Potential conflict of interest : Dr. Rumi received grants from Roche, Janssen, and Gilead. Dr. Colombo advises and received grants from Roche, Merck, Gilead, Bayers, and Bristol-Myers Squibb. He also advises Tibotec.
Polymorphisms in the interleukin-28B (IL28B) region are associated with spontaneous and treatment-induced viral clearance in hepatitis C virus (HCV) infection. Nevertheless, it is unknown whether genetic variation at the IL28B locus influences the natural history of chronic HCV infection. Thus, we asked whether an association between IL28B polymorphisms and liver fibrosis progression existed. We studied 247 consecutive patients with chronic HCV, an accurate estimate of the date of infection, and a liver biopsy performed before any treatment. No patient had a history of alcohol abuse or coinfection with other viruses. We assessed the role of rs8099917 and rs12979860 polymorphisms and the effect of host and environmental factors on fibrosis progression. Blood transfusion (75%) was the main modality of infection. Median age at infection was 21 years, and median interval between infection and liver biopsy was 25 years. One hundred twenty-nine patients (52%) were infected by HCV-1, 74 (30%) by HCV-2, 34 (14%) by HCV-3, and 10 (4%) by HCV-4. Bridging fibrosis/cirrhosis (Ishak ≥4) was detected in 24% of patients. Age at infection had a marked effect on fibrosis progression by both a linear model and Cox proportional-hazard regression (P < 2E-16). A 12.1% increase in the hazard of advanced fibrosis was estimated for each additional year at infection, suggesting that this was the major explanatory variable in this cohort. Male gender (P < 0.05), HCV genotype 3 (P < 0.001) and steatosis (P < 0.05) were also associated with faster fibrosis progression. Conversely, the two IL28B polymorphisms had no impact on fibrosis progression. Conclusion: In HCV patients with a known date of infection, IL28B genotype was not associated with fibrosis progression rate or with the risk of developing advanced liver fibrosis. (HEPATOLOGY 2011;)
Hepatitis C virus (HCV) infection is a global health care burden, with roughly 1%-2% of the European and U.S. populations being chronic carriers of the virus. Although HCV can lead to extrahepatic manifestations in a minority of patients, the prognosis of the disease is directly linked to the restless accumulation of fibrotic tissue in the liver, which, ultimately, alters the liver architecture and its vascularization, leading to the development of cirrhosis and its sequelae. Among the many factors known to substantially contribute to this process by determining a more rapid progressive course of the disease, emphasis has been put on a variety of environmental components, such as alcohol abuse and viral coinfections, and host risk factors, such as insulin resistance, obesity, and immunity.1, 2 More recently, HCV genotype 3 has been shown to be associated with accelerated fibrosis progression, compared to non-3 genotypes.3, 4 Although these factors do explain part of the extreme variability seen in fibrosis progression among HCV-infected patients, they do not completely account for these differences. Thus, the prediction of the rate of disease progression at an individual level is still quite inaccurate, often making impossible the selection of patients for anti-HCV treatment on the basis of the risk to progress to the advanced stages of hepatitis. Some evidences exist on the role of host genetics in modifying disease progression, because several single-nucleotide polymorphisms (SNPs) located in various genes, including interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and transforming growth factor beta (TGF-β), among others, have been associated with progression of liver fibrosis.2 A recent article supported these findings, providing evidence that a seven-SNP signature was associated with fibrosis progression in a large cohort of HCV-infected patients in Italy.5 Among the SNPs identified to play a major role in HCV infection, those located in the IL28B region on chromosome 19 have been strongly associated with spontaneous and treatment-induced viral clearance in patients of different ethnicity.6-10 Thus, patients with the T/T and C/T genotype at the rs12979860 SNP (nonresponder genotype) have been shown to exhibit a slower viral decline after pegylated interferon plus ribavirin (PEG-IFN/RBV) therapy, with the sustained virologic response (SVR) rates being significantly lower, compared to patients with the favorable C/C genotype.11 Although this discovery has radically changed the landscape of antiviral treatment for HCV patients, providing insights into the mechanisms of IFN hyporesponsiveness, the precise mechanisms behind this association still needs to be unraveled. In addition, whether there is a link between the clinical manifestation of chronic HCV infection and the patient IL28B genotype is still an open question.
Here, we asked whether a correlation between IL28B SNPs and fibrosis progression exists. To test this hypothesis, we studied 247 consecutive patients with a known date of HCV infection, whose liver fibrosis was staged by a percutaneous liver biopsy. We studied the effect of host and external factors on both fibrosis progression rate and advanced fibrosis stage.
A total of 247 participants with chronic HCV infection were included in this study. All participants were consecutively selected at the Center for Liver Disease at Maggiore Hospital (Milan, Italy). Inclusion criteria comprised the following: (1) HCV RNA serum positivity; (2) European descent; (3) an estimated date of HCV infection based on the first reported parenteral risk factor; (4) a diagnostic liver biopsy before antiviral treatment performed at least 4 years after the date of infection; and (5) no history of past or current alcohol use (>20g/day). Patients were excluded if they had any other cause of liver disease, including hepatitis B virus infection, human immunodeficiency virus (HIV) infection, Wilson's disease, hemochromatosis, or α1-antitrypsin deficiency. We also excluded patients with type 1 or 2 diabetes. Duration of disease was calculated by considering the time elapsed between the year of infection and the liver biopsy. Liver biopsies were evaluated by a single expert pathologist and scored using the Ishak system in separate reports for grading and staging. The score for staging ranged from 0 (no fibrosis) to 6 (cirrhosis). The study was approved by the Institutional Review Board of the Department of Internal Medicine at Maggiore Hospital. All patients gave their written informed consent to receive therapy and gave permission for use of their medical records.
DNA Extraction and Genotyping.
Genomic DNA (gDNA) was extracted from 1 mL of ethylenediaminetetraacetic acid (EDTA) whole blood. Briefly, GeneCatcher magnetic beads (Invitrogen, Carlsbad, CA) were used in a semiautomatic procedure on a FreedomEVO platform (Tecan, Männedorf, Switzerland). After final elution, gDNA concentration and purity were evaluated and the sample was further processed if A260/A280 ≥1.8 and A260/230 ≥1.5.
For rs8099917, genotype data were obtained from genome-wide profiling with the Human660W-Quad BeadChip (Illumina, San Diego, CA), after SNP calling with the default settings of Genome Studio software. Conversely, for rs12979860, TaqMan SNP genotyping assays were run on a 7900HT real-time polymerase chain reaction instrument (Applied Biosystems, Carlsbad, CA), following the manufacturer's instruction.
To quantitatively describe disease outcome, a fibrosis progression rate (FPR) was calculated by taking the ratio between the Ishak score (staging value) and the disease duration (in years). This calculation assumes that no significant liver fibrosis was present at the time of infection. A generalized linear model was formulated and fitted to the data, specifying the IL28B SNP genotypes and the covariates as explanatory variables, namely age at infection, gender, HCV genotype, the presence of moderate to severe liver steatosis (grade 2-3), the presence of liver necroinflammatory lesions (histological grading ≥9), and the body-mass index (BMI). A log10 transformation of the FPR was employed to obtain linearity. For cases having an Ishak = 0, the log10(FPR) was substituted with the lowest value observed. For SNP data, the minor allele was counted as follows: rs8099917 TT = 0, TG = 1, GG = 2; rs12979860 CC = 0, CT = 1, TT = 2. The model was checked through the regression diagnostic plots to verify normality, linearity of the data, and constant variance. The effect of the explanatory variables was considered significant if P < 0.05 and a final model was established with the significant terms only.
Additionally, survival analysis was performed to model the time taken for advanced fibrosis to occur. Here, advanced fibrosis was defined if Ishak ≥4. A Cox proportional-hazards regression model was fitted, and the covariates were considered significant if P < 0.05. The proportional hazard assumption was checked and a final model was proposed, considering the significant terms only.
All statistical analyses were performed in R,12 using the survival library for Cox regression.13
For the present analyses, 247 patients consecutively attending our center between September 2008 and March 2010 that fulfilled the strict selection criteria were selected. Patient characteristics are outlined in Table 1. The majority of the patients were infected with HCV genotype 1 (52%), although this study included subjects with HCV genotypes 1, 2, 3, and 4. Both males and females were well represented (52% males, 48% females). Median age at infection was 21 years, median disease duration was 25 years, and median age at biopsy was 47 years. Mean biopsy length was 26.3 mm. The FPR distribution resulted in being right-skewed, but approached a normal distribution after log10 transformation (see Supporting Information). The majority of the patients (87%) had minimal to mild histological activity (grade <9), whereas a minor fraction (13%) showed moderate to severe activity (grading ≥9). Moderate or severe steatosis (grade 2-3) was observed in 20% of the patients. Mean BMI was 25.3 kg/m2. The main reported risk factors for HCV infection were blood transfusions (75%) and the use of intravenous drugs (23%), with mother-to-child, needlestick, or sexual transmission as the other reported risks. In this cohort, 29 patients were age 0 at infection. Only 1 of these patients acquired the infection vertically, whereas the others received a blood transfusion at birth.
Table 1. Patient Characteristics, Stratified By HCV Genotype
Abbreviations: HCV, hepatitis C virus; SD, standard deviation; BMI, body mass index.
Median age at infection, years (range)
Median age at biopsy, years (range)
Median duration, years (range)
Mean biopsy length, mm (SD)
Fibrosis stage (Ishak)
Minimal to mild (<9)
Moderate to severe (≥9)
None to slight (0-1)
Moderate to severe (2-3)
Intravenous drug use
IL28B genotypes of patients with absent or mild fibrosis (Ishak <4) and patients with advanced fibrosis (Ishak ≥4) are shown in Table 2. Genotype frequencies did not differ significantly between the two groups, regardless of the analyzed SNP (rs8099917 or rs12979860). Moreover, SNP genotype frequencies did not deviate significantly from Hardy-Weinberg equilibrium expectation at a threshold of P = 0.01. The measured genotype frequencies are consistent to other published reports with HCV-infected patients.6, 7
To evaluate the contribution of genetic and nongenetic factors in the natural history of chronic HCV infection, we performed multiple analyses aimed at the definition of the individual contribution to fibrosis progression in the cohort of patients described above (see Patients and Methods). Here, we evaluated whether the genotype of rs8099917 and rs12979860 polymorphisms could influence fibrosis progression in the liver of HCV-infected patients. In a first analysis framework, we modeled the dependence of log10(FPR) on a linear combination of IL28B SNP genotypes along with different variables already reported to influence fibrosis progression. These were age at infection, patient gender, HCV genotype, the presence of steatosis, BMI, and inflammatory grading. An interaction term was specified between steatosis and viral genotype, to reflect that the effect of steatosis on fibrosis progression may be specific for different genotypes. The final model was chosen on the basis of the minimum Akaike information criterion. The results summarized in Table 3 show that age at infection was the variable most strongly and positively associated with fibrosis progression (P < 2E-16). Male gender (P = 0.012), HCV genotype 3 (P = 7.19E-04), and the presence of steatosis (P = 0.012) also resulted in being significantly associated with an accelerated rate of fibrosis progression. Importantly, the two IL28B SNPs had no significant effect on the outcome, and thus they were removed from the final model (Table 3). It is worth pointing out that HCV genotype 2 was associated with a slower rate of fibrosis progression (P = 0.034), as also suggested in a previous study.14 Single-term deletion analysis further confirmed the role of these explanatory variables. Figure 1 shows the remarkable effect of age at infection on rate of fibrosis progression. In contrast, no effect could be attributable to the host IL28B genotype. Notably, no impact on FPR was found, also assuming a dominant model of inheritance for the alleles, rs8099917 G or rs12979860 T, previously associated with treatment failure (data not shown). On the contrary, when these two SNPs were tested for association with therapy outcome, using data from 91 patients with HCV genotype 1 and available treatment information, the effect was readily detectable (see Supporting Information). Actually, rs12979860 genotypes CT and TT versus CC resulted in being associated with treatment failure (P < 0.01, odds ratio = 3.6, 95% CI = 1.3-10.2), in agreement with previous reports.
Table 3. Variables Significantly Affecting the Fibrosis Progression According to Linear Model
Estimate = effect size on log fibrosis progression rate; 95% CI = 95% confidence interval for the estimate. Hepatitis C virus (HCV) genotype 1 was considered the reference level.
Parameters estimated from the model without the interaction term between HCV genotype and steatosis.
Finally, we evaluated the role of explanatory variables, excluding individuals that acquired the infection at birth, to exclude possible confounding factors originating from the inclusion of a group of patients that acquired the infection in the setting of an immature immune system. In this subgroup of patients, we obtained the same estimates of the parameters and confirmed the lack of any effect by the two IL28B polymorphisms and the significant role of the nongenetic factors (Supporting Table 1).
In summary, the model outlined in Table 3—which includes patient gender, age at infection, viral genotype, and steatosis—explained an estimated 34% of the phenotypic variability, showing that patient age at infection has a major, highly significant role on fibrosis progression, in agreement with other reports.14-16 The estimated effect on fibrosis progression of each additional year at infection was a 2.8% (95% CI = 2.2%-3.4%) increase in FPR.
Cox Proportional-Hazard Model.
When using the FPR, we made the assumption of a linear fibrosis progression over time. To avoid biases imposed by the above assumption, we decided to use an alternative analysis approach. Thus, we also used a proportional-hazard survival model to analyze the relationship between the above-mentioned explanatory factors and the time it takes to develop bridging fibrosis or cirrhosis (Ishak ≥4). In agreement with the linear model, the significant variables found with this model were age at infection (P = 1.26E-13), male gender (P = 0.018), HCV genotype 3 (P = 0.004), and steatosis (P = 0.003), whereas the two IL28B polymorphisms had no effect on the estimated hazard of developing advanced fibrosis (Table 4; Fig. 2). From the estimates of the coefficients, we can derive the effect on the hazard. Thus, holding the other covariates constant, each additional year at infection produces a highly significant increase of the hazard of advanced fibrosis by a factor of 1.121 (95% confidence interval [CI] =1.088-1.155) or 12.1%. This effect is clearly evident when plotting the estimated survival functions for three representative infection times (0, 20, or 40 years of age, respectively; Fig. 2). Indeed, the three survival functions are well separated and their corresponding CIs do not overlap. Conversely, the corresponding estimated survival functions by IL28B genotype are clearly overlapping, underscoring the lack of any effect of these variables. Furthermore, the effect of HCV genotype, gender, and steatosis were also significant, but their estimates had wider CIs (Table 4).
Table 4. Variables Significantly Associated to the Hazard of Advanced Fibrosis According to the Cox Proportional-Hazard Model
Effect = eestimate
Estimate = effect size on the log-hazard; Effect = eestimate, or multiplicative effect on the hazard; 95% confidence interval for the effect; hepatitis C virus (HCV) genotype 1 was considered the reference level.
Parameters estimated from the model without the interaction term between HCV genotype and steatosis.
It is now well established that genetic variations in the region of the IL28B gene on chromosome 19, coding for IFN-λ3, are strongly associated with the achievement of treatment-induced or spontaneous viral clearance in individuals infected with HCV.11 In particular, numerous studies have shown that individuals with the C/C genotype at the rs12979860 SNP (i.e., “protective” or “responder” genotype) have higher rates of rapid, sustained virological response to treatment with the current standard of care (i.e. PEG-IFN/RBV), compared to those carrying the T allele (C/T and T/T genotypes). More recently, it has also been proposed that the poor response IL28B variants, in hepatitis C patients, are also associated with lower pretreatment low-density lipoprotein cholesterol levels,17, 18 hepatic steatosis,18, 19 and insulin resistance,19 compared to the “responder” genotype. However, it is still unclear whether the genetic variation at the IL28B locus affects the severity and pace of the progression of liver disease. Indeed, though some investigators found that the unfavorable rs12979860 T/T gene pattern was associated with worse liver fibrosis, others did not replicate this finding. Bitetto et al.20 looked at the link between the IL28B rs12979860 SNP and fibrosis stage in 548 individuals with chronic HCV infection, and found that carriage of the T/T genotype (n = 78) was associated with a higher staging score (Ishak 3 or 4), after adjusting for age, sex, BMI, alcohol intake, and diabetes mellitus, compared to the other two genotypes (n = 470; P < 0.02). In contrast, a smaller study from a different group—performed on 129 patients with HCV genotype 1—failed to find a similar association.21 Thompson et al. exploited the large patient population enrolled in the IDEAL study to investigate the relationship between IL28B polymorphisms and liver disease severity.22, 23 In the first analysis, 1,329 patients were genotyped and the researchers observed no relationship between IL28B rs12979860 and advanced fibrosis (METAVIR stage F3-F4). In the second analysis, however, the researchers did see a link between rs12979860 genotype, alanine aminotransferase (ALT) levels, and necroinflammatory activity, with C/C patients having higher pretreatment ALT values and more often moderate-to-severe (METAVIR stage A2-A3) necroinflammatory activity. Because elevated ALT and high histological grading are known to be associated with a faster fibrosis progression in patients with chronic hepatitis C, in principle, these findings could support a role of the IL28B polymorphisms as a determinant of disease severity.24
However, a limitation of all these studies is represented by the fact that the association between IL28B genetic variants and the presence of advanced fibrosis or cirrhosis was investigated without taking into consideration the time elapsed from the acquisition of the infection. For this reason, we decided to assess the potential association between IL28B polymorphisms and the rate of progression of liver fibrosis in a cohort of well-characterized patients for whom an accurate estimation of the date of infection could be obtained. Additionally, in the attempt to minimize the role of confounding factors in the interpretation of our data, we restricted our analysis to Caucasian patients only and excluded also patients with diabetes or those reporting past or current regular alcohol consumption. Using such strict inclusion criteria, we found that the host genetic background at the IL28B locus is not associated with the risk of developing advanced fibrosis. Conversely, we show that other factors have a strong impact on disease outcome, being strongly associated with fibrosis progression, as previously reported.1, 2
In a first effort to correlate disease progression with host and external variables, we modeled the fibrosis progression rate as a continuous outcome, considering the ratio between fibrosis level and disease duration. Although this approach might have been biased by the assumption that the rate of progression to cirrhosis remains constant over time,15 this method represents a way to consider the duration of the chronic disease within the model, instead of a simple split of the population in two groups on the basis of fibrosis score alone. In this model, we observed no effect of IL28B genotypes on fibrosis progression, whereas we confirm the well-established role of a number of other factors. Indeed, we detected a strong effect of age at infection on rate of disease progression, namely a 2.8% increase in the speed of disease progression for each additional year. This significant effect is manifested by the fact that patients infected perinatally have very slow progression of liver fibrosis. For those patients, mean progression is 0.049 FPR units, corresponding approximately to an increase of 2 Ishak points in 40 years, similarly to other reports.25, 26 Additionally, male gender and HCV genotype 3 resulted in being significantly associated with fast progression, compared to female gender and HCV genotype 1, respectively. Although there is general agreement on the faster disease observed in males, the role of HCV genotype has remained controversial for a long time. According to our data, patients with HCV genotype 3 have a faster disease progression, confirming other recent studies.3, 4 However, whether this association is directly mediated by the virus through the increased steatosis observed in patients with HCV genotype 3 or is a consequence of other external factors is still debated.27, 28 In our models, we included an interaction term between viral genotype and steatosis to account for the reasonable different influence of steatosis according to HCV genotype. Indeed, correcting for steatosis, we still detected an effect for the HCV 3 genotype, suggesting that the faster fibrosis progression observed in genotype 3 patients appears to be not completely explained by the presence of steatosis. Because patients with HCV genotype 3 infection acquired the virus, in most cases, during drug abuse in the 1970s-1980s, the confounding role of past alcohol use/abuse, even for a limited time period, cannot be completely ruled out as a relevant factor. Although, in our study, we did exclude patients reporting significant alcohol use in the past (>20g/day), we cannot completely rule out that our patients underreported alcohol consumption, especially if limited in time. Interestingly, viral genotype 2 was more weakly, but significantly, associated with a slower progression of liver fibrotic disease in our cohort. The same observation was described in a landmark article, where Poynard et al.14 reported that patients infected with genotype 2 had a slower rate of fibrosis progression relative to genotypes 1a or 1b, although the differences were not significant, presumably owing to the small number of patients with available genotypes.
To confirm the results of our analyses, in spite of the limitation of the model assumptions, we also used a Cox proportional-hazard regression to directly estimate the hazard of developing advanced fibrosis as a function of host and external factors. This analysis conveniently outputs the effect of the variables on the hazard, providing useful insight on the natural history of HCV infection. In this model, age at infection was the major contributor to the hazard of advanced fibrosis, with male gender, HCV genotype 3, and steatosis being significantly, but more weakly, associated with liver disease progression. Once again, the IL28B polymorphisms had no influence on the hazard of developing advanced fibrosis.
We do acknowledge that our study was not completely free of limitations, as we could not properly evaluate the influence of known accelerators of fibrosis progression, such as being overweight and past alcohol abuse.29 However, with respect to the influence of IL28B polymorphisms on disease progression, it seems unlikely that these factors might have been skewed toward one genotype to provide a significant bias. Moreover, assessing liver fibrosis through percutaneous biopsy does have some limitations, including sampling error bias, which accounts for differences in staging score of at least 1 point in up to 20% of cases when liver biopsies are performed in both lobes.30 Furthermore, a misdiagnosis of cirrhosis is seen in up to 30% of specimens.31 Nevertheless, despite all these caveats, liver biopsy is still considered the standard, if not the gold standard, for fibrosis staging.32
The correct identification of the time of infection is a critical point in determining the role of any predictor of disease progression in an acquired disease, such as chronic hepatitis C. Our study, from this point of view, was particularly solid, because, in most of our patients, the infection was acquired during a datable event, such as multiple blood transfusions or intravenous drug abuse. For this reason, we believe that our study provides important insights into the natural history of HCV infection and, specifically, into fibrosis progression and their relationship with host and external factors.
In conclusion, we show that the IL28B genotype does not have an effect on the risk of developing advanced fibrosis, whereas age at infection, male gender, and infection with HCV genotype 3 are confirmed to accelerate disease progression. This finding has important implications, as it opens additional questions on the role of host genetic factors in the modulation of disease progression. Further studies of the host genetic determinants associated with risk of liver disease progression in hepatitis C should represent a high priority of the scientific community, with the aim of both allowing a better understanding of disease pathogenesis and guiding an improved patient-selection process for eligibility to antiviral therapy.
The authors thank Prof. Mario Comelli for his special statistical support and help in writing the manuscript for this article.