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The impact of IL-6 and IL-28B gene polymorphisms on treatment outcome of chronic hepatitis C infection among intravenous drug users in Croatia

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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.
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The impact of IL-6 and IL-28B gene
polymorphisms on treatment outcome of
chronic hepatitis C infection among
intravenous drug users in Croatia
Zoran Bogdanovi
c
1
,
*, Ivana Marinovi
c-Terzi
c
2
,
*, Sendi Kuret
3
,
*,
Ana Jeronc
ˇi
c
4
,
*, Nikola Bradari
c
5
, Gea Forempoher
3
, Ozren Polas
ˇek
6
,
S
ˇimun AnCelinovi
c
3
and Janos
ˇTerzi
c
2
1Department of Internal Medicine, Division of Gastroenterology, Clinical Hospital Split, Split,
Croatia
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,
Split, Croatia
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.
ABSTRACT
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
Submitted 9May2016
Accepted 15 September 2016
Published 25 October 2016
Corresponding authors
Zoran Bogdanovi
c,
suzbog@gmail.com
Janos
ˇTerzi
c, janos.terzic@mefst.hr
Academic editor
Krystyna Da˛browska
Additional Information and
Declarations can be found on
page 11
DOI 10.7717/peerj.2576
Copyright
2016 Bogdanovi
c et al.
Distributed under
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
variation
INTRODUCTION
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).
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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
(Polas
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.
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Liver histology
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.
DNA extraction
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).
Statistical analysis
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
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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.
RESULTS
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
viral genotype.
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
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
status.
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%)
Notes:
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-
treatment group.
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%)
Note:
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
(
2
= 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.
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genotypes (Mantel-Haenszel 2 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%).
DISCUSSION
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.
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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
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(49%), thus strongly pointing towards the selective loss of CC homozygotes in the patient
population.
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
Bogdanovi
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.
CONCLUSIONS
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.
ACKNOWLEDGEMENTS
We thank our patients for taking part in this study, and Mrs. Sandra Vujevi
c for technical
assistance.
ADDITIONAL INFORMATION AND DECLARATIONS
Funding
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.
Grant Disclosures
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.
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
Zoran Bogdanovi
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.
Bogdanovi
c et al. (2016), PeerJ, DOI 10.7717/peerj.2576 11/16
Ivana Marinovi
c-Terzi
c performed the experiments.
Sendi Kuret performed the experiments, contributed reagents/materials/analysis tools.
Ana Jeronc
ˇi
c analyzed the data, wrote the paper, prepared figures and/or tables,
reviewed drafts of the paper.
Nikola Bradari
c contributed reagents/materials/analysis tools.
Gea Forempoher contributed reagents/materials/analysis tools.
Ozren Polas
ˇek contributed reagents/materials/analysis tools.
S
ˇimun AnCelinovi
c contributed reagents/materials/analysis tools.
Janos
ˇTerzi
c conceived and designed the experiments, analyzed the data, contributed
reagents/materials/analysis tools, wrote the paper, reviewed drafts of the paper.
Human Ethics
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.
Data Deposition
The following information was supplied regarding data availability:
The raw data has been supplied as Supplemental Dataset Files.
Supplemental Information
Supplemental information for this article can be found online at http://dx.doi.org/
10.7717/peerj.2576#supplemental-information.
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... Second, our findings in this study showed that miR-125b expression was considerably down regulated in patients after therapy. This finding is consistent with the findings of Dai et al. [15], who concluded that the IL-6/ STAT3 pathway up-regulates microRNA-125b expression in HCV and proposed that STAT3 or miR-125b management might be a therapeutic target for HCV infection. Previous reports [16][17][18] have revealed that miR-125b regulates inflammation and plays an important role in the development of various cancers. ...
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Abstract: The novel coronavirus-19 (SARS-CoV-2), has infected numerous individuals worldwide, resulting in millions of fatalities. The pandemic spread with high mortality rates in multiple waves, leaving others with moderate to severe symptoms. Co-morbidity variables, including hypertension, diabetes, and immunosuppression, have exacerbated the severity of COVID-19. In addition, numerous efforts have been made to comprehend the pathogenic and host variables that contribute to COVID-19 susceptibility and pathogenesis. One of these endeavours is understanding the host genetic factors predisposing an individual to COVID-19. Genome-Wide Association Studies (GWAS) have demonstrated the host predisposition factors in different populations. These factors are involved in the appropriate immune response, their imbalance influences susceptibility or resistance to viral infection. This review investigated the host genetic components implicated at the various stages of viral pathogenesis, including viral entry, pathophysiological alterations, and immunological responses. In addition, the recent and most updated genetic variations associated with multiple host factors affecting COVID-19 pathogenesis are described in the study.
... In contrast, a study showed that the GG genotype of the rs1800795 − 174 G/C polymorphism is associated with high serum levels of IL-6 and the likelihood of sustained virologic response (SVR) in patients co-infected with HCV and HIV [48]. Similarly, other studies have demonstrated that the CC genotype of the rs1800795 − 174 G/C polymorphism is associated with low production of IL-6 and an attenuated immune response against chronic HCV [49,50]. In contrast, it has also been reported that genetic polymorphisms of IL-6 in rs1800796 (-572 G > C) were not associated with HCV infection and development of hepatocellular carcinoma (HCC) in an Egyptian population [51]. ...
... In contrast, a study showed that the GG genotype of the rs1800795 − 174 G/C polymorphism is associated with high serum levels of IL-6 and the likelihood of sustained virologic response (SVR) in patients co-infected with HCV and HIV [48]. Similarly, other studies have demonstrated that the CC genotype of the rs1800795 − 174 G/C polymorphism is associated with low production of IL-6 and an attenuated immune response against chronic HCV [49,50]. In contrast, it has also been reported that genetic polymorphisms of IL-6 in rs1800796 (-572 G > C) were not associated with HCV infection and development of hepatocellular carcinoma (HCC) in an Egyptian population [51]. ...
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Background Emerged coronavirus disease 2019 (COVID-19) is a pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). Disease severity is associated with elevated levels of proinflammatory cytokines, such as interleukin-6 (IL-6). Genetic polymorphisms in the regulatory regions of cytokine genes may be associated with differential cytokine production in COVID-19 patients. This study aimed to investigate the association between three potentially functional single-nucleotide polymorphisms (SNPs) in the promoter region of IL-6 and the severity of susceptibility to COVID-19 in an Iranian population. Methods In total, 346 individuals (175 patients with severe COVID-19 and 171 patients with mild COVID-19) were recruited for this cohort study. Genomic DNA was extracted from peripheral blood leukocytes of patients to determine the genotypes of three selected SNPs (rs1800795 (-174 G>C), rs1800796 (-572 G>C), and rs1800797 (-597 G>A)) in the promoter region of the IL-6 gene using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Results There were no significant differences in the genotype or allele distribution of selected SNPs (rs1800795 (-174 G>C), rs1800796 (-572 G>C), and rs1800797 (-597 G>A)) in the promoter region of the IL-6 gene in patients with severe COVID-19 and patients with mild COVID-19. Discussion Our study indicated that these SNPs are not associated with COVID-19 severity in the Kurdish population from Kermanshah, Iran.
Chapter
SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causative agent of COVID-19 disease. This virus emerged in China and spread quickly worldwide, responsible for nearly 596 million infections and 6.4 million deaths globally. The different variants of concern (VOCs) of SARS-CoV-2 identified in different parts of the world are alarming for serious public health concerns. The VOCs place constraints on utilizing monoclonal antibodies to treat SARS-CoV-2 infection. The SARS-CoV-2 virus has a spherical envelope and single-stranded positive-sense RNA. It primarily encodes four structural proteins (S, E, M, and N) and numerous non-structural proteins (NSPs). Numerous vaccines that target SARS-CoV-2 various SARS-CoV-2 sites have recently been developed, and more are in works. The concerns regarding the newly emerging virus strains are still very high. The latest scientific information on disease susceptibility suggests that multiple genetic factors are associated with COVID-9 disease severity. The genetic architect of an individual contributes to the susceptibility and response to viral infection. The different VOCs contain various mutations and many of which are localized to the S protein. Mostly these mutations are responsible for the changes in viral behavior and pathogenesis. Several host genetic factors that are associated with illness susceptibility, severity, and immune response to SARS-CoV-2 have been covered in this book chapter.
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Background: The global pandemic of Coronavirus Disease 2019 (COVID-19) has resulted in significant fatality rates. Clinical outcomes for affected individuals range from being asymptomatic to severe illnesses requiring intensive care unit (ICU) admission. Among the various factors contributing to the variation in clinical outcomes, host genetics play a prominent role. Interleukin-6 (IL6), a key player in immune responses, has been identified as having a crucial impact on viral infections, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Specifically, certain variations known as single nucleotide polymorphisms (SNPs) in the IL6 promoter region have been found to significantly influence IL6 expression and the severity of viral infections. Materials and Methods: To explore the relationship between these genetic variations and COVID-19 in asymptomatic and ICU-admitted Kurdish patients, genetic sequencing was performed to determine the genotypes of nine IL6 SNPs. Results: The study findings revealed that although the proportion of the GG genotype of rs1800795 was slightly higher in asymptomatic COVID-19 cases, the difference was not statistically significant (chi2 = 2.666, p = 0.236). Notably, Kurdish patients displayed a uniform genetic makeup (monomorphic) for the dominant alleles of rs2069830 (C), rs142759801 (C), rs2069857 (C), rs2069829 (G), rs2234683 (G), rs13447446 (T), rs527770772 (C), and rs13447445 (C). Furthermore, patients carrying the haplotype GCGGCTCCC were found to have a 0.481-fold higher likelihood of being asymptomatic with COVID-19 (p = 0.016, OR = 0.481). Conclusions: This study demonstrates that the rs1800795 SNP is not statistically associated with COVID-19 at the genotype level. However, the presence of the dominant G allele of rs1800795 in the haplotype was found to be statistically associated with asymptomatic COVID-19 patients.
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Background Elevated levels of interleukin (IL)-6 and IL-17A have been linked to hyper inflammation in COVID-19 patients, and their levels are indicative of the progression of the disease. This study aimed to investigate whether single-nucleotide polymorphisms (SNPs) in IL-6 and IL-17A are linked to COVID-19 susceptibility and prognosis in Iranian patients. Methods The study enrolled 280 COVID-19 patients, divided into 140 non-severe and 140 severe cases. Genotyping for IL-6 rs1800795 and IL-17A rs2275913 was performed using tetra primer-amplification refractory mutation system-polymerase chain reaction (tetra-ARMS-PCR). IL-6 and IL-17A circulating levels were measured using enzyme-linked immunosorbent assay (ELISA). The study also investigated predictors of COVID-19 mortality. Results The rs1800795 GG genotype (78/140 (55.7%)) and G allele (205/280 (73.2%)) were significantly associated with a higher risk of severe COVID-19 infection (OR = 2.19, 95%CI: 1.35–3.54, P = .006 and OR = 1.79, 95%CI: 1.25–2.56, P < .001, respectively). The rs1800795 GG genotype was also significantly linked to disease mortality (OR = 1.95, 95%CI: 1.06–3.61, P = .04). In contrast, the rs2275913 GA genotype was found to be protective against severe COVID-19 (OR = 0.5, 95%CI: 0.31–0.80, P = .012), but no significant association was observed with disease mortality. Several predictors of COVID-19 mortality were identified, including INR ≥ 1.2 (OR = 2.19, 95%CI: 1.61–3.78, P = .007), D-dimer ≥ 565.5 ng/mL (OR = 3.12, 95%CI: 1.27–5.68, P = .019), respiratory rate ≥ 29 (OR = 1.19, 95%CI: 1.12–1.28, P = .001), IL-6 serum concentration ≥ 28.5 pg/mL (OR = 1.97, 95%CI: 1.942–2.06, P = .013), and IL-6 rs1800795 GG genotype (OR = 1.95, 95%CI: 1.06–3.61, P = .04). Conclusion The results of this study suggest that the rs1800795 GG genotype and G allele are associated with greater disease severity in COVID-19 patients, while the rs2275913 GA genotype is protective. These findings provide insights into the genetic and molecular mechanisms underlying COVID-19 and may inform the development of more effective therapies and prognostic tools for this disease.
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Single nucleotide polymorphisms (SNPs) in the proximity of the interleukin-28B (IL28B) gene can predict spontaneous resolution of hepatitis C virus (HCV) infection and response to interferon therapy. Screening for this polymorphism has become part of the standard criteria for the management of HCV-infected patients, hence the need for a rapid, cost-effective screening method. Here, we describe a rapid PCR-based test to screen for two IL28B SNPs (rs12979860 and rs8099917). We used this test to investigate IL28B polymorphism and prevalence in a cohort of French Canadian injection drug users who are part of a unique population known to have a strong genetic founder effect. This population had lower linkage disequilibrium between the two tested SNPs as compared to other cohorts (|d'| = 0.68, r = 0.59). The special genetic makeup should be considered in the management of HCV-infected patients within that population.
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Chinese translation Hepatitis C virus (HCV) infections occur worldwide and either spontaneously resolve or persist and markedly increase the person's lifetime risk for cirrhosis and hepatocellular carcinoma. Although HCV persistence occurs more often in persons of African ancestry and persons with genetic variants near interleukin-28B (IL-28B), the genetic basis is not well-understood. To evaluate the host genetic basis for spontaneous resolution of HCV infection. 2-stage, genome-wide association study. 13 international multicenter study sites. 919 persons with serum HCV antibodies but no HCV RNA (spontaneous resolution) and 1482 persons with serum HCV antibodies and HCV RNA (persistence). Frequencies of 792 721 single nucleotide polymorphisms (SNPs). Differences in allele frequencies between persons with spontaneous resolution and persistence were identified on chromosomes 19q13.13 and 6p21.32. On chromosome 19, allele frequency differences localized near IL-28B and included rs12979860 (overall per-allele OR, 0.45; P = 2.17 × 10-30) and 10 additional SNPs spanning 55 000 base pairs. On chromosome 6, allele frequency differences localized near genes for HLA class II and included rs4273729 (overall per-allele OR, 0.59; P = 1.71 × 10-16) near DQB1*03:01 and an additional 116 SNPs spanning 1 090 000 base pairs. The associations in chromosomes 19 and 6 were independent and additive and explain an estimated 14.9% (95% CI, 8.5% to 22.6%) and 15.8% (CI, 4.4% to 31.0%) of the variation in HCV resolution in persons of European and African ancestry, respectively. Replication of the chromosome 6 SNP, rs4272729, in an additional 745 persons confirmed the findings (P = 0.015). Epigenetic effects were not studied. IL-28B and HLA class II are independently associated with spontaneous resolution of HCV infection, and SNPs marking IL-28B and DQB1*03:01 may explain approximately 15% of spontaneous resolution of HCV infection. Office of AIDS Research, National Institutes of Health, and Frederick National Laboratory for Cancer Research.
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BACKGROUND/AIMS The cohort of Irish women infected with hepatitis C virus (HCV) genotype 1b via contaminated anti-D immunoglobulin in 1977 represent a unique homogenous group to investigate the natural course of HCV infection. METHODS The clinical status of 87 polymerase chain reaction (PCR) positive and 68 PCR negative women was investigated at diagnosis (1994/95) and after 4–5 years of follow up (21/22 years after inoculation). Other features investigated included: histological status/progression, psychosocial impact of HCV infection, extrahepatic manifestations, and HLA class II associations. RESULTS The most common symptoms reported were fatigue and arthralgia. Furthermore, 77% of women fell within the clinical range for psychological distress. A history of icteric hepatitis was reported in 20.6% of PCR negative and 3.4% of PCR positive women after inoculation (p=0.002). The mean histological activity index/fibrosis scores of PCR positive and negative women were 4.1 (1.4)/1.1 (1.3) and 2.1 (1.5)/0.15 (0.36) at diagnosis and 4.1 (1.2)/1.0 (1.0) in 44 PCR positive women after five years of follow up. Cirrhosis or hepatocellular carcinoma was not observed. The DRB1*01 allele was present in 28.8% of PCR negative and 8.7% of PCR positive women (p=0.004). The prevalence rates of mixed cryoglobulinaemia, sicca complex, positive thyroid autoantibodies, antinuclear antibody, rheumatoid factor, and antimitochondrial antibody in PCR positive women were 12.7%, 7.6%, 13.9%, 5.1%, 3.8%, and 3.8%. CONCLUSIONS A benign course of HCV infection with lack of disease progression was observed in women with chronic HCV, 22 years after inoculation. Acute icteric hepatitis and the HLA DRB1*01 allele were associated with viral clearance. Despite this favourable outcome, high levels of psychological distress and poor quality of life were present.