Relation of IL28B gene polymorphism with biochemical and histological features in hepatitis C virus-induced liver disease.
ABSTRACT Polymorphism at the IL28B gene may modify the course of hepatitis C virus (HCV) chronic infection. Our aim was to study the influence of IL28B rs12979860 gene polymorphism on the biochemistry and pathology of HCV-induced disease in the clinical course from mild chronic hepatitis C to hepatocellular carcinoma.
We have determined the rs12979860 single nucleotide polymorphism (SNP) upstream IL28B gene in two groups of patients with HCV-induced chronic liver disease: 1) 268 patients (159 men) with biopsy-proven chronic hepatitis C, to analyse its relation with biochemical, virological and histological features; and 2) 134 patients (97 men) with HCV-related hepatocellular carcinoma. The distribution of the analysed SNP in hepatocellular carcinoma patients was compared with that found in untreated chronic hepatitis C patients. All patients were white and most were Spaniards.
In multivariate analysis ALT values were higher (P = 0.001) and GGT values were lower (P<0.001) in chronic hepatitis C patients homozygotes for the major rs12979860C allele as compared with carriers of the mutated rs12979860T allele. Steatosis was more frequent (Odds ratio = 1.764, 95% C.I. 1.053-2.955) and severe (P = 0.026) in carriers of the rs12979860T allele. No relation was found between the analysed SNP and METAVIR scores for necroinflammation and fibrosis, and there were no differences in the distribution of the analysed SNP between hepatocellular carcinoma and untreated chronic hepatitis C patients.
The IL28B rs12979860 polymorphism correlates with the biochemical activity and the presence and severity of liver steatosis in chronic hepatitis C.
- SourceAvailable from: Mattias Mandorfer[show abstract] [hide abstract]
ABSTRACT: Evaluation of metabolic factors and elevated γ-glutamyltransferase (GGT) levels as independent predictors of treatment failure in a thoroughly documented cohort of HIV-/HCV-coinfected patients (HIV/HCV). Sixty-four HIV/HCV patients treated with pegylated interferon-α-2a plus ribavirin (PEGIFN + RBV) at the Medical University of Vienna within a prospective trial were included in this study. In addition, 124 patients with HIV/HCV from the AIFA-HIV and AHIVCOS cohorts were included as a validation cohort. Advanced liver fibrosis, GGT elevation, insulin resistance (IR) and low CD4+ nadir were defined as METAVIR F3/F4, GGT levels >1.5× sex-specific upper limit of normal, homoeostasis model assessment of insulin resistance >2 and CD4+ nadir <350 cells/μL, respectively. HCV-genotype 1/4 (OR26.3; P = 0.006), advanced liver fibrosis (OR20.2; P = 0.009), interleukin 28B rs12979860 non-C/C SNP (OR8.27; P = 0.02) and GGT elevation (OR7.97; P = 0.012) were independent predictors of treatment failure, while both IR (OR3.51; P = 0.106) and low CD4 + nadir (OR2.64; P = 0.263) were not independently associated with treatment failure. A statistically significant correlation between GGT elevation and prior alcohol abuse (r = 0.259; P = 0.039), liver steatosis (r = 0.301; P = 0.034) and low-density lipoprotein-cholesterol (r = -0.256; P = 0.041) was observed. The importance of GGT elevation as an independent predictor of treatment failure was confirmed in a validation cohort (OR2.76; P = 0.026). While GGT elevation emerged as an independent predictor of treatment failure in both the derivation and the validation cohort, no independent associations between metabolic factors and treatment failure were observed. Thus, our findings suggest that GGT elevation is an independent predictor of treatment failure in HIV/HCV that can easily be incorporated into predictive algorithms.Journal of Viral Hepatitis 01/2014; 21(1):33-41. · 3.08 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The IFNL3 genotype predicts the clearance of hepatitis C virus (HCV), spontaneously and with interferon (IFN)-based therapy. The responder genotype is associated with lower expression of interferon stimulated genes (ISGs) in liver biopsies from chronic hepatitis C patients. However, ISGs represent many interacting molecular pathways, and we hypothesised that the IFNL3 genotype may produce a characteristic pattern of ISG expression explaining the effect of genotype on viral clearance. For the first time, we identified an association between a cluster of ISGs, the metallothioneins (MTs) and IFNL3 genotype. Importantly, MTs were significantly upregulated (in contrast to most other ISGs) in HCV-infected liver biopsies of rs8099917 responders. An association between lower fibrosis scores and higher MT levels was demonstrated underlying clinical relevance of this association. As expected, overall ISGs were significantly downregulated in biopsies from subjects with the IFNL3 rs8099917 responder genotype (P=2.38 × 10(-7)). Peripheral blood analysis revealed paradoxical and not previously described findings with upregulation of ISGs seen in the responder genotype (P=1.00 × 10(-4)). The higher MT expression in responders may contribute to their improved viral clearance and MT-inducing agents may be useful adjuncts to therapy for HCV. Upregulation of immune cell ISGs in responders may also contribute to the IFNL3 genotype effect.Genes and Immunity advance online publication, 12 December 2013; doi:10.1038/gene.2013.66.Genes and immunity 12/2013; · 4.22 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Hepatitis C virus (HCV) infection is associated with insulin resistance (IR), although mechanisms leading to IR in these patients are not completely understood. The aim of this study was to evaluate the association of interleukin 28B (IL28B) and interleukin 28 receptor alpha (IL28RA) polymorphisms with IR among human immunodeficiency virus (HIV)/HCV-coinfected patients. We carried out a cross-sectional study on 203 patients. IL28B (rs8099917) and IL28RA (rs10903035) polymorphisms were genotyped by GoldenGate(®) assay. IR was defined as homeostatic model assessment (HOMA) values ≥3.00. Univariate and multivariate generalized linear models (GLM) were used to compare HOMA values and the percentage of patients with IR according to IL28B and IL28RA genotypes. In total, 32% (n = 65/203) of the patients had IR. IL28B rs8099917 TT was not significantly associated with HOMA values and IR. In contrast, rs10903035 AA was significantly associated with high HOMA values taking into account all patients (P = 0.024), as well as the subgroups of patients with significant fibrosis (P = 0.047) and infected with HCV genotype 3 (P = 0.024). Additionally, rs10903035 AA was significantly associated with IR (HOMA ≥3.00) in all patients (adjusted odds ratio (aOR) = 2.02; P = 0.034), in patients with significant fibrosis (aOR = 2.86; P = 0.039) and HCV genotype 3 patients (aOR = 4.89; P = 0.031). In conclusions, IL28RA polymorphism (rs10903035) seems to be implicated in the glucose homeostasis because AA genotype increases the likelihood of IR, but this association was different depending on hepatic fibrosis and HCV genotype.Journal of Viral Hepatitis 03/2014; 21(3):189-97. · 3.08 Impact Factor
Relation of IL28B Gene Polymorphism with Biochemical
and Histological Features in Hepatitis C Virus-Induced
Jose ´ A. Agu ´ndez1, Elena Garcı ´a-Martin2, Marı ´a L. Maestro3, Francisca Cuenca4, Carmen Martı ´nez1,
Luis Ortega5, Miguel Carballo6, Marta Vidaurreta3, Marta Agreda4, Gabriela Dı ´az-Zelaya5,
Avelina Sua ´rez7, Manuel Dı ´az-Rubio8, Jose ´ M. Ladero8*
1Department of Pharmacology, University of Extremadura, Ca ´ceres, Spain, 2Department of Biochemistry & Molecular Biology, University of Extremadura, Ca ´ceres, Spain,
3Genomics Unit, Service of Clinical Biochemistry, Hospital Clı ´nico San Carlos, Instituto de Investigacio ´n Sanitaria del Hospital Clı ´nico San Carlos (IdISSC), Madrid, Spain,
4Liver Unit, Service of Gastroenterology, Hospital Clı ´nico San Carlos, Instituto de Investigacio ´n Sanitaria del Hospital Clı ´nico San Carlos (IdISSC), Madrid, Spain, 5Service of
Pathology, Hospital Clı ´nico San Carlos, Instituto de Investigacio ´n Sanitaria del Hospital Clı ´nico San Carlos (IdISSC), Madrid, Spain, 6Laboratory of Molecular Genetics,
Hospital de Terrassa, Terrassa (Barcelona), Spain, 7Service of Clinical Microbiology, Hospital Clı ´nico San Carlos (IdISCC), Madrid, Spain, 8Liver Unit, Service of
Gastroenterology, Hospital Clı ´nico San Carlos, Department of Medicine, Medical School, Universidad Complutense, Instituto de Investigacio ´n Sanitaria del Hospital Clı ´nico
San Carlos (IdISSC), Madrid, Spain
Background/Aims: Polymorphism at the IL28B gene may modify the course of hepatitis C virus (HCV) chronic infection. Our
aim was to study the influence of IL28B rs12979860 gene polymorphism on the biochemistry and pathology of HCV-induced
disease in the clinical course from mild chronic hepatitis C to hepatocellular carcinoma.
Methods: We have determined the rs12979860 single nucleotide polymorphism (SNP) upstream IL28B gene in two groups
of patients with HCV-induced chronic liver disease: 1) 268 patients (159 men) with biopsy-proven chronic hepatitis C, to
analyse its relation with biochemical, virological and histological features; and 2) 134 patients (97 men) with HCV-related
hepatocellular carcinoma. The distribution of the analysed SNP in hepatocellular carcinoma patients was compared with
that found in untreated chronic hepatitis C patients. All patients were white and most were Spaniards.
Results: In multivariate analysis ALT values were higher (P=0.001) and GGT values were lower (P,0.001) in chronic hepatitis
C patients homozygotes for the major rs12979860C allele as compared with carriers of the mutated rs12979860T allele.
Steatosis was more frequent (Odds ratio=1.764, 95% C.I. 1.053–2.955) and severe (P=0.026) in carriers of the rs12979860T
allele. No relation was found between the analysed SNP and METAVIR scores for necroinflammation and fibrosis, and there
were no differences in the distribution of the analysed SNP between hepatocellular carcinoma and untreated chronic
hepatitis C patients.
Conclusion: The IL28B rs12979860 polymorphism correlates with the biochemical activity and the presence and severity of
liver steatosis in chronic hepatitis C.
Citation: Agu ´ndez JA, Garcı ´a-Martin E, Maestro ML, Cuenca F, Martı ´nez C, et al. (2012) Relation of IL28B Gene Polymorphism with Biochemical and Histological
Features in Hepatitis C Virus-Induced Liver Disease. PLoS ONE 7(5): e37998. doi:10.1371/journal.pone.0037998
Editor: Gulam Waris, Rosalind Franklin University of Medicine and Science, United States of America
Received March 7, 2012; Accepted April 27, 2012; Published May 29, 2012
Copyright: ? 2012 Agu ´ndez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Financial support: FIS PS09/00943, PS09/00469, and RETICS RD07/0064 from Fondo de Investigacio ´n Sanitaria, Instituto de Salud Carlos III, Spain
GR10068 from Junta de Extremadura, Spain. Financed in part with FEDER funds from the European Union. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
variable and difficult to define. Firstly, the proportion of infected
patients that clear the virus in the acute phase of the infection
broadly oscillates between 14% and 46% of cases . Long-term
follow up studies in patients with chronic hepatitis C have shown a
great variability in the rate of progression of the liver disease,
although after 2 or more decades many infected persons develop
of suffering from hepatocellular carcinoma (HCC). There are some
well-known host-related factors that are clearly associated with a
more rapid and severe progression of the liver disease, as when it is
acquired at an older age , and the metabolic syndrome complex
(non-alcoholic liver steatosis, obesity and type II diabetes mellitus)
[4,5]. There are some reports on host genetic factors related to the
risk of acute HCV infection becoming chronic  and to the rate of
progression in the chronic phase, as are some human leukocyte
antigen class II antigens [7,8] and polymorphisms in the genes of
transforming growth factor-b1 and angiotensin II .
PLoS ONE | www.plosone.org1 May 2012 | Volume 7 | Issue 5 | e37998
A genome wide association study (GWAS) has identified a non-
coding single nucleotide polymorphism (SNP), rs12979860, that
resides 3 kb upstream of the IL28B gene, located in the long arm
of chromosome 19, which encodes IFN-l3 . Two other
GWAS identified a second non-coding SNP, rs8099917, located
7.5 kb upstream of the IL28B start codo ´n [12,13]. Both SNPs are
in partial linkage disequilibrium and are part of an IL28B
haplotype that shows ethnic variations and is a strong determinant
of response (or lack of response) to therapy for HCV chronic
The mechanisms that regulate the influence of IL28B polymor-
phisms on the success rate of therapy in chronic hepatitis C remain
unknown. These genetic traits are not related with levels of
intrahepatic IL28B gene expression but the baseline expression of
interferon stimulated genes (ISGs) is significantly higher in patients
carrying the minor rs8099917G allele [16,17], that is in linkage
disequilibrium with the minor rs12979860T allele, thus letting a
narrower margin of response when HCV acutely infects the liver
or when interferon-based therapy is instituted in chronically
infected patients . These findings may explain why the
spontaneous clearance of HCV virus is more frequent in subjects
with the rs12979860CC genotype [19–21] and that this genotype
and the linked rs8099917TT genotype are predictors of SVR, as
unanimously confirm several studies [11–13,16,22–28].
In spite of the high number of studies mentioned above, data on
of untreated chronic HCVinfectionarescarce. Abe etal.found
that Japanese patients carrying the rs8099917TT genotype had
lower plasma levels of gamma glutamyl transferase (GGT) and
showed more severe necro-inflammation and fibrosis scores in liver
biopsies as compared to carriers of the minor G allele, but the weak
(0.01,p,0.05) association of the histological scores with the IL28B
polymorphism needs independent confirmation.
IL28B polymorphism-related differences in the baseline immune
response to HCV infection may influence the severity of liver
necro-inflammation, the main stimulus for fibrogenesis that is the
hallmark of an unfavourable course of chronic hepatitis C.
The aim of this study was to investigate the distribution of the
rs12979860 SNP upstream of the IL28B gene in a well-
phenotyped population of chronically HCV-infected patients
covering the whole spectrum of disease severity, from null-mild
fibrosis to hepatocellular carcinoma, to elucidate whether this
polymorphic trait may be related with the course of chronic HCV
Materials and Methods
Starting by October 1997 we collected and stored peripheral
blood for DNA extraction from all HCV-infected patients referred
to our Liver Unit who gave informed consent according with the
Declaration of Helsinki. The study was approved by the local
ethics committee of the Hospital Clı ´nico San Carlos, Madrid,
Spain. The study design included two series of patients: 1) patients
with a diagnosis of chronic hepatitis C and positive HCV viremia
who were scheduled for their first liver biopsy and naı ¨ve for
antiviral therapy; and 2) patients with a diagnosis of hepatocellular
carcinoma (HCC) on HCV-induced liver cirrhosis in accordance
with current criteria . All HCC patients were considered as
cirrhotic because most of them were diagnosed with liver cirrhosis
before the diagnosis of CHC and the clinical study of the
remaining ones was consistent with cirrhosis. Moreover, hepato-
cellular carcinoma is exceptional in HCV-induced non-cirrhotic
chronic liver disease . A blood sample was taken for standard
laboratory tests and DNA extraction on the same day of the liver
biopsy in patients of the group 1, and when a definite HCC
diagnosis was established in patients of the group 2. Patients with
HIV or active HBV infections were excluded. A database was
created to include clinical and demographical data, and baseline
biochemical, virological and histological results.
All patients provided informed consent according with the
Declaration of Helsinki. The study was approved by the local
ethics committee of the Hospital Clı ´nico San Carlos, Madrid,
Spain. This consent was verbal, after providing to patients with full
explanation of the objectives and methods of the study,
guarantying them absolute privacity in accordance with the
Spanish legal rules, that are more strict than those in other
European countries. This circumstance was communicated to the
Ethics Commmittee before obtaining its authorization. In
addition, the determination of the IL28B polymorphism is a
routine method at the clinical laboratory of our centre since
Quantitative analysis of HCV-RNA was performed with the
Cobas Amplicor HCV Monitor version 2.0 (Roche Molecular
Diagnostic). The detection range was 600 IU/mL to 8.56105IU/
mL. Starting from July 2005, viral RNA was extracted automat-
ically using Cobas AmpliPrep, and the viral load was detected
using Real-Time polymerase chain reaction (PCR) using Cobas
TaqMan (Roche Diagnostics) which has a detection range of
between 10 IU/mL and 26108IU/mL . Viral load was
classified as low (,400000 IU/ml) or high ($400000 IU/ml),
according with Wittho ¨ft et al.  HCV genotypes were
determined by a reverse hybridization assay (INNO-LiPA;
Innogenetics). The genotypes are assigned on the basis of sequence
variations in the 59untranslated region of HCV following gene
amplification using reverse transcription polymerase chain reac-
Liver biopsy specimens obtained from patients of group 1 were
examined by the same pathologist. Necroinflammation grade and
fibrosis stage were scored using the METAVIR system . To
perform some analysis, necroinflammation was classified as null-
significant (0–2) versus severe (3). Fibrosis was coded as non-mild
fibrosis (F0–F1) versus moderate-advanced fibrosis (F2–F4).
Steatosis of the liver was evaluated according with the Brunt
score : absent or grade 0 (,5% of hepatocytes affected), grade
1 (5–33% of hepatocytes affected), grade 2 (33–66% of hepatocytes
affected); and grade 3 (.66% of hepatocytes affected).
IL28B genotyping was carried out by means of custom TaqMan
Assay (Applied Biosciences Hispania, Alcobendas, Madrid, Spain)
designed to detect the rs12979860 SNP. The detection was carried
out by qPCR in an Eppendorf realplex thermocycler by using
fluorescent probes. The amplification conditions were as follows:
After a denaturation time of 10 min at 96uC, 45 cycles of 92uC
15 sec 60uC 90 sec were carried out and fluorescence was
measured at the end of every cycle and at endpoint. All samples
were determined by triplicate and genotypes were assigned both,
by the gene identification software (RealPlex 2.0, Eppendorf) and
by analysis of the reference cycle number for each fluorescence
curve, calculated by the use of CalQPlex algorithm (Eppendorf).
For technical validation purposes, the amplified fragments for
twenty individuals carrying the rs12979860 C/C and C/T
genotypes and eleven individuals with the T/T genotype were
sequenced, and in all cases the genotypes fully corresponded with
those detected with fluorescent probes.
IL28B Polymorphism in HCV Infection
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Continuous variables, expressed as mean (SD), were compared
with the Student’ t test or the Mann-Whitney U test, each when
adequate, depending on their Gaussian distribution. A p value
,0.05 was considered significant. Categorical variables were
compared with the x2or the Fisher exact tests, each when
appropriate, and the effect of differences was established by
calculating the odds ratio with the 95% confidence interval.
The variables different at a p value ,0.05 in the univariate
analysis were included in a multivariate analysis based on a
logistic regression model to identify which ones were indepen-
dently related to the determined SNP in the vicinity of the IL28B
Group 1 (Chronic Hepatitis C)
Two hundred and sixty eight patients were included. Patient
profiles are shown in Table 1. Most patients were men (59.3%). All
patients were Caucasian, mostly Spaniards. One hundred and
seven (39.9%) carried the rs12979860CC homozygous genotype,
whereas the remaining 161 were homo- (33 patients, 12.3%) or
heterozygotes (128 patients, 47.8%) for the T allele. The studied
polymorphism was in Hardy-Weinberg equilibrium.
Biochemical baseline data that showed differences at a p value
,0.05 when comparing carriers of the rs12979860CC genotype
with carriers of the rs12979860CT/TT genotypes were ALT
(0.012), AST/ALT ratio (p=0.002), GGT (p,0.001), and serum
cholesterol (p=0.022). In the multivariate analysis only lower
values for GGT (p,0.001) and higher values for ALT (p=0.001)
were related with the 12979860CC genotype. (Table 1)
Necroinflammatory grade and fibrosis stage were not related
with the rs12979860 genotype. (Table 2).
Liver steatosis was present in a greater percentage of carriers
of the T allele than in CC homozygotes (44.0% vs. 30.8%, Odds
ratio=1.764, 95% C.I.=1.053–2.955) and there was a signifi-
cant trend towards higher grades of steatosis in carriers of the T
allele (Chi2for trend=4.946, P=0.026). (Table 2). When the
analysis was restricted to patients infected by HCV genotype 1,
similar results were obtained (45.1% vs. 32.9%, Odds ra-
tio=1.618, 95% C.I.=0.925–2.834, and Chi2for trend=3.934,
METAVIR necroinflammatory grade was significantly corre-
lated at a P,0.001 for Spearman’s rho test with serum ALT
(alanine amino transferase) and GGT values, but not with the
remaining biochemical variables included in the analysis. META-
VIR fibrosis stage correlated at the same level of significance with
the inverse of platelet count and with serum AST (aspartate amino
transferase), ALT, GGT and ferritin values.
Group 2 (HCV-related Hepatocellular Carcinoma)
One hundred and thirty four patients (97 males, mean age at
diagnosis 67.4 years, SD 9.4, range 41–88) were included in this
group. All were white Spaniards. The diagnosis of HCC was based
on the pathological exam of surgical biopsy or cytological aspirate
in 73 patients, and according with current imaging criteria  in
the remaining 61. All patients in this group had detectable titers of
anti-HCV antibodies in serum. Only two patients had been
previously treated with interferon-ribavirin before developing
Fifty two patients (38.8%) carried the rs12979860CC homozy-
gous genotype, whereas the remaining 82 were homo- (16 patients,
11.9%) or heterozygotes (66 patients, 49.3%) for the T allele.
Former ethanol use $ 40 g/day was reported by 52 patients (50
men). The distribution of the analyzed SNP was very similar as
comparing these patients with moderate drinkers or teetotallers
(P.0.80 for each comparison, data not shown).
Table 1. Analysis of biochemical and virological parameters in relation with the rs12979860 genotype in 268 patients with chronic
T allele carriers
(161 cases)P (univariate)P (multivariate)
Age (yrs.) 47.2 (10.5)45.9 (10.4) 48.3 (10.5)0.076
Viral load #/.400.000 IU/mL/n.a.48/213/7 20/83/428/130/3OR=1.118
Viral genotype 1/non 1/n.a. 225/40/384/20/3141/20/0 OR=0.596
Hb (g/dL)15.1 (1.4)15.0 (1.4) 15.2 (1.4)0.399
Platelets (109/L)202 (59)202 (57)203 (61)0.830
Bilirubin (mg/dL) 0.83 (0.45)0.78 (0.36)0.87 (0.50)0.135
AST (IU)67 (48)71 (47)64 (48)0.224
ALT (IU)112 (94)130 (103) 99 (85)0.0120.001
AST/ALT0.69 (0.28)0.64 (0.24)0.73 (0.30)0.0020.593
GGT (IU) 79 (118)47 (42)100 (146)
Alkaline phosphatase (IU)111 (59)111 (54)111 (62)0.986
Cholesterol (mg/dL)178 (36)184 (40)173 (31)0.0220.102
Major allele: C. Minor allele: T.
n.a.: not available.
IL28B Polymorphism in HCV Infection
PLoS ONE | www.plosone.org3May 2012 | Volume 7 | Issue 5 | e37998
Comparative Analysis of HCV-infected Patients with and
To evaluate the possible relation between the analyzed IL28B
SNP and the stage of the HCV-induced liver disease, the CHC
patients in the chronic hepatitis group who had not received
antiviral therapy after performing the liver biopsy were compared
with the group of patients with hepatocellular carcinoma. No
significant differences were found between both groups in the
distribution of the studied IL28B gene SNP (Table 3).
The relation between genetic polymorphisms in the vicinity of
the IL28 gene and the response to pegylated interferon and
ribavirin combined therapy for hepatitis C has been clearly
demonstrated since the rs12979860 SNP was identified. Several
GWAS studies independently disclosed that the possession of any
of the homozygous genotypes rs12979860CC or rs8099917TT,
which are in strong linkage disequilibrium, greatly increases the
probability to obtain sustained viral response after antiviral
However, information on the possible influence of these
polymorphisms on the course of hepatitis C infection is presently
very limited and incomplete. It has been shown that the carriers of
the favourable genotypes have a higher rate of spontaneous
clearance of HCV during the acute phase of the infection [19–21].
In this study we have shown that GGT levels are lower in patients
with chronic hepatitis C that carry the rs12979860CC genotype
than in homo- or heterozygotes for the T mutated allele. Abe et al.
, in a cohort of 364 adult Japanese patients with chronic HCV
infection, reported a similar and highly significant (p=0.001)
association between the rs8099917TT genotype and GGT lower
values, a link that could be explained because both traits are
predictive of a favourable response to therapy.
In addition, we have found that rs12979860CC genotype is
associated with higher serum ALT than the remaining genotypes,
a finding that is in agreement with data of Thompson et al. 
obtained from a GWAS study that disclosed that only the
rs12979860 SNP was significantly associated with baseline ALT
levels. Serum ALT is considered as a marker of necroinflamma-
tory activity in the liver as confirms the analysis of our data that
shows a linear increase of ALT levels related to METAVIR
activity score. These findings are contradictory with those reported
by Abe et al.  who found that ALT and AST levels were lower
in carrier of the rs8099917TT genotype, although these authors
do not provide the level of significance for this association.
However, our results do not confirm the suggested relation
between the IL28B gene polymorphism and the histological
necroinflammatory activity reported by Abe et al.  and
Thompson et al. . Most patients included in the present study
were infected with HCV genotype 1. Hence, we have not been
able to confirm the interesting results reported by Bochud et al.
 linking the rs8099917G allele, that is associated with poor
response to therapy, with lower necroinflammatory activity
(p=0.04) and milder fibrosis (p=0.02) in patients infected with
non-1 HCV genotypes.
The relation between IL28B polymorphism and the stage of
fibrosis is controversial. Abe et al.  found higher fibrosis
METAVIR scores among carriers of the rs8099917TT genotype.
However, Di Marco et al.  in a group of 131 patients with
thalassemia major and chronic HCV infection who underwent a
liver biopsy reported that older age and the carrier state of the
minor alleles at rs12979860 and rs8099917 sites were associated
with more severe liver fibrosis (p,0.001 and p,0.005, respective-
ly). Falleti et al.  in a group of 629 HCV-positive patients,
disclosed that subjects with the rs12979860TT homozygous
genotype had a mean fibrosis score higher than the remaining
CC or CT genotypes (p, 0.05). Nevertheless, Thompson et al. 
reported that the rs12979860 polymorphism was not associated
with advanced hepatic fibrosis in CHC, a finding that is in
agreement with our results that are far from the statistical
significance for the determined SNP.
Table 2. Analysis of histological features in relation with the rs12979860 genotype in 268 patients with chronic hepatitis C.
T allele carriers
CC vs. T carriers
CT vs. TT
METAVIR necroinflammatory grade
1/16/40/50 3/30/68/60Chi2 LT=2.409
2/21/57/48 1/9/11/12 Chi2 LT=0.952
METAVIR fibrosis stage (0/1/2/3/4) 21/33/17/28/828/56/27/35/15Chi2 LT=0.0002
Steatosis (Brunt score 0/1/2/3/n.a.) 74/27/6/0/089/56/10/4/2 Chi2 LT=4.946
Steatosis (Brunt score) 0/.0/n.a. 74/33/089/70/2O.R.=1.764
Major allele: C. Minor allele: T.
Chi2LT: Chi square for linear trend.
Table 3. Distribution of the IL28B gene rs12979860 genotype
in untreated patients with chronic hepatitis C (CHC) and in
patients with HCV-related hepatocellular carcinoma (HCC).
CC37 (37.8)52 (38.8)
CT49 (50.0)66 (49.3)
TT11 (11.2)16 (11.9)P=0.979
T allele frequency 0.386 0.366 O.R.=0.985
IL28B Polymorphism in HCV Infection
PLoS ONE | www.plosone.org4May 2012 | Volume 7 | Issue 5 | e37998
Fabris et al.  analysed the distribution of rs12979860 C and
T alleles in a miscellaneous group of Italian patients with chronic
liver diseases, most HCV-related, including patients with mild
hepatitis (Ishak staging score # 2), cirrhosis and HCC. They
concluded that the T allele is more prevalent in HCV-induced
cirrhosis than in cirrhosis from other causes (p,0.005) but not
when comparing HCV-induced cirrhosis with milder hepatitis C
Hepatic steatosis is a frequent finding in chronic hepatitis C, as
a probable consequence of a disturbance of lipid metabolism .
Our study shows that steatosis is significantly more frequent in
CHC patients carrying the mutated T allele, in agreement with
two previous studies. Tillmann et al  analysed two indepen-
dent cohorts of patients with a global size of 325 patients with
rs12979860CC genotype patients (31.8%) and in 139 of 237
patients carrying the rs12979860T allele (58.6%). These differ-
ences were highly significant when each group was analysed
separately (as did the authors) or when considering them as a
unique group (Odds ratio=3.04, 95% C.I.=1.81–5.10). In the
second study  that included 153 Japanese patients, the analysis
of the rs8099917 genotype yielded similar results. A new aspect
disclosed in our study is that steatosis is not only more frequent,
but also more severe in patients carrying the mutated T allele.
Hepatocellular carcinoma (CHC) represents the most advanced
step in the natural course of chronic hepatitis C infection that
fortunately is reached only by a minority of patients. If the
hypothesis of the existence of any relationship between the IL28B
genetic polymorphism and the severity of HCV-induced liver
disease holds true, the most logical finding should be the existence
of differences in the genotype frequencies among the different
stages of the disease. We have found no differences in the
frequencies of the IL28B genotypes as comparing a group of HCV-
induced HCC patients with a group of 98 untreated patients with
CHC. Our results are coincidental with those reported by other
groups, both in Caucasian  and in Japanese [44,45]
populations. However, Asahina et al.  reported that carriers
of the rs8099917 TT genotype had a higher rate of sustained viral
response to interferon-based therapy (as expected) and that this
genotype was associated with a lower incidence of HCC, both in
was present in28 of 88
sustained viral responders and in non-responders. A direct effect of
the IL28B polymorphism on the risk of HCC may not be inferred
from these results, as SVR clearly improves the natural course of
chronic HCV infection [47,48] and, among non-responders, the
effect of the IL28B polymorphism was indirect and associated with
slightly lower levels of ALT and alpha-feto protein. Eurich et al.
, in a study of patients who received a liver graft due to severe
HCV-induced liver disease, found that the 12979860TT genotype
was more frequent in the 61 patients with HCC on the explanted
liver than in the 106 patients without HCC (p=0.041), suggesting
that the major C allele plays a protective role against the
development of HCC. In addition, Ren et al.  found that the
rs12979860 T allele was related to the susceptibility of both
chronic hepatitis B virus (HBV) infection and HBV-related HCC
in a group of Chinese patients. Thus, controversy remains about
the possible relation between the IL28B gene polymorphism and
the risk of developing HCC.
We conclude that the IL28B rs12979860CC genotype in
patients with chronic hepatitis C is related with higher serum
ALT, that is considered as a marker of hepatic necroinflammation,
and with lower values of GGT, which increase is a surrogate
marker of liver fibrosis and a negative predictive factor of response
to therapy . In addition, the carrier state of the rs12979860 T
allele is associated with greater frequency and severity of hepatic
steatosis in chronic hepatitis C. However, we have not found any
relation among this polymorphism and necroinflammatory grade
and fibrosis stage directly shown by the liver biopsy, neither with
the risk of developing HCV-related hepatocellular carcinoma.
We thank Ms. Gara Esguevillas, Ms. Isabel Salas and Ms. Debbie Benn for
their technical assistance.
Conceived and designed the experiments: JML JAA. Performed the
experiments: EG-M MLM CM MC MV. Analyzed the data: JML.
Contributed reagents/materials/analysis tools: FC LO MA AS GD-Z MD-
R. Wrote the paper: JML JAA.
1.Seeff LB (2002) Natural history of chronic hepatitis C. Hepatology 36: S35–S46.
Chen SL, Morgan TR (2006) The natural history of hepatitis C virus (HCV)
infection. Int J Med Sci 3: 47–52. [PMID: 16614742].
Seeff LB (2009) The history of the ‘‘natural history’’ of hepatitis C (1968–2009)
Liver Int 29 Suppl 1: 89–99. [PMID: 19207971].
Bissell DM (1999) Sex and hepatic fibrosis. Hepatology 29: 988–989. [PMID:
Williams MJ, Lang-Lenton M, Trent HCV study group (2011) Progression of
initially mild hepatic fibrosis in patients with chronic hepatitis C infection. J
Viral Hepat 18: 17–22. [PMID: 20088889].
Martı ´nez C, Garcı ´a-Martı ´n E, Ladero JM, Herra ´ez O, Ortega L, et al. (2007)
GSTT1 and GSTM1 null genotypes may facilitate hepatitis C virus infection
becoming chronic. J Infect Dis 195: 1320–1323. [PMID: 17397002].
Minton EJ, Smillie D, Neil KR, Irving WL, Underwood JC, et al. (1998)
Association between MHC class II alleles and clearance of circulating hepatitis
C virus. J Infect Dis 178: 39–44.[PMID: 9652421].
Singh R, Kaul R, Kaul A, Khan K (2007) A comparative review of HLA
associations with hepatitis B and C viral infections across global populations.
World J Gastroenterol 13: 1770–1787. [PMID: 17465466].
Wang H, Mengsteab S, Tag CG, Gao CF, Hellerbrand C, et al. (2005)
Transforming growth factor-beta1 gene polymorphisms are associated with
progression of liver fibrosis in Caucasians with chronic hepatitis C infection.
World J Gastroenterol 11: 1929–1936. [PMID: 15800982].
10. Kunzler S, Baumann M, Schirmacher F, Dries V, Bayer E, et al. (2001)
Prediction of progressive liver fibrosis in hepatitis C infection by serum and tissue
levels of transforming growth factor-b. J Virol 8: 430–437. [PMID: 11703574].
11. Ge D, Fellay J, Thompson AJ, Simon JS, Shianna KV, et al. (2009) Genetic
variation in IL28B predicts hepatitis C treatment-induced viral clearance.
Nature 461: 399–401. [PMID: 19684573].
12. Suppiah V, Moldovan M, Ahlenstiel G, Berg T, Weltman M, et al. (2009) IL28B
is associated with response to chronic hepatitis C interferon-a and ribavirin
therapy. Nature Genetics 41: 1100–1104. [PMID: 19749758].
13. Tanaka Y, Nishida N, Sugiyama M, Kurosaki M, Matsuura K, et al. (2009)
Genome-wide association of IL28B with response to pegylated interferon-a and
ribavirin therapy for chronic hepatitis C. Nature Genetics 41: 1105–1109.
14. Balagopal A, Thomas DL, Thio CL (2010) IL28B and the control of hepatitis C
virus infection. Gastroenterology 139: 1865–1876. [PMID: 20950615].
15. Afdhal NH, McHutchison JG, Zeuzem S, Mangia A, Pawlotsky JM, et al. (2010)
Hepatitis C pharmacogenetics: State of the art in 2010. Hepatology 53: 336–
345. [PMID: 21254181].
16. Honda M, Sakai A, Yamashita T, Nakamoto Y, Mizukoshi E, et al. (2010)
Hokuriku Liver Study Group. Hepatic ISG expression is associated with genetic
variation in interleukin 28B and the outcome of IFN therapy for chronic
hepatitis C. Gastroenterology 139: 499–509. [PMID: 20434452].
17. Urban TJ, Thompson AJ, Bradrick SS, Fellay J, Schuppan D, et al. (2010)
IL28B genotype is associated with differential expression of intrahepatic
interferon-stimulated genes in patients with chronic hepatitis C. Hepatology
52: 1888–1896. [PMID: 20931559].
18. Ahlenstiel G, Booth DR, George J (2010) IL28B in hepatitis C virus infection:
translating pharmacogenomics into clinical practice. J Gastroenterol 45: 903–
910. [PMID: 20635099].
IL28B Polymorphism in HCV Infection
PLoS ONE | www.plosone.org5 May 2012 | Volume 7 | Issue 5 | e37998
19. Thomas DL, Thio CL, Martin MP, Qi Y, Ge D, et al. (2009) Genetic variation
on IL28B and spontaneous clearance of hepatitis C virus. Nature 461: 798–801.
20. Montes-Cano MA, Garcı ´a-Lozano JR, Abad-Molina C, Romero-Go ´mez M,
Barroso N, et al. (2010) Interleukin-28B genetic variants and hepatitis virus
infection by different viral genotypes. Hepatology 52: 33–37. [PMID:
21. Tillmann HL, Thompson AJ, Patel K, Wiese M, Tenckhoff H, et al. (2010)
German Anti-D Study Group. A polymorphism near IL28B is associated with
spontaneous clearance of acute hepatitis C virus and jaundice. Gastroenterology
189: 1586–1592. [PMID: 20637200].
22. McCarthy JJ, Li JH, Thompson A, Suchindran S, Lao XQ, et al. (2010)
Replicated association between IL28B gene variant and a sustained response to
pegylated interferon and ribavirin. Gastroenterology; 138: 2307–2314. [PMID:
23. Thompson AJ, Muir AJ, Sulkowski MS, Ge D, Fellay J, et al. (2010) Interleukin-
28B polymorphism improves viral kinetics and is the strongest pre-treatment
predictor of sustained virologic response in genotype 1 hepatitis C virus.
Gastroenterology 139: 120–129. [PMID: 20399780].
24. Sta ¨ttermayer AF, Stauber R, Hofer H, Rutter K, Beinhardt S, et al. (2011)
Impact of IL28B genotype on the early and sustained virologic response in
treatment-naı ¨ve patients with chronic hepatitis C. Clin Gastroenterol Hepatol 9:
344–350. [PMID: 20728570].
25. Hayes CN, Kobayashi M, Akuta N, Suzuki F, Kumada H, et al. (2011) HCV
substitutions and IL28B polymorphisms on outcome of peg-interferon plus
ribavirin combination therapy. GUT 60: 261–267. [PMID: 21068134].
26. Rauch A, Kutalik Z, Descombes P, Cai T, Di Iulio J, et al. (2010) Swiss Hepatitis
C Cohort Study; Swiss HIV Cohort Study. Genetic variation in IL28B is
associated with chronic hepatitis C and treatment failure: a genome-wide
association study. Gastroenterology 138: 1338–1345. [PMID: 20060832].
27. Kurosaki M, Tanaka Y, Nishida N, Sakamoto N, Enomoto N, et al. (2011) Pre-
treatment prediction of response to pegylated-interferon plus ribavirin for
chronic hepatitis C using genetic polymorphism in IL28B and viral factors. J
Hepatol 54: 439–448. [PMID: 21129805].
28. Ladero JM, Garcı ´a-Martı ´n E, Ferna ´ndez C, Carballo M, Devesa MJ, et al.
(2012) Predicting response to therapy in chronic hepatitis C: An approach
combining interleukin-28B gene polymorphisms and clinical data. J Gastro-
enterol Hepatol 27: 279–285. [PMID: 21722179].
29. Abe H, Ochi H, Maekawa T, Hayes CN, Tsuge M, et al. (2010) Common
variation of IL28 affects gamma-GTP levels and inflammation of the liver in
chronically infected hepatitis C virus patients. J Hepatol 53: 439–443. [PMID:
30. Lencioni R (2011) Evolving strategies in the diagnosis of hepatocellular
carcinoma. J Hepatol 54: 184–186. [PMID: 20950887].
31. Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR.
Genome Res 6: 989–994. [PMID: 8908518].
32. Wittho ¨ft TH, Mo ¨ller B, Wiedmann KH, Mauss S, Link R, et al. (2007) Safety,
tolerability and efficacy of peginterferon alpha-2a and ribavirin in chronic
hepatitis C in clinical practice: The German Open Safety Trial. J Viral Hepat
14: 788–796. [PMID: 17927615].
33. The French METAVIR Cooperative Study Group (1994) Intraobserver and
interobserver variations in liver biopsy interpretation in patients with chronic
hepatitis C. Hepatology 20: 15–20. [PMID: 8020885].
34. Brunt E, Janney C, Di Bisceglie A, Neuschwander-Tetri B, Bacon B (1999)
Nonalcoholic steatohepatitis: a proposal for grading and staging the histological
lesions. Am J Gastroenterol 94: 2467–2474.[PMID: 10484010].
35. Thompson AJ, Clark PJ, Zhu M, Zhu Q, Ge D, et al. (2010) Genome wide-
association study identifies IL28B polymorphism to be associated with baseline
ALT and hepatic necro-inflammatory activity in chronic hepatitis C patients
enrolled in the IDEAL study. Hepatology 52 (Suppl). pp 1220A–1221A.
36. Bochud P-Y, Bibert S, Kutalik Z, Patin E, Guergnon J, et al. (2012) IL28B alleles
associated with poor hepatitis C virus (HCV) clearance protect against
inflammation and fibrosis in patients infected with non-1 HCV genotypes.
Hepatology 55: 384–94. [PMID: 22180014].
37. Di Marco V, Bronte F, Calvaruso V, Capra M, Borsellino Z, et al. (2011) IL28B
polymorphisms influence stage of liver fibrosis and spontaneous or interferon-
induced viral clearance in thamassemia patients with hepatitis C virus infection.
Haematologica [Epub ahead of print] [PMID: 22180419].
38. Falleti E, Bitetto D, Fabris C, Cussigh A, Fornasiere E, et al. (2011) Role of
interleukin 28B rs12979860 C/T polymorphism on the histological outcome of
chronic hepatitis C: relationship with gender and viral genotype. J Clin Immunol
31: 891–899. [PMID: 21747799].
39. Thompson AJ, Clark PJ, Fellay J, Muir AJ, Tillmann HL, et al. (2010) IL28B
genotype is not associated with advanced hepatic fibrosis in chronic hepatitis C
patients enrolled in the IDEAL study. Hepatology 52 (Suppl). pp 437A–438A.
40. Fabris C, Falleti E, Cussigh A, Bitetto D, Fontanini E, et al. (2011) IL-28B
re12979060 C/T allele distribution in patients with liver cirrhosis: Role in the
course of chronic viral hepatitis and the development of HCC. J Hepatol 54:
716–722. [PMID: 21146242].
41. Li JH, Lao XQ, Tillmann HL, Rowell J, Patel K, et al. (2010) Interferon-lambda
genotype and low serum low-density lipoprotein cholesterol levels in patients
with chronic hepatitis C infection. Hepatology 51: 1904–1911. [PMID:
42. Tillmann HL, Patel K, Muir AJ, Guy CD, Li JH, et al. (2011) Beneficial IL28B
genotype associated with lower frequency of hepatic steatosis in patients with
chronic hepatitis C. J Hepatol 55: 1195–2000. [PMID: 21703198].
43. Ohnishi M, Tsuge M, Kohno T, Zhang Y, Abe H, et al. (2012) IL28B
polymorphism is associated with fatty change in the liver of chronic hepatitis C
patients. J Gastroenterol [Epub ahead of print].[PMID: 22350701].
44. Miura M, Maekawa S, Kadokura M, Sueki R, Komase K, et al. (2011) Analysis
of viral amino acids sequences and the IL28B SNP influencing the development
of hepatocellular carcinoma in chronic hepatitis C. Hepatol Int [Epub ahead of
print]. [PMID: 22022823].
45. Joshita S, Umemura T, Katsuyama Y, Ichikawa Y, Kimura T, et al. (2011)
Association of IL28B gene polymorphism with development of hepatocellular
carcinoma in Japanese patients with chronic hepatitis C virus infection. Hum
Immunol 73; 298–300. [PMID 22245236].
46. Asahina Y, Tanaka K, Suzuki Y, Tamaki N, Hoshioka T, et al. (2011)
Association between IL28B gene variation and development of hepatocellular
carcinoma after interferon therapy in patients with chronic hepatitis C. J
Hepatol 54 (Suppl 1): S37.
47. Trapero-Maruga ´n M, Mendoza J, Chaparro M, Gonza ´lez-Moreno L, Moreno-
Monteagudo JA, et al. (2011) Long-term outcome of chronic hepatitis C patients
with sustained virological response to interferon plus ribavirin. World J
Gastroenterol 17: 493–498. [PMID: 21274379].
48. Maruoca D, Imazeki F, Arai M, Kanda T, Fujiwara K, et al. (2012) Long-term
cohort study of chronic hepatitis C according to interferon efficacy. J
Gastroentrol Hepatol 27: 291–299. [PMID: 21793911].
49. Eurich D, Boas-Knoop S, Bahra M, Neuhaus R, Somasundaram R, et al. (2012)
Role of IL28B polymorphism in the development of hepatitis C virus-induced
hepatocelular carcinoma, graft fibrosis, and posttransplant activiral therapy.
Transplantation 93: 644–649 [PMID: 22411462].
50. Ren S, Lu J, Du X, Huang Y, Ma L, et al. (2012) Genetic variation in IL28B is
associated with the development of hepatitis B-related hepatocellular carcinoma.
Cancer Immunol Immunother [Epub ahead of print]. [PMID: 22310928].
51. Cuenca F, Ferna ´ndez C, Devesa MJ, Lo ´pez-Alonso G, Mayol J, et al. (2010)
Predictive baseline criteria of primary therapeutic failure in chronic hepatitis C
genotype 1. Rev Esp Enferm Dig 102: 234–238. [PMID 20486745].
IL28B Polymorphism in HCV Infection
PLoS ONE | www.plosone.org6 May 2012 | Volume 7 | Issue 5 | e37998