influence of interleukin 12b (il12b) polymorphisms on spontaneous and treatment-induced recovery...
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Influence of interleukin 12B (IL12B) polymorphismson spontaneous and treatment-induced recovery from
hepatitis C virus infection
Tobias Mueller1, Andreas Mas-Marques2, Christoph Sarrazin3, Manfred Wiese4,Juliane Halangk5, Heiko Witt5, Golo Ahlenstiel6, Ulrich Spengler6, Uwe Goebel7,
Bertram Wiedenmann1, Eckart Schreier2, Thomas Berg1,*
1Charite, Campus Virchow-Klinikum, Medizinische Klinik mit Schwerpunkt, Hepatologie und Gastroenterologie, Universitatsmedizin Berlin,
Augustenburger Platz 1, 13353 Berlin, Germany2Robert Koch-Institut, Berlin, Germany
3Medizinische Klinik und Poliklinik—Innere Medizin II, Universitatskliniken des Saarlandes, Homburg/Saar, Germany4II. Klinik fur Innere Medizin, Stadtisches Klinikum St. Georg, Leipzig, Germany
5Klinik fur Padiatrie, Charite—Universitatsmedizin Berlin, Campus Virchow-Klinikum Berlin, Germany6Medizinische Klinik u. Poliklinik I, Allgemeine Innere Medizin, Universitat Bonn, Germany
74. Medizinische Klinik, Carl-Thiem-Klinikum, Cottbus, Germany
Background/Aims: Interleukin-12 (IL-12) governs the Th1-type immune response, affecting the spontaneous andtreatment-induced recovery from HCV-infection. We investigated whether the IL12B polymorphisms within the
promoter region (4 bp insertion/deletion) and the 3 0-UTR (1188-A/C), which have been reported to influence IL-12
synthesis, are associated with the outcome of HCV infection.
Methods: We analyzed 186 individuals with spontaneous HCV clearance, 501 chronically HCV infected patients, and
217 healthy controls. IL12B 3 0-UTR and promoter genotyping was performed by Taqman-based assays with allele-
specific oligonucleotide probes and PCR-based allele-specific DNA-amplification, respectively.
Results: The proportion of IL12B promoter and 3 0-UTR genotypes did not differ significantly between the different
cohorts. However, HCV genotype 1-infected patients with high baseline viremia carrying the IL12B 3 0-UTR 1188-C-allele showed significantly higher sustained virologic response (SVR) rates (25.3% vs. 46% vs. 54.5% for A/A, A/C and
C/C) due to reduced relapse rates (24.2% vs. 12% vs. zero % for A/A, A/C and C/C).
Conclusions: IL12B 3 0-UTR 1188-C-allele carriers appear to be capable of responding more efficiently to antiviral
combination therapy as a consequence of a reduced relapse rate. No association of IL12B polymorphisms and self-
limited HCV infection could be demonstrated.
q 2004 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Keywords: Antiviral therapy; HCV infection; Relapse rate; Single nucleotide polymorphism (SNP); Spontaneous
recovery; Treatment outcome; Virologic response
0168-8278/$30.00 q 2004 European Association for the Study of the Liver. Pub
doi:10.1016/j.jhep.2004.06.021
Received 7 January 2004; received in revised form 20 June 2004; accepted
25 June 2004; available online 10 July 2004
* Corresponding author. Tel.: C49-30-450-553072; fax: C49-30-450-
553903.
E-mail address: [email protected] (T. Berg).
1. Introduction
The host genetic background is likely to influence the
natural course of hepatitis C virus (HCV) infection. Recent
data confirm the pivotal role of a broad virus-specific
CD4(C) T-helper cell (Th1) response and strong type-1
cytokine release for spontaneous and treatment-induced
Journal of Hepatology 41 (2004) 652–658
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T. Mueller et al. / Journal of Hepatology 41 (2004) 652–658 653
recovery from HCV infection [1–4]. In this context, genetic
polymorphisms influencing interleukin-12 (IL-12) levels
may be crucial given the key immunoregulatory role of
IL-12 in polarizing T-cells towards a Th1-type profile and
suppressing Th2 T-cell differentiation [5]. Recent data
provided evidence that two polymorphisms within the gene
encoding the p40 subunit of IL-12 (IL12B)—a single
nucleotide polymorphism in the 3 0-UTR (3 0-UTR 1188-
A/C) [6,7] and a 4 bp insertion/deletion polymorphism at a
point 3 kb upstream of the transcriptional start site of the
IL12B promoter region [8,9]—influenced IL-12 synthesis
and may be functionally relevant in diseases with an altered
Th1/Th2 balance [8,10,11].
From that we hypothesized that HCV-infected patients
with IL12 genotypes shown to be associated with decreased
IL12 synthesis [8,9], have a diminished cellular immune
response, and this condition may affect the outcome of the
disease as well as the efficacy of antiviral treatment. In
contrast, IL12B 3 0UTR 1188-A-allele carriers—presumably
prone to increased IL-12 synthesis [10]—may respond more
favorably to HCV infection. We therefore evaluated the role
of IL12B polymorphisms in HCV infection by comparing
their distribution in patients with self-limited and chronic
HCV infection and also by defining the correlation with
treatment response.
Table 1
2. Patients and methodsCharacteristics of study participants with self-limited and chronic
HCV infection
Patients character-
istics
Course of HCV infection
Self-limited (nZ186) Chronic (nZ501)
Agea (years) 45 (23–74) 48 (23–79)
Gender (m/f)b (%
male)
31/155 (16.7%) 296/205 (59.1%)
HCV genotype
1 116 (62.4%) 313 (62.5%)
Non-1 – 191 (36.1%)
Unknown 70 (37.6%) 7 (1.4%)
Risk factor
Blood 134 (72%) 120 (24%)
IVDU – 119 (23.7%)
Othersc – 25 (5%)
Unknown 52 (28%) 237 (47.3%)
Liver histology (nZ458)d
Inflammation (grade) Not done
0–1 163 (35.6%)
2 232 (50.7%)
3 63 (13.8%)
Fibrosis Not done
0–1 151 (33%)
2 163 (35.6%)
3–4 144 (31.4%)
a Actual age at sampling time expressed as mean (range).b Male/female.c Occupational exposure, sexual transmission, skin tattoos, treatment for
bilharziosis.d Classification (grade, stage) according to the Scheuer score [14].
2.1. Patients
The present study was approved by the local ethics committee and allstudy participants gave written informed consent. We genotyped 186individuals with spontaneous HCV clearance, 501 chronically HCV-infected patients and 217 healthy control subjects. Among the individualswith self-limited HCV infection, 116 had been infected in 1978/1979 byadministration of HCV genotype-1-contaminated anti-D immunoglobulin[12,13]. In the remaining 70 individuals the diagnosis of spontaneous HCVclearance was made by the presence of positive HCV antibody titer butpersistent negative HCV RNA (PCR) and normal transaminase levelsduring the preceding 6 months.
Patients with chronic HCV infection were recruited from two differentliver centers across Germany. Chronic hepatitis C was diagnosed byelevated serum transaminase levels for at least 6 months and consistentlydetectable serum HCV RNA. All patients were anti-HCV positive and werenegative for hepatitis B surface antigen and antibodies to humanimmunodeficiency virus 1 and 2. Genotyping of HCV was performed byreverse hybridization assay (Inno LiPA HCV II, Innogenetics, Gent,Belgium). Among the patients with chronic hepatitis C, liver biopsy hadbeen performed in 458 cases (91.4%). Hepatic inflammation (grade) andfibrosis (stage) were classified according to the semiquantitative histo-logical score described by Scheuer et al. [14]. All patients had been treatedin prospective treatment studies with either standard interferon-alpha andribavirin (nZ289) or pegylated interferon-alpha and ribavirin (nZ212) for24 weeks in HCV genotype 2/3 infection and 48 weeks in HCV genotype 1infection. In total, 281 patients (56.1%) achieved a sustained virologicresponse (SVR), defined as negative HCV-RNA (qualitative PCR) 24weeks after termination of therapy. In contrast, 77 patients (15.4%) whoinitially responded to therapy failed to maintain viral clearance eitherduring treatment (breakthrough) or thereafter (relapse). These patients wereclassified as relapsed responders (RR). Finally, 143 patients (28.5%) did notrespond to therapy (NR), as documented by a viral decline of less than 2 logat treatment week 12 or positive HCV RNA (PCR) at treatment week 24.Therapy was therefore discontinued in these patients. Table 1 summarizes
relevant characteristics of patients with spontaneous HCV clearance andchronic HCV infection.
2.2. IL12B genotyping
IL12B 3 0-UTR genotyping was performed by using a Taqman-basedassay [15]. The following primers and probes were obtained from publishedsequences (GenBank AC011418) and synthesized by Applied Biosystems(Warrington, UK): 5 0-GGATCACAATGATATCTTTGCTGTATTT-3(3 0-UTR forward primer), 5 0-TGAGAGCTGGAAAATCTATACA-TAAATTAGC-3 0 (3 0-UTR reverse primer), 5 0-VIC-CATTTAGCATCTAACTATAC-3 0 (3 0-UTR 1188-A-allele specific probe) and 5 0-FAM-TTTAGCATCGAACTATAC-3 0 (30-UTR 1188-C-allele specific probe).The IL12B promoter locus was genotyped with the following primers:5 0-GTCAATGGGCATTTGGCTCATATTACC-3 0 (promoter forward pri-mer), 5 0-ATTGGTCCTTCTGTTTTGTCCTAATGTGGGGGCCACAT-TAGAG-3 0 (reverse specific primer for the 4 bp insertion alleleCTCTAA) and 5 0-TCTAATGTGGGGGCCACAGC-3 0 (reverse specificprimer for the 4 bp deletion allele GC). Allelic discrimination wasperformed by visualizing promoter alleles after electrophoresis on a 3.5%agarose gel and ethidium bromide staining.
2.3. Statistical analysis
The comparison of spontaneously resolved vs. chronic HCV infectionand treatment-induced sustained virological response vs. non-responseyielded to a statistical power ranging between 93 and 97% for testing adifference of 15% in the respective rates and 61 and 71% for testing adifference of 10%. Non-parametric data are expressed as mean values andtheir standard errors. Statistical analysis was primarily performed bycontingency tables using c2 statistics for categorial variables and two-sample t tests for continuous variables. The Mann–Whitney test andthe Kruskal–Wallis-test were used for comparison of median values.
T. Mueller et al. / Journal of Hepatology 41 (2004) 652–658654
All statistical calculations were performed with SPSS 11.0 software forWindows (SPSS, Inc, Chicago, IL). If not stated otherwise, all tests weretwo-sided and P-values lower than 0.05 were considered significant.
Fig. 1. Treatment response of HCV genotype 1-infected patients with
baseline hepatitis C viremia exceeding 800,000 IU/ml in relationship to
the IL12B 3 0UTR genotype (nZ156). 1SVR, sustained virologic
response: 25.3% (A/A), 46% (A/C), 54.5% (C/C); 2Relapse: 24.2%
(A/A), 12% (A/C), 0% (C/C); 3NR, non-response: 50.5% (A/A), 42%
(A/C), 45% (C/C); *SVR rates A/A vs. A/C vs. C/C PZ0.007; **RR
rates A/A vs. A/C vs. C/C PZ0.042.
3. Results
3.1. Comparison of frequency of IL12B polymorphism
in patients with self-limited and chronic hepatitis C virus
infection and healthy controls
Table 2 compares the distribution of IL12B polymorph-
isms in self-limited HCV infection to chronic HCV carriers
and healthy controls. Despite the difference in the overall
distribution of IL12B promoter genotypes between our two
cohorts of subjects with spontaneous HCV clearance
(PZ0.046; Table 2), none of the two groups varied
significantly in the frequency of both the IL12B promoter
and 3 0UTR genotypes compared to chronic HCV carriers
and healthy controls. In particular, we neither found an
under-representation of the heterozygous promoter geno-
type in the total group of 186 patients with spontaneous
HCV clearance nor an over-representation in chronically
HCV infected patients (PZ0.661; Table 2). Similarly, the
IL12B 3 0-UTR 1188-A/C polymorphism was equally
distributed with respect to genotypes (PZ0.734, Table 2)
and alleles (PZ0.522, data not shown) within the three
study cohorts. In addition, our analysis did not detect a
haplotype with a special risk for developing a chronic HCV
carrier state (data not shown).
3.2. Treatment response with respect to the IL12B genotypes
The distribution of IL12B promoter genotypes did not
differ greatly within the three treatment response groups,
arguing against an important role with respect to treatment
outcome. In particular, patients heterozygous for the IL12B
promoter region achieved similar SVR rates compared to
both homozygous carriers for the 4 bp insertion and the 4 bp
deletion allele (Table 3a). In contrast, patients with C-allele
containing genotypes (1188-A/C, 1188-C/C) showed a
tendency to clear the virus more frequently (PZ0.226)
based on a non-significant decrease in relapse rates,
(PZ0.138). When we restricted our analysis to patients
with HCV genotype 1 infection and a baseline virus load
exceeding 800,000 IU/ml—both well established negative
predictive parameters for treatment outcome—patients with
IL12B 3 0-UTR 1188-C allele containing genotypes showed
significantly increased SVR rates compared to others
(PZ0.007) as a consequence of a significant decrease in
relapse rates (PZ0.042) (see Table 3b and Fig. 1).
However, in type 1-infected patients with low-level
baseline viremia, this kind of association was not observed
(Table 3b). This discrepancy may be explained by the
overall more favorable treatment outcome in type 1-infected
patients with low-level viremia, thereby outweighing the
relevance of the genetic factors.
The treatment response was also significantly better in
HCV genotype 3-infected patients with C-allele containing
IL12B 3 0UTR genotypes when the initial virus load in these
patients did not exceed 800,000 IU/ml (Table 3b). Of note, a
relapse occurred only in HCV type 3-infected patients with
C-allele negative IL12B 3 0UTR genotype (PZ0.072).
On the allelic level, we found a similar non-significant
trend associating the IL12B 3 0-UTR 1188-C allele with
increased SVR (61.1% compared to 54.5% SVR for the
A-allele; PZ0.075). This observation became significant
when we analyzed only HCV type 1-infected patients with
high viral load (48.6% SVR for the C-allele versus 29.6%
SVR for the A-allele; odds ratio 2.25; 95% CI 1.314–3.386;
PZ0.003). In contrast, both IL12B promoter alleles did not
influence the outcome of therapy independent of our
stratification criteria (data not shown).
Of note, all these observations were independent from
the different combination therapy regimens applied, which
included either pegylated interferon-a or standard inter-
feron-a (data not shown).
3.3. IL12B polymorphism and severity of histological liver
damage
Among 458 liver biopsies obtained from patients with
chronic HCV infection, 144 (31.4%) were staged with
advanced fibrosis (stage 3–4) and 63 (13.8%) were graded
with severe inflammation (grade 3). The IL12B promoter
polymorphism neither influenced the degree of hepatic
inflammation nor the fibrosis stage (Table 4). The IL12B
3 0UTR polymorphism also had no influence on the grade of
hepatic inflammation.
A significantly higher rate of advanced liver fibrosis
(stage 3–4) was observed in patients bearing the rare
homozygous IL12B 3 0UTR 1188-C/C genotype (nZ31)
Table 2
Distribution of IL12B genotypes in patients with self-limited and chronic HCV infection and healthy controls
IL12B genotypes Self-limited HCV infection Chronic HCV
infection
Healthy controls
Anti-Da (nZ116) Othersb (nZ70) Total (nZ186) (nZ501) (nZ217)
Promoterc
4bp(C)/4bp(C) 25 (21%) 22 (31%) 47 (25%) 130 (26%) 51 (24%)
4bp(C)/4bp(K) 60 (52%)d 26 (38%) 86 (46%) 231 (46%) 113 (52%)
4bp(K)/4bp(K) 31 (27%) 22 (31%) 53 (29%)e 140 (28%) 53 (24%)
3 0 UTR
1188-A/A 77 (66%) 40 (57%) 117 (63%) 297 (59%) 137 (63%)
1188-A/C 28 (24%)f 27 (39%) 55 (30%)g 169 (34%) 68 (31%)
1188-C/C 11 (10%) 3 (4%) 14 (7%) 35 (7%) 12 (6%)
a Subjects infected in 1978/1979 by administration of HCV genotype-1-contaminated anti-D immunoglobulin [12,13].b Subjects from different liver centers with positive HCV antibody titer but persistent negative HCV RNA (PCR) and normal transaminase levels for more
than 6 months prior to study inclusion.c 4bp(C)ZIL12B promoter allele with 4 bp insertion (CTCTAA), 4bp(K) IL12B promoter allele with 4 bp deletion (GC).d PZ0.046 for the comparison of IL12B promoter heterozygosity in anti-D patients (52%) compared to 38% heterozygosity in Others.e PZ0.661 for the comparison of overall IL12B promoter genotype frequencies in the total group of patients with self-limited HCV infection compared to
chronic HCV carriers and healthy controls.f PZ0.112 for the comparison of IL12B 3 0UTR genotype frequencies in Anti-D patients compared to Others.g PZ0.734 for the comparison of overall IL12B 3 0UTR genotype frequencies in the total group of self-limited HCV infection compared to chronic HCV
carriers and healthy controls.
T. Mueller et al. / Journal of Hepatology 41 (2004) 652–658 655
compared to other genotypes (PZ0.039, Table 4). Male
carriers of the homozygous IL12B 3 0UTR 1188-C/C type
(nZ20) with a course of the disease lasting for more than
5 years showed higher stages of liver fibrosis when
compared to the IL12B 3 0UTR 1188-A/A and -A/C males
with the same duration of the disease (70% stage 3–4 in C/C
genotypes vs. 36.1% in non-C/C [nZ269; PZ0.005]).
Analyzing only patients with mild (stage 0–1/grade 0–1) or
cirrhotic (stage 4) HCV disease showed no differences in the
distribution pattern of the IL12B 3 0UTR polymorphism
(data not shown).
Finally, the positive effect of IL12B 3 0UTR 1188-C
allele-containing genotypes with regard to treatment out-
come was independent of the stages of fibrosis prior to
therapy (SVR rate for A/A vs. A/C vs. C/C with stage 0–1
was 52.9% vs. 61.8% vs. 77.8% [PZ0.24] compared to
Table 3a
IL12B polymorphism and overall treatment response in chronic HCV infect
IL12B polymorphism
genotype
(n) Treatment respon
SVR
Promotera
4bp(C)/4bp(C) (130) 58.5
4bp(C)/4bp(K) (231) 55.8
4bp(K)/4bp(K) (140) 54.3
3 0 UTR
1188-A/A (297) 53.2
1188-A/C (169) 59.2
1188-C/C (35) 65.7b
SVR, sustained virologic response; RR, relapsed response; NR, non-response.a 4bp(C) IL12B promoter allele with 4 bp insertion (CTCTAA), 4bp(K) IL12b PZ0.226 for the comparison of SVR IL12B 3 0UTR 1188-C/C vs. non-C/C.c PZ0.138 for the comparison of RR IL12B 30UTR 1188-C/C. vs. non-C/C.
57.8% vs. 61.1% vs. 71.4% [stage 2; PZ0.635] and 47.7%
vs. 53.5% vs. 66.7% [stage 3–4; PZ0.39]).
4. Discussion
Our data clearly exclude a major influence of IL12B
polymorphisms on the natural course of acute HCV
infection. The overall genotype distribution and allele
frequency of both IL12B polymorphisms did not differ
between patients with self-limited and chronic HCV
infection as well as healthy controls. In particular, neither
heterozygosity for the IL12B promoter polymorphism nor
homozygosity for the IL12B 3 0-UTR 1188-C allele
conferred an increased risk for the development of chronic
ion
se (%)
RR NR
14.6 26.9
13.9 30.3
18.6 27.1
17.5 29.3
13.6 27.2
5.7c 28.6
B promoter allele with 4 bp deletion (GC).
Table 3b
IL12B 30UTR polymorphism and treatment response according to the HCV genotype and baseline viremia level prior to combination therapy
Patient groups (n) IL12B 3 0UTR genotypes (A/A-A/C-C/C) and treatment response (n[% of total number of genotypes A/A, A/C,C/C])
SVR RR NR
A/A A/C C/C A/A A/C C/C A/A A/C C/C
HCV genotype 1
Totala (313) 71 (37%) 47 (47%) 9 (45%) 44 (23%) 15 (15%) 2 (10%) 77 (40%) 39 (39%) 9 (45%)
Low virus loada,b (143) 44 (49%) 21 (45%) 2 (33%) 21 (23%) 8 (17%) 2 (33%) 25 (28%) 18 (38%) 2 (33%)
High virus loada,c (156) 24 (25%) 23 (46%) 6 (55%)d 23 (24%) 6 (12%) 0 (0%) 48 (51%) 21 (42%) 5 (46%)
HCV genotype 2
Total (41) 24 (92%) 11 (92%) 3 (100%) 1 (4%) 0 (0%) 0 (0%) 1 (4%) 1 (8%) 0 (0%)
Low virus load (16) 9 (82%) 5 (100%) –e 1 (9%) 0 (0%) – 1 (9%) 0 (0%) –
High virus load (19) 11 (100%) 4 (80%) 3 (100%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (20%) 0 (0%)
HCV genotype 3
Total (131) 61 (85%) 41 (82%) 9 (100%) 6 (8%) 6 (12%) 0 (0%) 5 (7%) 3 (6%) 0 (0%)
Low virus load (54) 24 (80%)f 20 (100%) 4 (100%) 4 (13%) 0 (0%) 0 (0%) 2 (7%) 0 (0%) 0 (0%)
High virus load (69) 34 (87%) 19 (76%) 5 (100%) 2 (5%) 5 (20%) 0 (0%) 3 (8%) 1 (4%) 0 (0%)
SVR, sustained virologic response; RR, relapsed response; NR, non-response.a Total number of patients varies from subsequent subgroups due to missing baseline HCV RNA qunatification for some patients.b Baseline HCV RNA %800,000 IU/ml.c Baseline HCV RNA O800,000 IU/ml.d PZ0.007 for the comparison of SVR A/A vs. A/C vs. C/C.e No patient within this group.f PZ0.025 or the comparison of SVR A/A vs. A/C vs. C/C.
T. Mueller et al. / Journal of Hepatology 41 (2004) 652–658656
HCV infection despite recent reports showing impaired
IL-12 production within these genotypes [8,10].
These findings contradict a previous report describing an
over-representation of heterozygous IL12B 3 0-UTR allele
carriers in individuals with a history of spontaneous HCV
clearance compared to chronic HCV carriers [16]. We inves-
tigated twice as many patients as Cramp and colleagues [16]
which could explain the observed differences. Ethnical and
environmental factors and the possibility of spurious allelic
associations must also be taken into consideration [17–19].
This latter point might be responsible for the observed small
differences in the promoter genotype frequency observed in
our cohort of patients with spontaneous HCV clearance
(see Table 2). In addition, it is noteworthy that our study
population with spontaneous HCV clearance derived from
the homogenous and well-documented cohort of young
Table 4
Hepatic inflammation grade and fibrosis stage in relation to IL12B polymorp
IL12B genotype (n) Inflammation (grade)a (%)
0–1 2 3
Promotorb
4bp(C)/4bp(C) (115) 36.5 50.4 1
4bp(C)/4bp(K) (214) 36 53.3 1
4bp(K)/4bp(K) (129) 34.1 46.5 1
3 0 UTR
1188-A/A (275) 34.2 54.3 1
1188-A/C (152) 38.2 50 1
1188-C/C (31) 35.5 48.4 1
a Classification (grade, stage) according to the Scheuer score [14].b IL12B promoter allele with 4 bp insertion (CTCTAA), 4bp(-) IL12B promotec Comparison of fibrosis stage 3-4 in IL12B 3 0 UTR 1188-C/C (48%) vs. stage 3
Mueller T et al. IL12B polymorphisms in HCV infection 26.
women who were infected during a single-source outbreak of
hepatitis C in Eastern Germany in 1979 [12,13], thereby
excluding a major influence of environmental factors or HCV
genotype dependent host-virus interactions.
An additional aim of our study was to determine whether
IL12B polymorphisms affect the individual response to
interferon-based combination therapy. The IL12B promoter
polymorphism plays a rather minor role in determining the
treatment outcome given comparable SVR rates for all three
IL12B promoter genotypes. In contrast, we found evidence
that the IL12B 3 0UTR polymorphism may influence the
treatment outcome. Analysis of treatment data from the total
group of 501 patients showed a non-significant trend for
rising SVR rates in C-allele positive IL12B 3 0UTR
genotypes. This observation became significant when we
stratified our patients for HCV genotype and its replication
hisms in 458 chronic HCV-infected patients
Fibrosis (stage)a (%)
0–1 2 3–4
3 35.7 34.8 29.6
0.7 33.2 35.5 31.6
9.4 30.2 36.4 33.3
4.5 31.6 37.1 31.3
1.8 36.2 35.5 28.3
6.1 29 22.6 48.4c
r allele with 4 bp deletion (GC).
-4 in non-1188-C/C (30.2%): odds ratio 2.17; 95% CI 1.39-4.512; pZ0.039.
T. Mueller et al. / Journal of Hepatology 41 (2004) 652–658 657
status. In particular, HCV genotype 1-infected patients with
a high baseline virus load (well established negative
predictive parameters) responded significantly better to
therapy when they carried at least one IL12B 3 0UTR 1188-
C-allele. This increase in SVR was due to diminished
relapse rates, whereas the frequency of non-response
remained unaffected. IL12B 3 0UTR 1188-C-allele carriers
with HCV type 3-infection (without high baseline HCV
RNA levels) were similarly protected from a relapse.
The reduction in relapse rates seen in IL12B 3 0UTR
1188-C-allele carrying patients may be due to a more
pronounced activation of immunological defense mechan-
isms which may be closely associated with this genotype.
Association between the C-allele and the postulated
improved immune response may be due to an increased
IL12 production which in turn may be influenced by the
treatment type.
Our findings are in accordance with recent reports
showing an association of the 3 0-UTR 1188-C allele with
increased IL-12 production. This not only refers to chronic
HCV carriers [16] but also to healthy blood donors [20],
indicating a possible linkage between higher IL-12 levels
and treatment response [21,22]. However, our data contrast
a previous report describing an association between the
IL12B 3 0UTR 1188-A allele and increased IL-12 production
[10], a finding which has recently been questioned [23].
A more refined analysis of our data also indicates that
patients with the rare homozygous IL12B 3 0-UTR 1188-C/C
genotype may have an increased risk for severe liver
fibrosis. In accord with the concept of the IL-12-induced
immune stimulation in these patients, a higher inflammatory
response might lead to increased fibrosis rates [21]. Thus,
the IL12B 3 0UTR genotype might influence intrahepatic
IL-12 expression levels in chronic HCV infection, where
non-parenchymal cells (i.e. infiltrating lymphocytes and
macrophages) and hepatic stellate cells have been demon-
strated to be IL-12 immunoreactive [24]. This finding
appears to be particularly interesting considering that these
cells play a key role in HCV-induced liver fibrosis [25].
However, more information is required in this respect in
view of the small number studied (nZ31) and the absence
of a clear pro-fibrotic gene dosage effect (31% vs. 28% vs.
48% advanced fibrosis for IL12B 3 0UTR 1188-A/A, -A/C
and -C/C, respectively, Table 4).
In conclusion, we found no association between poly-
morphisms within the promoter region and the 3 0-UTR of
the IL12B gene and self-limited HCV infection. However,
increased SVR rates were observed in those high risk
patients (highly replicative HCV type 1 infection) who
carried at least one IL12B 3 0-UTR 1188-C allele.
Acknowledgements
Supported in part by the German BMBF Network of
Competence for Viral Hepatitis (Hep Net).
References
[1] Diepolder HM, Zachoval R, Hoffmann RM, Jung MC, Gerlach T,
Pape GR. The role of hepatitis C virus specific CD4C T lymphocytes
in acute and chronic hepatitis C. J Mol Med 1996;74:583–588.
[2] Cramp M, Carucci P, Rossol C, Chokshi S, Maertens G, Williams R,
et al. Hepatitis C virus (HCV) specific immune responses in anti-
HCV positive patients without hepatitis C viraemia. Gut 1999;44:
424–429.
[3] Gerlach JT, Diepolder HM, Jung MC, Gruener NH, Schraut WW,
Zachoval R, et al. Recurrence of hepatitis C virus after loss of virus-
specific CD4 (C) T-cell response in acute hepatitis C. Gastroenter-
ology 1999;117:1012–1014.
[4] Day CL, Lauer GM, Robbins GK, McGovern B, Wurcel AG,
Gandhi RT, et al. Broad specificity of virus-specific CD4C T-helper-
cell responses in resolved hepatitis C virus infection. J Virol
2002;12584–12595.
[5] Trinchieri G. Interleukin-12 and the regulation of innate resistance
and adaptive immunity. Nat Rev Immunol 2003;3:133–146.
[6] Huang D, Cancilla MR, Morahan G. Complete primary structure,
chromosomal localization and definition of polymorphisms of the
gene encoding the human interleukin-12 p40 subunit. Genes Immun
2000;1:515–520.
[7] Hall MA, McGlinn E, Coakley G, Fisher SA, Boki K, Middleton D,
et al. Genetic polymorphism of IL-12 p40 gene in immune-mediated
disease. Genes Immun 2000;1:219–224.
[8] Morahan G, Huang D, Wu M, Holt BJ, White GP, Kendall GE, et al.
Association of IL12B promoter polymorphism with severity of atopic
and non-atopic asthma in children. Lancet 2002;360:455–459.
[9] Morahan G, Boutlis CS, Huang D, Pain A, Saunders JR,
Hobbs MR, et al. A promoter polymorphism in the gene encoding
interleukin-12 p40 (IL12B) is associated with mortality from
cerebral malaria and with reduced nitric oxide production. Genes
Immun 2002;3:414–418.
[10] Morahan G, Huang D, Ymer SI, Cancilla MR, Stephen K,
Dabadghao P, et al. Linkage disequilibrium of a type 1 diabetes
susceptibility locus with a regulatory IL12B allele. Nat Genet 2001;
27:218–221.
[11] Tsunemi Y, Saeki H, Nakamura K, Sekiya T, Hirai K, Fujita H, et al.
Interleukin-12 p40 gene (IL12B) 3 0-untranslated region polymorph-
ism is associated with susceptibility to atopic dermatitis and psoriasis
vulgaris. J Dermatol Sci 2002;30:161–166.
[12] Tabaki A, Wiese M, Maertens G, Depla E, Seifert U, Liebetrau A,
et al. Cellular immune responses persist and humoral responses
decrease two decades after recovery from a single-source outbreak of
hepatitis C. Nature 2000;6:578–582.
[13] Wiese M, Berr F, Lafrenz M, Porst H, Oesen U. Low frequency of
cirrhosis in a hepatitis V (genotype 1b) single-source outbreak in
Germany: a 20-year multicenter study. Hepatology 2000;32:91–96.
[14] Scheuer PJ. Classification of chronic viral hepatitis: a need for
reassessment. J Hepatol 1991;13:327–374.
[15] Livak KJ. Allelic discrimination using fluorogenic probes and the
5 0nuclease assay. Genet Anal 1999;14:143–149.
[16] Cramp ME, Demaine AG, Houldsworth A, Kaminski E, Metzner M,
Rossol S. IL-12 gene polymorphism and outcome of HCV infection.
Hepatology 2003;38 (suppl 1):314 (abstract).
[17] Bataller R, North KE, Brenner DA. Genetic polymorphisms and the
progression of liver fibrosis: a critical appraisal. Hepatology 2003;37:
493–503.
[18] Grimes DA, Schultz KF. Bias and causal associations in observational
research. Lancet 2002;359:248–252.
[19] Cardon LR, Palmer LJ. Population stratification and spurious allele
association. Lancet 2003;361:598–604.
[20] Seegers D, Zwiers A, Strober W, Pena AS, Bouma G. A TaqI
polymorphism in the 3 0UTR of the IL-12 p40 gene correlates with
increased IL-12 secretion. Genes Immun 2002;7:419–423.
T. Mueller et al. / Journal of Hepatology 41 (2004) 652–658658
[21] Quiroga JA, Martin J, Navas S, Carreno V. Induction of
interleukin-12 production in chronic Hepatitis C virus infection
correlates with the hepatocellular damage. J Infect Dis 1998;178:
247–251.
[22] Amaraa R, Mareckova H, Urbanek P, Fucikova T. Production
of interleukins 10 and 12 by activated peripheral blood
monocytes/ macrophages in patients suffering from chronic
hepatitis C virus infection with respect to the response to
interferon and ribavirin treatment. Immunol Lett 2002;83:
209–214.
[23] Dahlman I, Eaves IA, Kosoy R, Morrison VA, Heward J, Gough SCL,
et al. Parameters for reliable results in genetic association studies in
common disease. Nat Genet 2002;30:149–150.
[24] Leifeld L, Cheng S, Ramakers J, Dumoulin FL, Trautwein C,
Sauerbruch T, et al. Imbalanced intrahepatic expression of interleukin
12, interferon gamma, and interleukin 10 in fulminant hepatitis B.
Hepatology 2002;36:1001–1008.
[25] Bataller R, Paik YH, Lindquist JN, Lemasters JL, Brenner DA.
Hepatitis C virus core and nonstructural proteins induce fibrogenic
effects in hepatic stellate cells. Gastroenterology 2004;126:529–540.