The impact of IL-6 and IL-28B gene
polymorphisms on treatment outcome of
chronic hepatitis C infection among
intravenous drug users in Croatia
*, Ivana Marinovi
*, Sendi Kuret
*, Nikola Bradari
, Gea Forempoher
, Ozren Polas
1Department of Internal Medicine, Division of Gastroenterology, Clinical Hospital Split, Split,
2Department of Immunology, Univeristy of Split, School of Medicine, Split, Croatia
3Department of Pathology, Clinical Hospital Split, Split, Croatia
4Department of Research in Biomedicine and Health, Univeristy of Split, School of Medicine,
5Department of Infectious Diseases, Clinical Hospital Split, Split, Croatia
6Department of Public Health, Univeristy of Split, School of Medicine, Split, Croatia
*These authors contributed equally to this work.
Background: Several genes and their single nucleotide polymorphisms (SNPs) are
associated with either spontaneous resolution of hepatitis C infection or better
treatment-induced viral clearance. We tested a cohort of intravenous drug users
(IVDU) diagnosed with chronic hepatitis C virus (HCV) for treatment response and
its association with the SNPs in the interleukin-6 (rs1800795-IL6) and the
interleukin-28B (rs12979860-IL28B) genes.
Methods: The study included 110 Croatian IVDU positive for anti-HCV antibody.
Genotyping was performed by polymerase chain reaction (PCR) based approach.
Patients were treated by standard pegylated-interferon/ribavirin and followed
throughout a period of four years, during which sustained virological response
(SVR) was determined. All data were analysed with statistical package SPSS 19.0
(IBM Corp, Armonk, NY, USA) and PLINK v1.07 software.
Results: Patients showed a signiﬁcantly better response to treatment according to the
number of copies of the C allele carried at rs1800795-IL6 (P = 0.034). All but one of
the patients with CC genotype achieved SVR (93%), whereas the response rate of
patients with GG genotype was 64%. The association of rs1800795-IL6 with SVR
status remained signiﬁcant after further adjustment for patients’ age, ﬁbrosis
staging, and viral genotype (OR 2.15, 95% CI 1.16–4.68, P = 0.019). Distributions of
allele frequencies at the locus rs12979860-IL28B among the study cohort and the
underlying general population were suggestive of a protective effect of CC genotype
in acquiring chronic hepatitis C in the Croatian IVDU population.
Discussion: The rs1800795-IL6 polymorphism is associated with positive response
to treatment in IVDU patients positive for HCV infection. A protective role of
rs12979860-IL28B CC genotype in acquiring chronic hepatitis C is suggested for
Croatian IVDU population.
How to cite this article Bogdanovic et al. (2016), The impact of IL-6 and IL-28B gene polymorphisms on treatment outcome of chronic
hepatitis C infection among intravenous drug users in Croatia. PeerJ 4:e2576; DOI 10.7717/peerj.2576
Accepted 15 September 2016
Published 25 October 2016
Additional Information and
Declarations can be found on
c et al.
Creative Commons CC-BY 4.0
Subjects Gastroenterology and Hepatology, Immunology, Infectious Diseases, Medical Genetics
Keywords Hepatitis C, Intravenous drug users, IL-6, IL-28, Sustained virological response, Genetic
It is estimated that chronic hepatitis C virus (HCV) infection affects nearly 170 million
individuals worldwide (Averhoff, Glass & Holtzman, 2012;World Health Organization,
2014). It poses as one of the most important and growing threats to public health.
Being an intravenous drug user (IVDU) is one of the most important risk factors in
acquiring this infection.
Only a minority of patients are able to clear the virus, and so do not run the risk of
developing HCV induced end liver damage. The majority of patients will develop either
acute or chronic hepatitis that will require treatment. PegIFN-a2a in combination with
ribavirin was used as a standard treatment in patient that did not meet exclusion criteria.
Since 2011, a few generations of direct-acting antivirals (DAAs) were approved for
HCV treatment (Zeuzem et al., 2011). Each of three DAAs subgroups can be used as a
component of a triple combination regimen with PegIFN-a and ribavirin, yielding
sustained viral response (SVR) rates of 60–100%. Response rate depends on the DAA
used, the HCV genotype, the presence of detectable pre-existing amino acid substitutions
conferring resistance to the DAA used and the severity of liver disease (European
Association for the Study of the Liver, 2014). With three new HCV DAAs approved,
IFN-free combinations are reserved for patients with advanced liver disease (ﬁbrosis
METAVIR score F3 or F4).
Among the different and diverse list of factors that inﬂuence the therapeutic response,
the host’s cytokines play a very important role. The cytokine levels are directly inﬂuenced
by certain gene polymorphisms located within their coding or regulatory regions
(Wilson et al., 1997).
Among others, IL-6 is reported to be elevated in chronic HCV infection compared to
healthy controls (Malaguarnera et al., 1997). The low-producing interleukin-6 genotype
IL-6 CC (IL-6 rs1800795 G174C) was associated with spontaneous clearance of HCV
in patients infected by contaminated blood products (Barrett et al., 2001). On contrary,
high producing interleukin-6 genotypes of the rs1800795 174G/C polymorphism (i.e.,
GG or GC genotypes) were associated with a greater likelihood of SVR in patients
coinfected with HCV and HIV (Nattermann et al., 2007).
The human interleukin-28B gene encodes interferon lambda-3 (IFN-lambda-3).
Interferon lambda (IFN-) has demonstrated antiviral activity against HCV genotype 1
in vivo (Pagliaccetti & Robek, 2010) and in vitro (Muir et al., 2010). It has been shown
that a single nucleotide polymorphism (SNP) of IL28B gene (IL-28B rs12979860 C/T)
predicts hepatitis C treatment induced viral clearance (Ge et al., 2009;Halfon et al., 2011;
Lin et al., 2011;Lindh et al., 2011;Luo et al., 2013) and is associated with spontaneous
resolution of hepatitis C infection (Duggal et al., 2013;Kurbanov et al., 2011;Shi et al.,
2012;Thomas et al., 2009;Tillmann et al., 2010).
c et al. (2016), PeerJ, DOI 10.7717/peerj.2576 2/16
Here, we studied whether interleukin-28B or interleukin-6 (IL-6) promoter SNP
affects the response to the PegIFN-a2a/ribavirin antiviral treatment in IVDU patients
diagnosed with chronic hepatitis C. We determined IL-6 promoter and IL-28B gene
polymorphisms in a cohort of 110 patients with chronic hepatitis C. All IVDU positive
patients diagnosed with chronic hepatitis C were treated with a standard protocol of peg-
interferon alpha-2a and ribavirin. Rates of SVR were compared between IL-6 and IL-28B
wild type, heterozygous and homozygous genotypes.
SUBJECTS AND METHODS
The study samples
The sample of 112 patients was recruited from the outpatient hepatology unit at the
Split Medical Center, Croatia and was followed from September 2007 until November
2013. The sample was drawn from the total of 947 HCV positive patients but only
112 individuals were diagnosed, biopsied, genotyped, treated and followed for a period of
four years. Two patients were excluded from further analysis due to an ambiguous SVR
status, possible reuse of intravenous drugs and re-infection with a different viral strain,
resulting in a cohort of 110 patients. All enrolled patients had an alcohol consumption of
less than 14 units per week and other common forms of chronic liver disease were
excluded in all cases. Patients were Caucasians with the median (interquartile ranges
(IQR)) age at diagnosis of 40 (35–45) years in the SVR group and 41.5 (39–47) in the
non-responder (NR) group.
We also used the data from the 10,001 Dalmatians biobank as the source of population-
based sample of the underlying, general Croatian population. All controls were apparently
healthy subjects with no record of addiction, risky behaviors or detected HCV infection
ˇek, 2013;Rudan et al., 2009).
Patients’ data were collected as a part of standard clinical procedure and the informed
consent was obtained prior to participating in the study in all cases. The Ethics Committee
of the Clinical Hospital Split approved the study (No: 2181-147-06-01/01-M.J).
Diagnosis and treatment of HCV infection
A third generation enzyme immunoassay (ELISA; Abbott Diagnostics, Wetzlar, Germany)
was used to test all subjects for HCV speciﬁc antibodies. Reverse-transcriptase polymerase
chain reaction (RT-PCR) assay (Amplicor; Roche Diagnostic Systems, New Jersey, USA)
was used to test for HCV RNA in all subjects, in order to determine SVR. PegIFN-a
was used as a subcutaneous injection of 180 mg (or less if dose reduction was needed),
once a week. Depending on patient’s body weight (75kg), a total of 1,000 or 1,200 mg
of ribavirin was administered, in divided doses.
SVR was deﬁned as undetectable HCV RNA 12 weeks or 24 weeks after treatment
completion as assessed by a sensitive molecular method with a lower level of detection
15 IU/ml. NRs were patient who did not meet the SVR.
Our patients were followed up for four years and we were able to identify relapses.
Patients that relapsed during the follow up time were included in NR group.
c et al. (2016), PeerJ, DOI 10.7717/peerj.2576 3/16
Percutaneous liver biopsy was performed at the time of initial diagnosis and at the
beginning of the treatment, using the Trucut biopsy technique (Sterylab, Rho MI, Italy)
following informed consent. Inﬂammation was graded using a histological activity index
(HAI) (Knodell et al., 1981) and ﬁbrosis (Ishak et al., 1995). Every ﬁfth biopsy was
independently validated by two pathologists. A minority of patients was not biopsied
either due to secondary coagulopathy or refusal to sign the informed consent and to
participate in the procedure.
A salting out technique was used to extract DNA from whole blood or using the QIAmp
DNA midi prep kit, (Qiagen Ltd., Crawley, UK). The obtained DNA was used for IL-6
promoter and IL-28B genotyping. During this process, all the RNA was removed by
incubating the digested preparation with 1.5 ml ribonuclease A (Boehringer Mannheim
UK Ltd, East Sussex, UK) per 400 ml of nuclear lysate.
IL-6 and IL28B genotyping
A 175 base pair (bp) fragment of the human IL-6 gene spanning the promoter IL-6
G-174C region was ampliﬁed with gene speciﬁc primers (Roche Diagnostics, Alameda,
California, USA). The resulting PCR fragments were analyzed with hybridization probes
labeled with LightCycler Red 640. The genotypes were identiﬁed by running a melting
curve with speciﬁc melting points (Tm) (Tib Molbiol GmbH, Berlin, Germany).
Genetic polymorphism in a SNP located near the IL-28B gene (rs12979860) was
determined by enzymatic digestion of the PCR product. DNA fragments were ampliﬁed
by using speciﬁc primers. Primer sequences were 5′-GCCTGTCGTGTACTGAACCA-3′,
and 5′-GCTCAGGGGTCAAATCACAGAAG-3′, a PCR product of 143 bp was digested
with HhaI enzyme and the resulting fragments of 27, 38, 65 and 78 bp were separated on
20% acrylamide gel followed by silver staining (Fig. 1).
Data were analysed with the statistical package SPSS 19.0 (IBM Corp, Armonk, NY, USA)
and PLINK v1.07 software (Purcell et al., 2007). Absolute numbers and percentages
were used to describe categorical data, whereas median and IQR were used to describe
quantitative data. One sample binomial test was used to assess distributions of sex,
and response to the treatment. The association of response-to-treatment with sex or the
viral genotype in infected patients was estimated with the Pearson’s chi square test.
Furthermore, nonparametric Mann-Whitney test was used to assess age differences, or
differences in severity of ﬁbrosis between the response groups.
Genetic association tests were mostly performed within PLINK software by using
the case and control design. We deﬁned cases as the group of patients that achieved SVR.
Full model association tests were run in PLINK for each SNP using either chi-square or,
when appropriate, Fisher exact test; and the best-ﬁt model was identiﬁed. Full model
included basic allelic, Cochran-Armitage trend, genotypic, dominant gene action and the
c et al. (2016), PeerJ, DOI 10.7717/peerj.2576 4/16
recessive gene action tests. Additionally, the difference in distribution of IL28B CC carriers
vs. CT+TT carriers across the response groups was estimated by the Pearson’s chi
square test using the SPSS v19 software (IBM, Armonk, NY, USA) since the recessive
model in PLINK tests only the minor allele and in both our samples (patients and the
underlying, healthy general population) the allele C at the IL28B locus was the dominant
allele. All signiﬁcant p-values yielded by genetic tests were further controlled by empirical
p-values which were generated by the permutation procedure. Cochran-Mantel-Haenszel
statistics was used to test whether the predictive power of the SNP markers was
independent of viral genotypes detected in infected patients. Associations of the SVR
status with both SNPs were further evaluated by multivariate logistic regression while
accounting for covariates/factors: age, viral genotype, stage of ﬁbrosis.
The achieved post hoc power for genetic association tests was calculated with Genetic
Power Calculation software (http://pngu.mgh.harvard.edu/∼purcell/gpc/), whereas
Gpower version 3.1.7 (Universita
¨t Kiel, Kiel, Germany) was used to assess the achieved
power for comparison of genotype frequencies between the two population samples.
A total of 110 IVDU patients with elevated liver function tests were diagnosed with a
chronic hepatitis C, treated by standard interferon 2-alfa/ribavirin protocol and followed
over at least one year for a SVR. The majority of treated patients (78 out of 110, or
71%) achieved SVR (one sample binomial test, p < 0.001). Men were signiﬁcantly more
prevalent in our sample than women: 82 (75%) vs. 28 (25%) (one sample binomial test,
p < 0.001). Demographic and clinical characteristics of the sample are presented in
Table 1. As shown in Table 1, NRs were signiﬁcantly older than responders. We found
no association of the SVR status with the sex, the initial Ishak score ﬁbrosis stage, or the
Distributions of patients’ genotypes across response-to-treatment groups are shown for
both SNP loci, rs1800795-IL6 and rs12979860-IL28B, in Table 2.
Figure 1 PCR electrophoresis results for rs12979860 genotyping. M, marker; ns, non speciﬁc band;
CC, CT, and TT–rs12979860 genotypes.
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With regard to genetic variation at the rs1800795-IL6 locus, we determined Hardy-
Weinberg equilibrium in both response groups (exact test, p-values from 0.392 to 0.819).
Overall, the genetic association tests indicated that the addition of allele ‘C’ has protective
effect and increases the chance of achieving SVR. Speciﬁcally, several genetic association
tests conﬁrmed the association between rs1800795-IL6 polymorphism and SVR status,
with the Cochran-Armitage trend test providing the best model ﬁt (= 4.477, df = 1,
p = 0.034, empirical p = 0.039). Patients with rs1800795-IL6 CC genotype had
signiﬁcantly better SVR (14 out of 15, 93%) compared to those with GC (37 out of
52, 71%) or GG (27 out of 43, 63%) genotypes (Fig. 2). The achieved post-hoc power of
this association test was high: 90% (calculated under the additive model, at type I error
rate of 0.05 and with responders/NRs ratio of 78/32).
After controlling for viral genotypes, the association of rs1800795-IL6 polymorphism
with SVR status remained signiﬁcant (Mantel-Haenszel 2 2
= 4.483, p = 0.034,
with odds ratio of 1.99, 95% CI 1.05–3.78). The association was further evaluated by
multivariate logistic regression analysis while accounting for covariates: age, viral
genotype, and ﬁbrosis (F 3). The result of regression analysis conﬁrmed that the
Table 1 Demographic and clinical characteristics of IVDU patients by the response-to-treatment
SVR, N = 78 NR, N = 32 Statistics
Sex, N (%)
Men 56 (72%) 26 (81%) Chi square test, p = 0.301
Women 22 (28%) 6 (19%)
Age, median (IQR) 40 (35–45) 41.5 (39–47) Mann-Whitney, p = 0.041
Fibrosis stage, N (%)
Mean Ishak score
3 (2.0–3.0) 2 (1.0–3.0) Mann-Whitney, p = 0.077
Mean Knodell score
7 (5.0–9.0) 6 (5.0–9.0) Mann-Whitney, p = 0.803
Viral genotype, N (%)
1 51 (73%) 19 (27%) Chi square test, p = 0.706
3 27 (68%) 13 (33%)
SVR, sustained viral response; NR, non responders.
Liver ﬁbrosis was scored by Ishak ﬁbrosis score (0–6) as previously described (Ishak et al., 1995).
Histology activity index by Knodell score (0–18) in chronic active hepatitis (Knodell et al., 1981).
Table 2 Distributions of rs1800795-IL6 and rs12979860-IL28B genotypes, by the response-to-
SNP Genotype SVR, N = 78 NR, N = 32 Total
IL-28B, N (%) CC 22 (28%) 8 (25%) 30 (27%)
CT 50 (64%) 19 (59%) 69 (63%)
TT 6 (8%) 5 (16%) 11 (10%)
IL-6, N (%) GG 27 (35%) 16 (50%) 43 (39%)
GC 37 (47%) 15 (47%) 52 (47%)
CC 14 (18%) 1 (3%) 15 (14%)
SVR, sustained viral response; NR, non responders.
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addition of allele ‘C’ increased the chance of achieving SVR (OR 2.45, 95% CI 1.13–5.30,
p = 0.023). Besides the rs1800795-IL6 polymorphism, age was the only covariate that
signiﬁcantly affected SVR status, with each additional year slightly decreasing the
chance of SVR response (signiﬁcance at the level of 0.1, OR 0.95, 95% CI 0.89–1.01,
p = 0.096).
The genotype frequencies at the rs12979860-IL28B locus met the Hardy-Weinberg
expectations in nonresponders (exact test, p = 0.473), and deviated from the Hardy-
Weinberg equilibrium in SVR cases (i.e. SVR responders, p = 0.004). When we analysed
the genotype frequencies at the rs12979860 locus in the IVDU cohort, and in the
underlying, apparently healthy, general-population sample (n = 531 individuals; HWE,
p = 0.999), we observed that the frequencies of CC, CT and TT genotypes in our cohort
were: 27, 63, and 10%; whereas the corresponding frequencies in the population-based
sample were 49, 42, and 9% (Fig. 3). The ﬁnding demonstrated a large and signiﬁcant
reduction of CC genotype (z-score test for proportions, p < 0.001), and a signiﬁcant
increase in frequency of heterozygous TC genotype (p < 0.001) in the IVDU cohort of
chronic HCV patients compared to the underlying, apparently healthy population. The
frequencies of TT genotype were comparable between the groups (p = 0.741). The post
hoc power for detecting medium size effect in genotype frequencies between these two
population samples was 99%.
With regard to the SVR responder and NR groups, we did not, however, observe a
signiﬁcant association of SVR response with the allele-carrier groups: CC and CT+TT
= 0.118, df = 1, p = 0.732); although the frequency of CC genotype was in fact
somewhat higher in responders (by 3%). The result persisted after we controlled for viral
Figure 2 The percentage of patients with SVR by the rs1800795-IL6 genotypes. n, total number of
patients with each genotype.
c et al. (2016), PeerJ, DOI 10.7717/peerj.2576 7/16
genotypes (Mantel-Haenszel 2 2
= 0.630, p = 0.427) and after multivariate logistic
regression using age, viral genotype and ﬁbrosis stage as additional predictors. It should
be noted, however, that the achieved power for this test, under the dominant genetic
model: CT+TT vs. CC, type I error rate of 0.05, and observed responders/NRs ratio
was very low: 9%. This was in contrast to a priori power calculation that was based on
Ge’s data (Ge et al., 2009) which showed that in treatment-naive, chronic HCV type 1
patients of European-American origin, as little as 20 patients were enough to achieve
80% of power for the rs12979860-IL28B association test on the response to therapy. Also,
the observed penetrance of NR phenotype in our cohort of 29% was considerably lower
than the penetrance in Ge’s cohort of 45% (z-score test for proportions, p = 0.001;
achieved power 80%).
This study investigated the role of IL-6 and IL-28B gene polymorphisms on SVR in IVDU
patients diagnosed with chronic hepatitis C infection. The treatment was conventional
and included Peg IFN combined with ribavirin for either 48 weeks (genotype 1) or
24 weeks (genotype 3). We deﬁned sustained virological response (SVR) as an absence of
detectable virus at the end of follow up evaluation and or disease relapses according to
the standard deﬁnitions (Ghany et al., 2009).
Interleukin-6 was originally discovered as a protein that caused the ﬁnal differentiation
of B cells into immunoglobulin secreting cells (Muraguchi et al., 1988). Additional
work showed that IL-6 and its receptor—sIL-6Rasuppress neutrophil recruitment at site
of acute inﬂammation, making way for the inﬂux of monocytes as the inﬂammatory
Figure 3 The distribution of rs12979860-IL28B genotypes among: IVDU with chronic HCV (in
black bars), and the underlying (base), apparently healthy population (in gray bars). Shown are
percentages with accompanying 95% CI. Statistically signiﬁcant difference, p < 0.05.
c et al. (2016), PeerJ, DOI 10.7717/peerj.2576 8/16
response is sustained (Kopf et al., 1994). IL-6 is well known pro-inﬂammatory cytokine
with pro-tumorigenic potential (Grivennikov et al., 2009) and is emerging as a key
regulatory signal in the development of the newly described pro-inﬂammatory effectors
T-cell subset, so called Th17 cells (Harrington et al., 2005). The IL-6 rs1800795 G allele has
been also associated with higher degrees of liver necroinﬂammation (Falleti et al., 2010)
and ﬁbrosis (Cussigh et al., 2011).
In our study, allele C at rs1800795-IL6—a SNP in the IL-6 gene promoter, was
associated with SVR (OR 2.45, 95% CI 1.13–5.30, p = 0.023). The genotype that
confers the highest degree of protection in terms of achieving SVR, rs1800795-IL6 CC,
demonstrated an overwhelming lower relapse rate in HCV treated patients (1 out of
15 patients, 7%). Similar results were reported for Italian non-IVDU HCV infected
patients thus corroborating the importance of our ﬁndings (Cussigh et al., 2011).
According to prior studies, the CC genotype appears to be associated with signiﬁcantly
lower level of plasma IL-6, whereas the GG and GC genotypes appear to have higher
levels of plasma IL-6 (Fishman et al., 1998;Lapi
nski, 2001). This implies a possible
connection of IL-6 status with the therapy outcome. The putative low producing IL-6
phenotype may play a protective role against chronic hepatitis C infection by helping
to clear the viral particles during standard therapy. Chronic hepatitis C patients with
rs1800795-IL6 CC genotype and lower IL-6 serum level may have an attenuated adoptive
immune response, directed away from damaging, pro-inﬂammatory and autoimmune to
predominately suppressive and anti-viral inﬂammatory response.
An IL-28B gene SNP is located 8 kb upstream of the start codon of IL-28B gene that
encodes IFN-a member of type III IFN family. IFN-interacts with a transmembrane
receptor to induce a potent antiviral response (Donnelly & Kotenko, 2010;Fox, Sheppard &
O’Hara, 2009;Li & Huang, 2007). The antiviral activity is mediated through the activation
of the either JAK-STAT (IFN a,and ) or MAPK (IFN aand ) pathways (Arslani et al.,
2013). There is a strong association of genetic variations in IL-28B gene with response
to therapy (Ge et al., 2009;Suppiah et al., 2009;Tanaka et al., 2009), and with spontaneous
HCV clearance (Duggal et al., 2013). In our cohort of IVDU patients diagnosed with
chronic hepatitis C, no association between the therapy outcome and the SNP in the
IL-28B gene, rs12979860-IL28B was identiﬁed, but the study was largely underpowered to
draw a solid conclusion from this test. Nevertheless, our data still do support the
involvement of the rs12979860-IL28B CC genotype in both of these patho-physiological/
immunological processes: spontaneous HCV clearance and the response to therapy.
Firstly, when we compared our cohort of IVDU patients suffering from chronic HCV
with the population-based sample taken from the apparently healthy, underlying
population; the frequency of the favorable rs12979860-IL28B CC genotype which has
been also associated with the spontaneous clearance of HCV, was largely decreased
in the patient group. Conversely, the frequencies of genotypes considered to have a
neutral effect on acquiring HCV infection/response to therapy were either increased in
patients (TC) or showed no difference between the samples (TT). In other words, the
protective genotype rs12979860-IL28B CC was found at much lower frequency in infected
IVDU individuals with chronic HCV (27%) than in the underlying, healthy population
c et al. (2016), PeerJ, DOI 10.7717/peerj.2576 9/16
(49%), thus strongly pointing towards the selective loss of CC homozygotes in the patient
In line with our ﬁnding, Ge
´linas et al. (2013) have observed signiﬁcantly higher
prevalence of the responder genotype rs12979860 CC in a group of IVDU who were
spontaneous resolvers from a HCV infection than in a baseline population of IVDU users;
suggesting a dilution of CC genotype in chronic IVDU HCV patients. Additionally, also
supporting our ﬁndings, Ezzikouri et al. (2013) found that patients who had cleared HCV
spontaneously were from 2.7 to 4.7 times more likely to carry CC genotype than the
TC, or the TT genotype, respectively, while Montes-Cano et al. (2010) observed the CC
genotype in 73% of individuals with spontaneous resolution of HCV infection versus only
46% in individuals with the persistent infection.
Secondly, our results still suggest the positive effect of rs12979860-IL28B CC genotype
in acquiring SVR. In particular, the distribution of rs12979860-IL28B genotypes in the
cohort of IVDU chronic patients signiﬁcantly deviated from HWE-P only in the group of
responders, whereas in NRs, and in healthy controls the rs12979860-IL28B genotypes
followed the HWE. Since the case (SVR responder) genotypes will only be in HWE
under the multiplicative genetic model (Lewis & Knight, 2012), the departure from this
equilibrium, if found exclusively in cases, can be expected in relatively small samples of
patients over a range of genetic models and is indicative of the actual association to the
trait under study (Wittke-Thompson, Pluzhnikov & Cox, 2005). In addition, we have
observed somewhat higher percentage of CC genotype in SVR cases, although this
percentage did not reach a statistical signiﬁcance.
Similar to our results, Seaberg et al. (2015) also did not ﬁnd an association between
rs12979860-IL28B genotypes and the spontaneous clearance of HCV in men who have
sex with men. There are obviously a large number of factors: demographic, viral, and
human genetic factors; which inﬂuence HCV viremia and the results on distribution of
particular SNPs should be interpreted in a larger context. The fact that the frequency
of rs12979860 CC genotype varies in different ethnic groups or geographical areas
adds to this complexity. In particular, several studies have estimated that East Asians
had a high percentage of CC genotype, whereas the frequency of this genotype was
intermediate in Europeans and minor frequency in African cohorts (Ge et al., 2009;
Thomas et al., 2009).
Concerning the geographic variability of rs12979860 CC genotypes, previous studies
on Caucasian patients who were infected with the HCV viral genotype 1 estimated the
prevalence of CC genotypes to be between 35 and 39% (Cavalcante et al., 2012;Ge et al.,
2009;Montes-Cano et al., 2010;Nattermann et al., 2011). The percentage of CC genotypes
observed in our patients infected with the same viral genotype was 27% (95% CI 19–36%)
which is somewhat lower, suggesting that the impact of IL-28B polymorphism on
acquiring of infection or spontaneous clearance of HCV might be more prominent in the
Croatian population. In addition, it seems that compared to Ge et al.’s (2009) cohort of
chronically infected HCV patients, penetrance of nonresponders in our IVDU cohort
exhibited considerably lower value. This might indicate that the genetic background of the
Croatian population is such that both the HCV spontaneous clearance and the response to
c et al. (2016), PeerJ, DOI 10.7717/peerj.2576 10/16
therapy in chronically infected IVDU is more pronounced in this population. Having in
mind that the population differences in rates of spontaneous clearance have also been
proposed for IVDU patients (Fischer et al., 2004;Miedzinski & Taylor, 2008), the impact of
IL-28B polymorphism on the spontaneous clearance of HCV in the Croatian population
should be investigated in more details in order to increase our knowledge on the
therapeutic effectiveness of PegIFN-a2a/ribavirin on rs12979860 genotypes in different
populations, particularly in high-HCV-risk IVDU population.
In conclusion, we have identiﬁed that the rs1800795-IL6 CC genotype is associated with
signiﬁcantly better SVR to the standard Peg IFN and ribavirin treatment in IVDU/HCV
patients. Also, ﬁndings point towards a strong protective role of rs12979860-IL28B CC
genotype in acquiring chronic hepatitis C infection in the Croatian IVDU population.
Finally, among all covariates, age is the most important, where every additional year
slightly decreases the chance of SVR response. Further prospective and large scale clinical
studies are necessary to conﬁrm our results before we can prospectively identify IVDU and
HCV patients for whom therapy is likely to be successful.
We thank our patients for taking part in this study, and Mrs. Sandra Vujevi
c for technical
ADDITIONAL INFORMATION AND DECLARATIONS
The project was funded by the Croatian Ministry of Science, grant number 258-2160800-
0333 to S
ˇ.A. J.T. was supported by the Croatian Science Foundation grant No: IP-2014-09-
1904 and the Croatian Ministry of Science, Sport and Education grant number
216-000000-3348. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
The following grant information was disclosed by the authors:
Croatian Ministry of Science: 258-2160800-0333.
Croatian Science Foundation: IP-2014-09-1904.
Croatian Ministry of Science, Sport and Education: 216-000000-3348.
The authors declare that they have no competing interests.
c conceived and designed the experiments, performed the
experiments, analyzed the data, contributed reagents/materials/analysis tools, wrote
the paper, prepared ﬁgures and/or tables, reviewed drafts of the paper.
c et al. (2016), PeerJ, DOI 10.7717/peerj.2576 11/16
c performed the experiments.
Sendi Kuret performed the experiments, contributed reagents/materials/analysis tools.
c analyzed the data, wrote the paper, prepared ﬁgures and/or tables,
reviewed drafts of the paper.
c contributed reagents/materials/analysis tools.
Gea Forempoher contributed reagents/materials/analysis tools.
ˇek contributed reagents/materials/analysis tools.
c contributed reagents/materials/analysis tools.
c conceived and designed the experiments, analyzed the data, contributed
reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper.
The following information was supplied relating to ethical approvals (i.e., approving body
and any reference numbers):
The Ethics Committee of the Clinical Hospital Split No: 2181-147-06-01/01-M.J.
The following information was supplied regarding data availability:
The raw data has been supplied as Supplemental Dataset Files.
Supplemental information for this article can be found online at http://dx.doi.org/
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