (2). In Egypt, there is a predominance of subtype 4a (55%). Because a significant number of patients will fail to respond or will have significant adverse effects, it is of major interest for both patient care and economic approach to predict nonresponse (3).
Cytokines represent a large family of molecules, including the chemokine, interleukins (ILs), IFNs, and members of the tumor necrosis factor family, all of which play an important role in the initiation and regulation of immune responses. Therefore, they may affect susceptibility to and/or the natural course of HCV infection. Many studies show that the immunity level of the host correlates with relevant gene polymorphisms, especially with SNPs in the promoter region that regulate gene expression. The gene polymorphisms probably determine the outcome of the infection. Several novel cytokines of the IL-10–like cytokine family have been discovered, including IFN-λs (4). IFN-λs are related to IL-10 and other members of the IL-10–like family, which have been shown to confer hepatoprotection (5,6). IL-10 is a multifunctional cytokine with potent immunoregulatory and anti-inflammatory properties. It prevents the release and function of a number of proinflammatory cytokines.
The role of polymorphism in IL-10 in clearance of HCV infection has been investigated, but some of these results are conflicting (7). IL-10 is also a T helper 2 (TH2)–type cytokine, and it has been suggested that an imbalance of the TH1/TH2 immune response could influence the clinical outcome and disease progression (8).
Allelic variants in the IL-28B gene have gained major scientific interest because several genome-wide association studies identified a panel of SNPs on chromosome 19q13 to be strongly associated with treatment-induced and spontaneous clearance of hepatitis. Genome-wide association studies have identified genetic variations near the IL-28B gene, which are strongly associated with spontaneous and treatment-induced clearance of HCV infection (9).
Generally, studies evaluating the natural history of HCV infection in Egyptian pediatric patients with HCV are lacking. Therefore, the aim of the present study was to investigate the relation of SNPs in IL-10 (−1082, −819, −592) and IL-28B (rs12979860) genes in Egyptian pediatric patients with chronic HCV infection with their efficacy in predicting the response to PEG-IFN-α/RBV therapy.
This study is a cohort study of 34 Egyptian pediatric patients (23 boys and 11 girls) of mean age 13. 8 ± 1.7 years (mean ± standard deviation), with chronic HCV-4 patients who attended the outpatient's clinic of the hepatology unit at Abou El Reesh Hospital, Cairo University. The patients are the entire cohort of patients treated with PEG-IFN/RBV from August 2011 to August 2012. In addition, 20 healthy children, 14 boys and 6 girls, ages 13.25 ± 1.8 years, were evaluated as controls. An informed consent was obtained from mothers of all enrolled children in this study. The study protocol was approved by the ethical committee of the Faculty of Medicine, Cairo University and conformed to the ethical guidelines of the 1975 Declaration of Helsinki. The criteria used to determine hepatitis C therapy were in accordance with international guidelines. Chronic HCV infection was diagnosed by the persistence of anti-HCV and HCV RNA in serum for >6 months before therapy. Exclusion criteria included evidence of hepatitis B surface antigen (HBsAg) and human immunodeficiency virus antibodies and autoimmune liver disease by presence of ANA (anti-nuclear antibodies) titer >1/160.
All pediatric patients were treated with a subcutaneous injection of PEG-IFN-α2b (60 μg · m−2 · week−1) once per week in combination with a weight-adjusted dose of oral RBV (15 mg · kg−1 · day−1) for 48 weeks. According to the response to treatment, patients were classified into 2 groups: responders (20 patients) and nonresponders (14 patients). SVR is defined as an undetectable HCV RNA upon completion of treatment and 24 weeks thereafter on repeated quantitative reverse transcription polymerase chain reaction (RT-PCR) testing. Our regimen of treatment is similar to that of Wirth et al (10).
Blood Sampling and Laboratory Assays
Venous blood samples (∼5 mL) were collected from patients as well as controls before the start of the treatment. Serum was separated and used for the assessment of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, direct bilirubin, HCV-RNA, HCV-specific antibody titers, albumin, and α-fetoprotein. Another portion of blood was collected in vacutainer tubes containing citrate to separate plasma used for assay of prothrombin time. Assays were performed using Roche Hitachi Chemistry Analyzer (Roche Diagnostics, Indianapolis, IN). A third portion of blood collected in ethylenediaminetetraacetic acid tubes was stored at −80°C until molecular assays.
Viral RNA was extracted using viral RNA extraction kit (Qiagen, Millipore Corp, Billerica, MA) and stored at −80°C. DNA was extracted from blood in ethylenediaminetetraacetic acid tubes for genotyping of IL-10 and IL-28B. The serum HCV-RNA level was measured using a real-time (RT)-PCR Kit (TaqMan assay reagents and Ambion; the RNA Company-one step RT-PCR kit, Applied Biosystems, Carlsbad, CA). The HCV genotype was determined on the basis of the sequence of the core region. Hepatitis B surface antigen, hepatitis B core antigen, and anti-HCV were assessed by routine methods using commercially available assays. Thyroid function tests (T3, T4, thyroid-stimulating hormone ) were performed in all patients before receiving treatment using the Immulite analyzer (Siemens, Munich, Germany). ANA and anti-DNA were done using commercially available immunofluorescence kits.
Genotyping of SNPs in IL-28 rs12979860 and IL-10 (−1082, −592, −819)
Genomic DNA was prepared from peripheral blood lymphocytes using the QIAamp DNA minikit (Qiagen) following the manufacturer's instructions. The 3 SNPs at the IL-10 gene promoter −592, −819, and −1082 sites were detected using primers that amplify a short fragment of DNA containing the polymorphism. The primers’ sequences were F: 5′-CCTAGGTCACAGTGACGTGG-3′ and R: 5′-GGTGAGCACTACCTGACTAGC-3′ for detection of IL-10-592 (A/C), F:5′-TCATTCTATGTGCTGGAGATGG-3′ and R:5′ TGGGGGAAGTGGGTAAGAGT-3′ for detection of IL-10-819 (T/C), and F: 5′-CTCGCTGCAACCCAACTGGC-3′ and R: 5′-TCTTACCTATCCCTACTTCC-3′ for detection of IL-10 −1082 (G/A). The digestion products of all alleles were visualized on a 3% ethidium bromide–stained agarose gel for visualization on a ultraviolet transilluminator. For rs12979860, SNP was screened using TaqMan SNP genotyping assays (Applied Biosystems) in Qiaplex thermal cycler.
Data were statistically described in terms of mean ± standard deviation, frequencies (number), and percentages when appropriate. Comparison of numerical variables between the study groups was done using the Student t test for independent samples in comparing 2 groups when normally distributed and the Mann-Whitney U test for independent samples when not normally distributed. Comparison of numerical variables between more than 2 groups was done using 1-way analysis of variance test with post hoc multiple 2-group comparisons in comparing normal data and the Kruskal-Wallis test when data were not normal. For comparing categorical data, the χ 2 test was performed. The Fisher exact test was used instead when the expected frequency was <5. Hardy-Weinberg equilibrium was tested for the studied genes. Accuracy was represented using the terms sensitivity and specificity. Receiver operating characteristic analysis was used to determine the optimum cutoff value for the studied prognostic markers. P values <0.05 were considered statistically significant. All statistical calculations were done using SPSS (SPSS Inc, Chicago, IL) version 15 for Microsoft Windows.
Six months after treatment with INF, children with chronic hepatitis C (CHC) were classified into nonresponders (11) and responders (12). No statistically significant difference was found with regard to age; it was 13 ± 1.5 years in nonresponders versus 13.8 ± 1.7 years in responders (P = 0.1). Also, the sex was 10 males and 4 females in nonresponders versus 13 males and 7 females in responders (P = 0.2). The baseline (pretreatment) liver enzymes alanine aminotransferase and aspartate aminotransferase were 62.6 ± 27.2 and 62.5 ± 38.4 in nonresponders and 80.2 ± 42.6 and 82.5 ± 57.5 in responders, respectively (P = 0.1 and 0.2, respectively) and HCV RNA viral load was <600,000 IU/mL in both groups with no significant difference (P = 0.3). Also, no significant difference was found regarding route of infection in both groups as blood transfusion. There were no patients with previous malignancies or immune deficiencies.
The distribution of SNPs in IL-28B gene (rs12979860) in the HCV group was 15 of 34 for CC, 14 of 34 for CT, and 5 of 34 for TT (Table 1) (11,12). No statistically significant difference in SNPs in the IL-28B gene (rs12979860) of hepatitis C patients and controls was observed: CC was 10 of 20, CT was 7 of 20, and TT was 3 of 20 (P = 0.897). Meanwhile, there was a statistically significant difference in the prevalence of SNPs in the IL-28B gene (rs12979860) between nonresponders and responders with regard to genotypes (CC, CT, and TT), P = 0.04, and the alleles (C and T), P = 0.02 (Table 1). With a statistically significant difference between responders and nonresponders regarding CC genotype and CT/CC genotypes (P = 0.02), CC genotype patients are 2 times more likely to develop SVR than those with CT/TT genotypes (Table 1).
The distribution of SNP of IL-10 (−1082) in the studied group was 5 of 34 for GG, 15 of 34 for GA, and 14 of 34 for AA. No statistically significant difference was detected between hepatitis C patients and controls with regard to genotypes. Table 2 showed no statistically significant difference in the prevalence of SNP of IL-10 (−1082) between nonresponders and responders with regard to genotypes (GG, GA, and AA), or the alleles (G and A) (P > 0.05). Also, there was no significant difference regarding the genotype GG and genotypes GA/AA (11,12).
The distribution of SNP of IL-10 (−592) in the diseased group was 6 of 34 for CC, 17 of 34 for CA, and 11 of 34 for AA. There was no statistically significant difference between hepatitis C patients and controls with regard to genotypes. Table 3 showed no statistically significant difference in the prevalence of SNP of IL-10 (−592) between nonresponders and responders with regard to genotypes (CC, CA, and AA) or regarding alleles (P > 0.05). Also, no statistically significance difference was shown between nonresponders and responders regarding CC genotype and the CA/AA genotypes (P > 0.05).
The distribution of SNP of IL-10 (−819) in the studied group was 13 of 34 for TT, 6 of 34 for CT, and 15 of 34 for CC (Table 4). No statistically significant difference was found between hepatitis C patients and controls with regard to genotypes (TT, CT, CC). Table 4 showed a statistically significant difference in the prevalence of SNP in the IL-10 (−819) gene between nonresponders and responders with regard to genotypes (CC, CT, TT; P = 0.04) and alleles (P = 0.01). A statistically significant difference was shown between nonresponders and responders with regard to genotypes CC and CT/TT. There was a strong association (odds ratio 7.5) between TT genotype with response.
Egypt has the highest prevalence of adult HCV infection in the world (15%–25%). The main (90%) HCV genotype is type 4 (13). Many immunological proteins may play a role in the response to HCV antiviral treatment, in particular, IFNs. Polymorphisms in several genes have been shown to be associated with HCV clearance or persistence (14). Studies have indicated that SNPs in the IL-10 gene may influence the response to IFN-α treatment in patients with chronic HCV (15).
An SNP (rs12979860) was shown to associate strongly with a >2-fold difference in response to HCV drug treatment (9). There is a paucity of data and published studies regarding IL-10 and IL-28B SNPs and response to HCV drug treatment in children with HCV-4.
The present study was conducted on 54 pediatric subjects divided into 2 groups: group I, which included 34 patients with CHC virus who received PEG-IFN-α-2b /RBV, and group II (controls), which included 20 healthy individuals. At the end of treatment, 20 were responders (58.8%) and 14 (41.2%) were nonresponders. International studies showed that SVR rates vary according to the genotype of HCV.
Schwarz et al (14) did studies on children with different HCV genotypes; their results regarding genotype 1 showed that SVR rates ranged from 44% to 59%, whereas regarding genotypes 2/3, their results were >90%. Also, the results of Sokal et al (15) coincided with ours because they found that PEG-IFN-α-2a and RBV treatment allowed them to achieve SVR in 57% of pediatric patients with genotypes 1, 4, 5, and 6. Other studies showed that of patients with genotype 4 treated with PEG-IFN-α and RBV, 63% had SVR (2), which reflects that our results on pediatric population with genotype 4 yielded nearly the same results as an adult population of the same genotype.
The present study revealed a statistically significant difference between responder and nonresponder groups (P = 0.04) with regard to SNP IL-28B (rs12979860). The CC genotype had higher percentages in responders than in nonresponders (60% and 21.4%, respectively), whereas genotypes CT/TT were 40% in responders and 78.6% in nonresponders (P = 0.02). There is a higher frequency for C allele (75%) than that for T allele (25%) in patients who responded to HCV therapy (P = 0.02). These findings agreed with Thomas et al (16) who did their studies on adults of European and African ancestry. They showed that patients with the CC genotype were associated with better treatment response and were 3 times more likely to clear HCV relative to patients of European and African ancestry with the CT and TT genotypes.
Our results are consistent with multiple studies that have shown that adult patients with CHC carrying the C/C genotype have a 2-fold greater chance of SVR than patients with the CT/TT genotypes (9,17,18). The underlying mechanism for this finding is not clear because rs12979860 CC does not seem to negatively influence the replication of HCV, at least in untreated patients. This SNP has strong linkage disequilibrium with a nonsynonymous coding variant in the IL-28 gene. Thus, it is possible that changes in rs12979860 genotype are associated with abnormalities in the IFN-λ3 signal transduction pathway, although functional data are lacking (9). IFN-λ1, which inhibits HCV replication, increases the levels of INF-stimulated genes and enhances the antiviral effect of INF-α (12). It is conceivable that IFN-λ3, a closely related cytokine with activity against other viruses comparable with that of IFN-λ1, works in a similar way against HCV (19).
Our findings for IL-28B gene (rs12979860) in pediatric population are in agreement with other studies of the same gene on adults, and accordingly hypothesize that the underlying mechanism of action of IL-28B in HCV infection and the role of its polymorphism may be the same in both adults and children.
In the present study, we studied the SNPs in the promoter region of IL-10 at positions −1082(G/A), −592(C/A), and −819(C/T) in the Egyptian pediatric population. Results showed no statistically significant difference with regards to the percentage distribution of genotypes and allele frequencies between responders and nonresponders in SNPs −1082(G/A) and −592(C/A). Meanwhile, SNP at −819(C/T) revealed statistically significant difference between responders and nonresponders (P = 0.04).
Patients with genotypes −819 CT/TT develop SVR, as opposed to those with the −819 CC genotype (odds ratio 7.5), which suggests a protective value of −819 T allele whether in the homozygous or heterozygous form.
Because of scarceness in the studies of IL-10 SNPs at −1082(G/A), −592(C/A), and −819(C/T) in pediatric population, we compared our results with those of adults. Analysis of IL-10 polymorphism and its association with HCV susceptibility have produced ambiguous results (20,21). Our results were in accordance with some studies in which no association has been made between clearance of HCV infection and genotypes distribution at SNPs positions −1082G/A and −592C/A genotypes (22–24); however, their results were contrary to ours regarding −819 C/T because they found no association between it and HCV clearance.
Some studies revealed a higher prevalence of SNPs of IL-10 −592AA and −819TT in the responders than in the nonresponders group (25). In another study, the −1082GG genotype appeared 2 times more frequently in responders compared with nonresponders, whereas −1082GA was more common in the nonresponders (26). Likewise, although not statistically significant, it has been demonstrated that the −1082GG was associated with the responder status (27,28).
Afzal et al (29) studied 100 Pakistani adult patients with HCV and found a higher frequency of GG genotype of IL-10 SNP at −1082 in patients with HCV than in the healthy controls (17% vs 3%); −1082 genotype GA was more common in controls than in patients with HCV (93% vs 75%), suggesting a possible association with the protection against HCV infection. They found −1082 AA genotype occurs more frequently in patients than in controls; these results agree with our study in which 5 patients harbored −1082GG genotype versus 3 in the control group. Also, there were more patients with −1082AA genotype than controls with same genotype (14 vs 8), respectively. Our results may not have been statistically significant because of the sample size. The Afzal et al (29) study showed a statistically nonsignificant higher frequency of the −819CC genotype in patients with HCV than healthy controls. Likewise, our results showed 15 patients with HCV with −819CC versus 7 healthy subjects.
A higher occurrence of the IL-10-819C allele was observed in patients with HCV compared with controls (29). In turn, a meta-analysis of 13 studies regarding hepatitis C in Asian and European populations demonstrated a weak association of IL-10 SNP at the −592A allele with HCV infection in Asian populations and proved no correlation for alleles at positions −1082 and −819 (30). Genotype distribution analysis indicated that the IL-10-1082GG genotype is more common in patients with HCV than in controls (31), which suggests that individuals who have this genotype are more susceptible to chronic HCV infection. Similar results have been reported in analyses of IL-10-1082G/A regarding sex susceptibility to CHC. It has been revealed that this genotype is associated with a diminished risk of persistent HCV and the −1082AA with an increased risk of persistent HCV, respectively (22). It has been reported that IL-10 −1082(G/A) alleles (28) and genotypes (32) are not differently distributed between patients and controls. This is similar with regard to the −819(C/T) allele and genotype as well as the IL-10 −592(C/A) allele (32). Regarding the effect of cytokine gene polymorphism on the histological activity index in HCV-infected individuals, patients with the homozygous G allele at IL-10 SNP at −1082 were seen to be more prone to necroinflammatory activity, whereas patients with IL-10 SNP −819 TT were associated with a less severe form of liver fibrosis. According to our results, individuals with IL-10 SNPs at −819 TT were also less susceptible to disease.
These differing results appear to be caused by several factors. First, studies included populations from different corners of the world, and IL-10 variants are distributed differently in various ethnic populations. Second, the structure of examined populations (age and sex proportion, HCV genotypes) and crucial factors affecting SVR, such as fibrosis, HCV genotype, sex, type of therapy (monotherapy/combined treatment) and its duration, should be taken into account. Third, contradictory results may be influenced by different environmental/cultural backgrounds in populations. Because it is suggested that some often-omitted factors may affect IL-10 production, they may play a role in hepatitis C pathogenesis. For instance, it has been reported that smoking and increased BMI are likely to be associated with decreased production of IL-10 (33). Finally, discrepancies between the presented results may be caused by a relatively small sample size.
The rate of SVR after PEG-IFN/RBV was 80% (12/15) in rs12979860 CC subjects, compared with 42% (8/19) in CT/TT genotypes, and the figures for SVR according to −819 IL-10 SNPs were 84.6% (11/13) in TT subjects and 42.9% (9/21) in those showing CC/CT genotypes. The CC genotype of IL-28B and TT genotype of IL-10 at −819 can be used for predicting response before treatment with PEG-IFN/RBV therapy.
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