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.
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ABSTRACT: To assess the role of IL28B rs 12979860 polymorphism in predicting response to treatment in genotype 4 (G4) Egyptian patients, and to evaluate the role of alpha fetoprotein (AFP) in increasing the predictive strength of IL28B rs 12979860 polymorphism to predict response to treatment. One hundred thirty 7 HCV patients were genotyped for IL28B rs 12979860 by polymerase chain reaction-restriction fragment length polymorphism technique. The presence of the C allele of IL28B rs 12979860 was associated with response to treatment, while the T allele was associated with failure of response to treatment. AFP is associated with IL28B rs 12979860 SNP genotypes at cut off 2.68 and 4.5 ng/mL individually. Response rate was 1.3 and 1.6, 3 times higher in CC, CT, and TT respectively in patients below AFP 4.5 ng/mL than in patients above it. IL28B rs 12979860 polymorphism is strongly associated with treatment induced response to treatment. AFP (cut off 4.5 mg/mL) increases the predictive power of IL28B in response to treatment.Journal of interferon & cytokine research: the official journal of the International Society for Interferon and Cytokine Research 03/2014; · 1.63 Impact Factor
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ABSTRACT: The primary goal of HCV therapy is to achieve a sustained virological response (SVR). Many host and viral factors influence the treatment response. Cytokines play an important role in the defense against viral infections, where successful treatment of hepatitis C depends on a complex balance between pro- and anti-inflammatory responses. In the present study, we investigated the relationship between the presence and percentage of some cytokines (IL-28, IFN-γ, and TNF-α) regarding different clinicopathological parameters including response to therapy in chronic HCV patients using immunohistochemical technique. This study was carried out on 64 chronic HCV patients (34 responders and 30 non-responders). Of cases, 54% showed IL-28 expression, which was associated with low AST (p = 0.002) and low HAI score (p = 0.006). Of cases, 67 and 45% showed IFN-γ and TNF-α expression, respectively, where the median percentage of TNF-α expression was higher in grade II spotty necrosis compared to grade I. Some inflammatory cytokines expressed by intrahepatic inflammatory cells in chronic HCV patients promote inflammation and injury (pro-inflammatory) such as TNF-α. Other cytokines aid in resolving inflammation and injury (anti-inflammatory) such as IL-28. The balance between these cytokines will determine the degree of inflammatory state. None of the investigated cytokines proved its clear cut role in affecting response to therapy, however, their levels varied between responders and non-responders for further investigations to clarify.Apmis 08/2014; · 2.07 Impact Factor
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ABSTRACT: Genetic variation of interleukin-28B (IL-28B) rs12979860 T/C polymorphism is associated with the immune response to interferon (IFN) therapy, which is applied in the treatment of chronic viral hepatitis induced by hepatitis B virus (HBV) and hepatitis C virus (HCV). These chronic liver diseases could progress to end-stage liver diseases, such as hepatocellular carcinoma (HCC). The aim of this study was to clarify whether there exists a causal association between IL-28B rs12979860 T/C polymorphism and development of HCC. In a meta-analysis of six studies with 850 cases and 811 controls, we summarized the data on the association between IL-28B rs12979860 T/C polymorphism and HCC risk and calculated ORs and 95 % CIs to estimate the association strength. We observed that IL-28B rs12979860 T/C polymorphism was positively associated with overall HCC risk (TT vs. CC: OR = 2.38; 95 %, 1.60-3.55; TT vs CT + CC: OR = 1.79; 95 %, 1.23-2.60). In the stratified analysis by ethnicity, the robust association retained in Caucasians with higher risk among TT carriers relative to the CC carriers. A similar trend was found in the studies of healthy controls when data were stratified by source of controls. The combined data suggest that IL-28B rs12979860 T/C polymorphism seems to augment the risk of developing HCC, especially in Caucasians.Tumor Biology 05/2014; · 2.52 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: email@example.com
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.
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