IJC International Journal of Cancer
The toll-like receptor 2 (TLR2) -196 to -174 del/ins polymorphism affects viral loads and susceptibility to hepatocellular carcinoma in chronic hepatitis C Hans-Dieter Nischalke1*, Martin Coenen1*, Cordula Berger1, Katharina Aldenhoff1, Tobias Mu¨ller2, Thomas Berg2, Benjamin Kra¨mer1, Christian Ko¨rner1, Margarete Odenthal3, Falko Schulze3, Frank Gru¨nhage4, Jacob Nattermann1, Tilman Sauerbruch1 and Ulrich Spengler1 1
Department of Internal Medicine I, University of Bonn, Bonn, Germany Medical Clinic for Hepatology and Gastroenterology, Medical University Charite´ Campus, Virchow–Klinikum Berlin, Berlin, Germany 3 Institute for Pathology, University of Cologne, Cologne, Germany 4 Department of Medicine II, Saarland University Hospital, Homburg, Germany 2
Chronic hepatitis C virus (HCV) infection is a major risk factor for hepatocellular carcinoma (HCC). HCV proteins core and NS3 can bind to toll-like receptor 2 (TLR2) and trigger inflammatory responses. Polymorphisms in the TLR2 gene predispose to various forms of malignancy but have not been studied in HCV-associated HCC. Here, we investigated whether single nucleotide polymorphisms (SNPs), rs4696480, rs5743708, rs5743704 and the -196 to -174 del/ins polymorphism of the TLR2 gene affect the risk for HCC in chronic hepatitis C. The study involved 189 and 192 HCV genotype 1 infected patients with and without HCC, respectively, as well as 347 healthy controls. TLR2 alleles were determined by hybridization probe assays and allele-specific short fragment polymerase chain reaction on a LightCycler system. All TLR2 polymorphisms matched the Hardy– Weinberg equilibrium in each study group. Although TLR2 SNPs showed no effect, the frequency of the TLR2 -196 to -174 del allele was significantly higher in patients with HCV-associated HCC (22.5%) than in HCV-infected patients without HCC (15.6%, p 5 0.016) and healthy controls (15.3%, p 5 0.003). HCV-infected carriers of a TLR2 -196 to -174 del allele had significantly higher HCV viral loads than TLR2 -196 to -174 ins/ins homozygous patients (p 5 0.031). Finally, in carriers of the TLR2 -196 to -174 del allele, stimulation of monocytes resulted in significantly lower TLR2 expression levels and interleukin-8 (IL-8) induction than in individuals with the TLR2 -196 to -174 ins/ins genotype (p < 0.05). Our data suggest the TLR2 -196 to -174 del allele to affect HCV viral loads and to increase the risk for HCC in HCV genotype1-infected patients.
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Chronic hepatitis C virus (HCV) infection is a major cause of chronic liver disease leading to progressive hepatic fibrosis with long-term progression to cirrhosis and hepatocellular carcinoma (HCC).1 Host genetic, environmental and viral factors appear to play an important role in determining the progression of chronic HCV infection to liver cirrhosis and
Key words: TLR2, -196 to -174 del/ins polymorphism, HCV, viral load, HCC Grant sponsor: Deutsche Krebshilfe; Grant number: 107865; Grant sponsor: BONFOR; Grant number: O-107.0097; Grant sponsor: Deutsche Forschungsgemeinschaft (SFB); Grant number: TR57 TP12; Grant sponsor: H.W. and J. Hector Foundation; Grant number: M42 *H.-D.N. and M.C. contributed equally to this work DOI: 10.1002/ijc.26143 History: Received 29 Oct 2010; Accepted 23 Mar 2011; Online 15 Apr 2011 Correspondence to: Hans-Dieter Nischalke, Department of Internal Medicine I, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany, Tel: þ49-228-287-51416, Fax: þ49-228-287-51419, E-mail:
[email protected]
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HCC. The underlying molecular mechanisms, however, remain only poorly understood. Several HCV proteins have been shown to upregulate mitogenic processes, thus exerting direct oncogenic effects.2,3 On the other hand, HCV constituents also stimulate inflammatory pathways, e.g., via triggering of toll-like receptors (TLRs). TLRs play a critical role in innate immune responses. They recognize conserved molecular patterns expressed by microbial pathogens and trigger inflammatory responses including activation of transcription factors and production of pro-inflammatory cytokines such as interleukin (IL)-6 and IL-8, which may promote tumor development.4 TLR2 recognizes various bacterial components, and we and others have recently shown that TLR2 is triggered by HCV proteins core and NS3 leading to activation of inflammatory cells.5,6 Moreover, transgenic mice expressing the HCV core protein have been shown to ultimately develop HCC.7 The human TLR2 gene is located on chromosome 4q32. Genetic studies on the TLR2 gene have identified a number of polymorphisms which have been shown to affect host defense and disease progression.8,9 A 22-bp nucleotide deletion at position -196 to -174 of the untranslated 50 -region is associated with
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Table 1. Demographics and distribution of genetic TLR2 variants
Total number
HCV/HCC
HCV without HCC
Healthy controls
189
192
347
Median age, years (range)
61 (38–82)
50.5 (18–86)
58.5 (20–94)
Gender (male/female)
57.1%/42.9%
54.4%/45.6%
51.4%/47.6%
Mean ?-GT 6 SD
117.1 6 101.5
88.9 6 123.6
–
Mean AST 6 SD
124.3 6 181.6
58.3 6 42.7
–
Mean ALT 6 SD
106.9 6 206.3
87.4 6 68.6
–
Mean HCV viral load (IU 6 SD)
1,684,952 6 2,554,530
2,259,991 6 3,198,149
–
ins/ins
115 (60.8%)
138 (71.9%)
248 (71.5%)
ins/del
63 (33.3%)
48 (25.0%)
92 (26.5%)
del/del
11 (5.9%)
6 (3.1%)
7 (2.0%)
TT
44 (23.3%)
58 (30.2%)
95 (27.4%)
TA
95 (50.3%)
88 (45.8%)
162 (46.7%)
AA
50 (26.4%)
46 (24.0%)
90 (25.9%)
GG
174 (92.1%)
175 (91.1%)
319 (91.9%)
GA
15 (7.9%)
17 (8.9%)
28 (8.1%)
AA
–
–
–
CC
175 (92.6%)
178 (92.7%)
175 (89.9%)
CA
14 (7.4%)
14 (7.3%)
14 (10.1%)
AA
–
–
–
TLR polymorphisms TLR -196 to -174 del/ins
TLR -16934 A>T (rs4696480)
TLR2 Arg753Gln (rs5743708)
reduced transcriptional activity compared to the wild type allele in luciferase reporter assays.10 This polymorphism has already been shown to be associated with an increased risk of noncardiac gastric cancer and susceptibility to cervical cancer.11,12 As TLR2 is directly triggered by HCV proteins, we hypothesized that polymorphisms in the TLR2 gene may contribute to differences in the course of chronic HCV infection, in particular progression to HCC. In this study, we investigated the influence of TLR2 single nucleotide polymorphisms (SNPs), -16934 A > T (rs4696480), Arg753Gln (rs5743708), Pro631His (rs5743704) and the TLR2 -196 to -174 del/ins polymorphism on HCV replication and susceptibility for HCV-associated HCC.
Material and Methods Study population and sample collection
A total of 197 patients with HCV-associated HCC (57.1% male; median age 61 years, range 38–82 years, 192 HCVinfected patients without HCC (54.4% male; median age 51 years, range 18–86 years) and 347 healthy controls (51.4% male; median age 58.5 years, range 20–94 years) were included in this study. As the risk of HCC appears to be C 2011 UICC Int. J. Cancer: 130, 1470–1475 (2012) V
particular prominent in HCV genotype 1 infection,13,14 exclusively HCV genotype 1 infected patients had been recruited from the Bonn and Berlin University Departments of Gastroenterology as well as Cologne University Pathology services. Healthy control samples were obtained from blood donors who volunteered to participate in the study. Characteristics of all patients are listed in Table 1. Informed consent was obtained from each patient and the study conformed to the ethical guidelines of the Helsinki Declaration as approved by the local ethics committees.
Diagnosis of HCV infection
HCV antibodies were detected with a microparticle enzyme immunoassay (Axsym; Abbott) and confirmed by dot immunoassay (Matrix; Abbott). HCV RNA was detected with a nucleic acid purification kit (QIAamp Viral Kit; Qiagen, Hilden, Germany), followed by reverse transcription and nested polymerase chain reaction. Quantitative determination of HCV loads was done by branched DNA technology (Chiron, Emeryville, CA). HCV genotype was determined by the Innolipa II line probe assay (Innogenetics, Zwijndrecht, Belgium).
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TLR Pro631His (rs5743704)
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TLR2 -196 to -174 del/ins polymorphism
Figure 1. (a) Analysis of the melting temperature of amplified PCR products from an individual homozygous for the TLR2 22bp insertion (solid line), a carrier homozygous for the deletion (dot and dashed line) and a carrier of the heterozygous genotype (dashed line). The dotted line indicates the negative control (H2O). (b) The frequency of the deletion allele in HCV/HCC patients in comparison to the HCV patients without HCC and healthy controls.
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Genotyping of the TLR2 gene
Determination of TLR2 -196 to -174 del/ins polymorphism was performed by LightCycler real-time PCR using the following oligonucleotide primers: CTC GAA GGC AGC GAG AAA (forward), TAC CTG GGA GAA CTC CGA (reverse). Samples were set up in a final volume of 10 ll, containing 1 ll of DNA solution, 2 ll of LightCycler FastStart DNA MasterPLUS SYBR Green I Mix (Roche Molecular Biochemicals, Mannheim, Germany) and 0.5 lM of each primer (Tib MolBiol, Berlin, Germany). The cycling conditions were as follows: initial denaturation at 95 C for 10 min, followed by 45 cycles of denaturation at 95 C for 3 sec, annealing at 60 C for 5 sec and extension at 72 C for 10 sec. Fluorescence was monitored at the end of each extension phase at 72 C. After completion of the PCR, a melting curve of the amplification products was plotted by denaturation at 95 C for 5 sec, holding the sample at 70 C for 10 sec and then slowly heating the sample to 95 C with a ramp rate of 0.1 C/sec as well as continuous fluorescence acquisition. The primers were designed to amplify gene segments of different length (54bp and 77bp), depending on the presence or absence of the deletion mutation in the template DNA. The 77-bp fragment, corresponding to the insertion allele, had a melting temperature of 89 C. In contrast, the 54-bp PCR product, indicating the deletion allele, had a melting temperature of 85 C. This difference in melting temperatures enabled us to easily distinguish the two alleles15 (Fig. 1a).
Determination of TLR2 SNPs rs4696480 and rs5743708 was performed by LightCycler real-time PCR using the following oligonucleotide primers and hybridization probes: TLR2 rs4696480: AAC AGA AAT TTA TCC ATT CAT GGT T (sense), AGC AGT TTA TTG TGA GAA TGA GTT T (antisense), CCA GAT GAC CCT CAC CAG ATG C-FL (sensor) and LC-Red640-GCC CTT CAA TCT TGG ACT TCC CAG ACT CC-p (anchor); TLR2 rs5743708: AAA GCT CCC AGC AGG AAC AT (sense), GAC TTT ATC GCA GCT CTC AGA TTT AC (antisense), CAA GCT GCA GAA GAT AAT GAA CAC CAA G-FL (sensor) and LC Red640CCT ACC TGG AGT GGC CCA TGG ACG-p (anchor) (all TIB MOLBIOL, Berlin, Germany). The TLR2 SNP Pro631His (rs5743704) was determined by a LightSNiP assays according to recommendations of the manufacturer (TIB MOLBIOL, Berlin, Germany). In vitro induction of TLR2 expression
A total of 500 ll of whole blood was incubated in a humidified atmosphere at 37 C with 5% CO2 for 16 hr with 5 lg/ ml recombinant HCV NS3 protein (eBioscience, Boston, MA), 1 lg/ml peptidoglycan (PGN; Sigma, Deisenhofen, Germany) and phosphate buffered saline (PBS) (unstimulated control), respectively. For analysis of TLR2 expression on monocytes, 100-ll whole blood was stained with phycoerythrin-conjugated monoclonal TLR2 antibody (clone T2.5, eBioscience, Boston, MA) and counterstained with antiC 2011 UICC Int. J. Cancer: 130, 1470–1475 (2012) V
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CD14-PerCP using a Lyse/Wash Procedure (BD Bioscience). Phycoerythrin-conjugated mouse IgG1 isotype control antibody (eBioscience) served as a control reference. Dead cells were excluded by counter-staining with DAPI (40 ,6-diamidino-2-phenylindole) (BD Bioscience). Results are expressed as normalized median fluorescence intensity (nMFI): nMFI ¼ [median fluorescence intensity (TLR2 stained sample) median fluorescence intensity (isotype control)]/median fluorescence intensity (isotype control). In vitro induction of interleukin-8
Supernatants from stimulation experiments (buffer control, NS3 and PGN) as described above were prediluted and analyzed in a commercially available IL-8 ELISA as recommended by the manufacturer (Gen-Probe Diaclone, Besancon, France).
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distribution of TLR2 -196 to -174 del/ins alleles was not significantly different between HCV-infected patients without HCC and healthy controls. We next assessed whether the TLR2 -196 to -174 del/ins polymorphism had an influence on clinical parameters of HCV infected patients. We did not find any significant relationship between the TLR2 -196 to -174 del/ins polymorphism and hepatic inflammation, stage of fibrosis or levels of alanine transaminase (ALT) (113 vs. 84 U/l), aspartate transaminase (AST) (95 vs. 68 U/l) and gamma-glutamyltransferase (111 vs. 91 U/l). However, HCV patients carrying the TLR2 -196 to -174 deletion allele had significantly higher viral loads (2,823,000 6 3,837,000) compared to patients with the homozygous TLR2 -196 to -174 ins/ins genotype (1,899,400 6 2,819,700; p ¼ 0.031 Mann-Whitney test) (Fig. 2a). In vitro induction of TLR2 expression
Statistical analysis
Genotype frequencies were determined and tested for consistency with Hardy–Weinberg equilibrium using an exact test. Allele and genotype frequencies were compared between cases and controls by Pearson’s goodness-of-fit v2 test and Armitage’s trend test, respectively (http://ihg2.helmholtzmuenchen.de/cgi-bin/hw/hwa1). Differences between groups were analyzed by t-test and Mann-Whitney U test as appropriate. Statistical analysis was performed with SPSS 18.0 (SPSS, Munich, Germany). Data are given as means 6 SD, unless stated otherwise.
Results
The TLR2 del/ins polymorphism is located in the promoter region of the TLR2 gene. Therefore, we studied in vitro if TLR2 del/ins alleles are associated with differential induction of TLR2 expression. To this end, we stimulated peripheral blood of genetically characterized healthy individuals with recombinant HCV-NS3 protein and PGN and determined TLR2 expression on monocytes as well as induced levels of IL-8. As shown in Figure 2b, carriers of the TLR2 deletion allele achieved significantly lower TLR2 expression on their monocytes after stimulation with HCV-NS3 (p ¼ 0.023) and PGN (p ¼ 0.009) than individuals carrying the TLR2 -196 to -174 ins/ins genotype. In line with TLR2 expression on monocytes levels of induced IL-8 were significantly lower in supernatants from carriers of the TLR2 deletion allele (Fig. 2c).
TLR2 (-196 to -174 del/ins) gene polymorphism and HCC
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Discussion TLRs play a key role in the activation of the innate immune response and induce transcription factors, which induce proinflammatory cytokines. Here, we demonstrate that in chronic genotype 1 hepatitis C the TLR2 -196 to -174 del allele is associated with increased viral loads and an enhanced risk to develop HCC; the TLR2 -196 to -174 del allele was significantly more frequent in patients with HCV-associated HCC than in healthy controls and in HCV-infected patients without HCC. Importantly, the distribution of TLR2 -196 to -174 del/ins alleles was identical in healthy controls and HCV infected patients without HCC. Thus, our data indicate a pivotal role of the TLR2 -196 to -174 del/ins polymorphism concerning the risk to develop HCC in chronic hepatitis C. Various studies emphasize a critical role of TLR genetics in host immune responses and disease pathogenesis, and recent studies suggest a role of TLR gene polymorphisms also for malignant diseases.9,16 The TLR2 -196 to -174 del/ins polymorphism has further been shown to be associated with cervical cancer,11 an increased risk of noncardiac gastric cancer12 and the severity of intestinal metaplasia in gastroduodenal disease. Importantly, the TLR2 -196 to -174 del/ins polymorphism has been recently proposed to reflect differential
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SNPs -16934 (rs4696480), Arg753Gln (rs5743708), Pro631His (rs5743704) and the -196 to -174 del/ins gene polymorphism were analyzed in all 728 subjects. The distribution of TLR2 genotypes is shown in Table 1. All genotype frequencies matched the Hardy–Weinberg equilibrium. Whereas the distribution of alleles in the three SNPs did not indicate any significant variation between our study groups, the frequency of the -196 to -174 deletion allele was significantly higher in HCC patients than in healthy controls (22.5% vs. 15.3%, odds ratio (OR) 1.609, 95%-confidence interval (CI) 1.171– 2.212; p ¼ 0.0032) and HCV-infected subjects without HCC (22.5% vs. 15.6%, OR 1.567, 95%-CI 1.086–2.260; p ¼ 0.016), respectively (Fig. 1b). Homozygous carriers of the TLR2 -196 to -174 deletion had approximately a threefold increased risk for HCC as compared to homozygous carriers of the TLR2 196 to -174 ins/ins genotype (OR 3.389, 95%-CI 1.281–8.976; p ¼ 0.0096), when compared to healthy controls. Moreover, carriers of the TLR2 -196 to -174 del allele had approximately a 1.5-fold increased risk for HCC than individuals with a homozygous TLR2 -196 to -174 ins/ins genotype as compared to healthy control (OR 1.6130, 95%-CI 1.064– 2.330; p ¼ 0.012) and patients with hepatitis C alone (OR 1.645, 95%-CI 1.034–2.349; p ¼ 0.023). Importantly, the
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Figure 2. (a) Comparison of viral loads between patients carrying the TLR2 -196 to -174 deletion allele and patients showing the TLR2 -196 to -174 ins/ins genotype. As detected by branched DNA technology, patients with the deletion allele had a significantly higher viral load as compared to patients carrying the TLR2 -196 to -174 ins/ins genotype (p ¼ 0.007 Mann-Whitney test). (b) TLR2 expression on monocytes in carriers of the deletion allele (black
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columns, n ¼ 7) and in carriers homozygous for the insertion allele (gray columns, n ¼ 7). To study the induction of TLR2 expression, 500 ll whole blood was incubated for 16 hr with HCV NS3 protein (5 lg/ml; right columns), peptidoglycan (1 lg/ml; middle) and PBS (unstimulated; left columns), respectively. Results are expressed as normalized median fluorescence intensity (nMFI), which was calculated as nMFI ¼ [median fluorescence intensity (TLR2-stained sample) median fluorescence intensity (isotype control)]/median fluorescence intensity (isotype control). (c) IL-8 induction in whole blood from carriers of the deletion allele (black columns, n ¼ 7) and from carriers homozygous for the insertion allele (gray columns, n ¼ 7). To study IL-8 induction, 500-ll whole blood was stimulated for 16 hr with HCV NS3 protein (5 lg/ml; right columns), peptidoglycan (1 lg/ml; middle) and PBS (unstimulated; left columns), respectively. IL-8 was measured in the supernatants after centrifugation using a commercially available IL-8 ELISA (Gen-Probe Diaclone).
TLR2 -196 to -174 del/ins polymorphism
trans-activation of TLR2 promoter constructs.10 To check, if this polymorphism affects TLR2 expression on immune-competent cells, we performed in vitro stimulation experiments with PGN and HCV-NS3, which upregulate TLR2 expression on monocytes and macrophages. These experiments clearly indicated impaired upregulation of TLR2 to be associated with the TLR2 -196 to -174 del allele. In line with this finding, induction of IL-8, a cytokine upregulated in response to TLR2 triggering, was also reduced in carriers of the TLR2 196 to -174 del allele. Thus, our findings confirm that differential in vitro trans-activation of TLR2 promoter constructs as shown by Noguchi et al.10 corresponds to functional differences, when cells from patients carrying different TLR2 22bp del/ins variants are stimulated. Taken together, these findings support the concept that reduced TLR2 signaling leads to less effective immune responses facilitating viral replication with increased HCV counts in the serum. Our observations indicate that the TLR2 -196 to -174 del allele might enhance the risk for HCV-induced HCC probably because of reduced TLR2 signaling, whereas increased TLR2 expression appears to predispose to HCV-associated lympho-proliferative disease and B cell lymphoma.6 Thus, our findings suggest divergent roles of TLR2 signaling in each of the two HCV-associated malignant diseases. Increased secretion of cytokines such as IL-6 following TLR2 stimulation is likely to act as growth factor for lymphoma development, whereas low level TLR2 expression and production of inflammatory cytokines associated with the TLR2 196 to -174 del allele seems to allow for increased viral loads thus contributing to an increased risk to develop HCC.17 Recently, increased serum levels of ALT and AST were found to be significantly associated with HCC.18,19 However, in our study group, these parameters were only slightly increased, and thus, in the face of considerable individual variation, we failed to detect any significant relationship between the TLR2 -196 to -174 del/ins polymorphism and any liver enzyme serum levels. Unexpectedly, we could also not find any correlation between the TLR2 -196 to -174 del/ ins polymorphism and stages of liver fibrosis. However, the role of fibrosis concerning pathogenesis of HCC is still discussed controversially.20–22 Our analysis of the functional role of TLR2 -196 to -174 del/ins alleles with respect to TLR2 expression was based on in vitro stimulation studies. However, our in vitro findings are supported by increased TLR2 expression reported for mononuclear cells in the peripheral blood23 and livers of HCV-infected patients.24 It is noteworthy that we found an identical distribution of TLR2 -196 to -174 del/ins alleles in healthy controls and HCVinfected patients without liver cancer. Thus, the polymorphism does not appear to affect the host’s susceptibility to acquire HCV infection or its transition from an acute to chronic infection. Taken together, our data suggest that the TLR2 polymorphism is an important risk factor modifying the outcome of HCV infection rather than the risk of HCV infection itself. C 2011 UICC Int. J. Cancer: 130, 1470–1475 (2012) V
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In summary, our results provide first evidence for the TLR2 -196 to -174 del/ins polymorphism as a novel host factor affecting HCV replication and thus the risk for HCC. As this polymorphism might enable the easy identification of HCV genotype 1 infected patients with an increased risk of liver cancer, it should be further prospectively evaluated as a prognostic marker. Ultimately, such studies may also offer an
opportunity to find out how TLR-mediated signals might be exploited as future therapeutic and prophylactic targets.
Acknowledgements This study was funded by the Deutsche Krebshilfe to H.D.N. and U.S., the BONFOR to M.C., the Deutsche Forschungsgemeinschaft to J.N. and U.S. as well as the H.W. and J. Hector Foundation to J.N.
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serum immunoglobin E levels for Toll-like receptor polymorphisms in a Japanese population. Clin Exp Allergy 2004;34: 177–83. Pandey S, Mittal RD, Srivastava M, Srivastava K, Singh S, Srivastava S, Mittal B. Impact of Toll-like receptors [TLR] 2 (-196 to -174 del) and TLR 4 (Asp299Gly, Thr399Ile) in cervical cancer susceptibility in North Indian women. Gynecol Oncol 2009;114:501–5. Tahara T, Arisawa T, Wang F, Shibata T, Nakamura M, Sakata M, Hirata I, Nakano H. Toll-like receptor 2 -196 to 174del polymorphism influences the susceptibility of Japanese people to gastric cancer. Cancer Sci 2007;98:1790–4. Bruno S, Crosignani A, Maisonneuve P, Rossi S, Silini E, Mondelli MU. Hepatitis C virus genotype 1b as a major risk factor associated with hepatocellular carcinoma in patients with cirrhosis: a seventeen-year prospective cohort study. Hepatology 2007; 46:1350–6. Raimondi S, Bruno S, Mondelli MU, Maisonneuve P. Hepatitis C virus genotype 1b as a risk factor for hepatocellular carcinoma development: a meta-analysis. J Hepatol 2009;50:1142–54. Nischalke HD, Nattermann J, Lichterfeld M, Woitas RP, Rockstroh JK, Sauerbruch T, Spengler U. Rapid determination of the Delta32 deletion in the human CC-chemokine receptor 5 (CCR5) gene without DNA extraction by lightcycler real-time polymerase chain reaction. AIDS Res Hum Retroviruses 2004;20: 750–4. El-Omar EM, Ng MT, Hold GL. Polymorphisms in Toll-like receptor genes and risk of cancer. Oncogene 2008;27: 244–52. Ishihara T, Kaito M, Takeuchi K, Gabazza EC, Tanaka Y, Higuchi K, Ikoma J, Watanabe S, Sato H, Adachi Y. Role of UGT1A1 mutation in fasting hyperbilirubinemia. J Gastroenterol Hepatol 2001;16:678–82.
18. Benvegnu L, Alberti A. Risk factors and prevention of hepatocellular carcinoma in HCV infection. Dig Dis Sci 1996;41 (Suppl):49S–55S. 19. Sato A, Kato Y, Nakata K, Nakao K, Daikoku M, Ishii N, Matsumoto T, Iseki K, Mazume H, Nagataki S. Relationship between sustained elevation of serum alanine aminotransferase and progression from cirrhosis to hepatocellular carcinoma: comparison in patients with hepatitis B virus- and hepatitis C virus-associated cirrhosis. J Gastroenterol Hepatol 1996;11: 944–8. 20. Bege T, Le Treut YP, Hardwigsen J, Ananian P, Richa H, Campan P, Garcia S. Prognostic factors after resection for hepatocellular carcinoma in nonfibrotic or moderately fibrotic liver. A 116-case European series. J Gastrointest Surg 2007; 11:619–25. 21. Lawson A, Hagan S, Rye K, Taguri N, Ratib S, Zaitoun AM, Neal KR, Ryder SD, Irving WL. The natural history of hepatitis C with severe hepatic fibrosis. J Hepatol 2007;47:37–45. 22. Lok AS, Seeff LB, Morgan TR, di Bisceglie AM, Sterling RK, Curto TM, Everson GT, Lindsay KL, Lee WM, Bonkovsky HL, Dienstag JL, Ghany MG, et al. Incidence of hepatocellular carcinoma and associated risk factors in hepatitis C-related advanced liver disease. Gastroenterology 2009;136: 138–48. 23. Riordan SM, Skinner NA, Kurtovic J, Locarnini S, McIver CJ, Williams R, Visvanathan K. Toll-like receptor expression in chronic hepatitis C: correlation with pro-inflammatory cytokine levels and liver injury. Inflamm Res 2006; 55:279–85. 24. Mozer-Lisewska I, Sluzewski W, Kaczmarek M, Jenek R, Szczepanski M, Figlerowicz M, Kowala-Piaskowska A, Zeromski J. Tissue localization of Toll-like receptors in biopsy specimens of liver from children infected with hepatitis C virus. Scand J Immunol 2005;62:407–12.
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