TRAIL receptor I (DR4) polymorphisms C626G and A683C are associated with an increased risk for hepatocellular carcinoma (HCC) in HCV-infected patients
© Körner et al; licensee BioMed Central Ltd. 2012
Received: 30 August 2011
Accepted: 8 March 2012
Published: 8 March 2012
Tumour surveillance via induction of TRAIL-mediated apoptosis is a key mechanism, how the immune system prevents malignancy. To determine if gene variants in the TRAIL receptor I (DR4) gene affect the risk of hepatitis C virus (HCV)-induced liver cancer (HCC), we analysed DR4 mutations C626G (rs20575) and A683C (rs20576) in HCV-infected patients with and without HCC.
Frequencies of DR4 gene polymorphisms were determined by LightSNiP assays in 159 and 234 HCV-infected patients with HCC and without HCC, respectively. 359 healthy controls served as reference population.
Distribution of C626G and A683C genotypes were not significantly different between healthy controls and HCV-positive patients without HCC. DR4 variants 626C and 683A occurred at increased frequencies in patients with HCC. The risk of HCC was linked to carriage of the 626C allele and the homozygous 683AA genotype, and the simultaneous presence of the two risk variants was confirmed as independent HCC risk factor by Cox regression analysis (Odds ratio 1.975, 95% CI 1.205-3.236; p = 0.007). Furthermore HCV viral loads were significantly increased in patients who simultaneously carried both genetic risk factors (2.69 ± 0.36 × 106 IU/ml vs. 1.81 ± 0.23 × 106 IU/ml, p = 0.049).
The increased prevalence of patients with a 626C allele and the homozygous 683AA genotype in HCV-infected patients with HCC suggests that these genetic variants are a risk factor for HCC in chronic hepatitis C.
KeywordsTRAIL receptor I DR4 Apoptosis Polymorphism C626G (rs20575) A683C (rs20576) HCV HCC Cancer
Nearly 3% of the world population, app. 180 million people, suffer from hepatitis C virus infection (HCV) thus representing a global health problem . In most of the cases exposure to HCV results in chronic viral persistence. Progression of chronic hepatitis C leads to liver fibrosis and cirrhosis and is associated with an increased risk to develop hepatocellular carcinoma (HCC) [2, 3]. HCC has become the third leading cause of cancer-related death worldwide [4–6], and in Western Countries chronic hepatitis accounts for the majority of HCCs. HCV proteins can interact with tumour suppressor proteins as well as with proteins involved in cell-cycle control, and these interactions may promote the development of abnormal cells in the liver.
Tumour development is normally prevented by the immune system, which eliminates transformed cells via induction of apoptosis by tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) [7, 8]. Binding of TRAIL to its cognate death receptors DR4 and DR5 triggers activation of the apoptotic cascade, formation of apoptotic bodies and eventually to depletion of the apoptotic cell. DR4 and DR5 are members of the tumour necrosis factor super family and are characterized by the existence of an extra-cellular cysteine-rich binding domain as well as an intra-cellular death domain essential for the transmission of the apoptotic stimulus.
Genetic alterations in death receptors might compromise apoptotic cell signalling and therefore contribute to the development of tumour cells. Several studies suggested an increased risk for cancer associated with single nucleotide polymorphisms (SNPs) in the DR4 gene [9–13]. However, the potential influence of DR4 gene mutations on the development of HCC has not been investigated so far. Therefore, we analysed the effect of the DR4 polymorphisms C626G (Thr209Arg, rs20575) and A638C (Glu228Ala, rs20576) on the occurrence of HCC in patients chronically infected with HCV.
Design and study population
Patient characteristics and distribution of DR4 genotypes
HCV(+) all without HCC
HCV(+) Cirrhosis (-) Without HCC
HCV(+) Cirrhosis (+) without HCC
HCV(+) Cirrhosis (+) with HCC
HBV(+) With HCC
Median age (range)
Male Gender (%)
HCV infection (%)
HCV genotype 1 (%)
HCV viral load(10 6 IU/ml) a)
2.04 ± 0.25
2.10 ± 0.29
1.86 ± 0.48
1.88 ± 0.36
HBV infection (%)
BMI > 30 (%)
rs20575 (C626G) b)
Deviation from Hardy-Weinberg equilibrium c)
p = 0.74
p = 0.29
p = 0.43
p = 0.49
p = 0.27
p = 0.59
rs20576 (A683C) b)
Deviation from Hardy-Weinberg equilibrium c)
p = 0.88
p = 0.58
p = 0.49
p = 1.0
p = 0.17
p = 1.0
Cirrhosis was diagnosed either by liver biopsy, transient elastography (stiffness > 15 kPa), and signs of portal hypertension (splenomegaly, esophageal varices, ascites). The diagnosis of HCC was made by contrast enhanced magnetic resonance imaging and computed tomography according to recently established diagnostic criteria [EASL 2009 and AASLD 2010 guidelines].
Informed consent was obtained from all patients. The study conformed to the ethical guidelines of the Helsinki declaration and had been approved by the University of Bonn ethics committee (reference number: 019/07).
Diagnosis of HCV Infection
HCV antibodies were detected with a micro particle 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).
Genomic DNA was extracted from 200 μl EDTA-blood using the QIAamp Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Determination of the DR4 gene polymorphisms C626G (rs20575) and A683C (rs20576) were performed by LightCycler real time PCR (Roche, Mannheim, Germany) using commercial LightSNiP (SimpleProbe) assays from TIB-MolBiol (Berlin, Germany) according to the manufacturer's recommendations.
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 (χ2) test and Armitage's trend test, respectively (http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl).
Statistical analysis was performed with SPSS 18.0 (SPSS, Munich, Germany). Data are given as means ± SD, unless stated otherwise. Differences between groups were analyzed by t-test and Mann-Whitney-U test as appropriate.
To take into account potentially confounding risk factors of cirrhosis (age, gender, HBV infection, alcohol and obesity), univariate comparisons (ANOVA and chi2-statistics) followed by forward conditional logistic regression were performed. Parameters with univariate effects at p < 0.1 were entered into the multivariate analysis with p < 0.05 for inclusion and p > 0.1 for exclusion as selection criterion for parameters in the final statistical model.
Prevalence of carriers with a 626C allele (genotypes 626CG and CC) (79.9%) and 626C allele frequency (52.5%) were significantly increased in the patients with HCV-associated HCC (Figure 1A). The 626C allele frequency in patients with HCV-associated HCC was significantly different only from that in healthy controls (OR = 1.556, 95%CI: 1.192-2.031, p = 0.001).
Importantly, the increased prevalence of carriers with a 626C allele reached statistical significance both with respect to healthy controls (OR = 2.106, 95% CI: 1.348-3.290, p = 0.001) and to HCV-infected patients without HCC (OR = 1.670, 95% CI: 1.034-2.696, p = 0.034) (Figure 1A).
The distribution of A683 > C variants did not differ between healthy controls and HCV-infected patients without HCC irrespective from the presence of cirrhosis (Table 1). Patients with HCV-associated HCC had the highest frequency of the 623A allele (86.5%), which was significantly increased as compared to healthy controls (A allele frequency 77.3%, OR = 1.856 95% CI: 1.291-2.667, p = 0.0006) and HCV-infected patients without HCC (A allele frequency 77.6%, OR = 1.828, 95% CI: 1.243-2.686, p = 0.002). This deviation was strong enough to achieve statistical significance even when HCV-infected subgroups with and without cirrhosis were compared separately (cirrhosis: 77.3% A allele frequency, OR = 1.852, 95% CI: 1.126-3.045, p = 0.013; without cirrhosis: 77.7% A allele frequency, OR = 1.816, 95% CI: 1.201-2.746, p = 0.004).
Regression analysis for risk factors of HCC among patients with hepatitis C genotype 1
95% Confidence interval
HCV viral load (106 IU/ml)
Obesity (BMI > 30)
C626G genotype ([CC+CG] vs. GG)
A683C genotype (AA vs. [AC+CC])
Combined 626C and 683AA risk genotype vs. all other combinations
95% Confidence interval
Combined 626C and 683AA risk genotype
To check, if our findings were specific for hepatitis C, we performed a similar analysis in our group of HCC patients, who had chronic hepatitis B. However, unlike hepatitis C-associated HCC the distributions of genetic DR4 variants in HBV-associated HCC, healthy controls and HCV-positive patients without HCC were not significantly different (Table 1). In particular, the simultaneous combination of a 626C allele with the 683AA genotype was observed in rather similar frequencies in the groups with HBV-associated HCC (50.0%), hepatitis C without HCC (49.4%) and healthy controls (47.0%) while the difference in frequency between HBV-and HCV-associated HCC (65.4%) was significant (OR = 1.891, 95% CI: 1.020-3.506, p = 0.042).
Emerging evidence suggest an important role of TRAIL for control and elimination of HCV infection. TRAIL has been implicated in the death of HCV-infected but not normal liver cells . In HCV infection its expression on natural killer cells is up-regulated by interferon, and is inversely correlated to HCV-RNA serum levels [15, 16]. TRAIL can also induce cell death in hepatic tumour cells but expression of TRAIL receptors on human hepatocellular carcinoma is variable and frequently down-regulated . Nevertheless, several studies suggest TRAIL receptor-mediated apoptosis to play a role in the elimination of tumour cells in human hepatocellular carcinoma [18, 19].
In this study we found that the TRAIL receptor I wild type with threonine at amino acid position 209 (626C) and alanine at position 228 (638A) is associated with an increased risk of HCC in patients with chronic hepatitis C. Of note, it was particularly the combination of the risk genotypes 626CC or 626CG with the homozygous 683AA genotype that was identified as a new independent predictor for HCC. Interestingly, this association of DR4 genetic variants and the risk to develop HCC was evident only for patients with chronic hepatitis C but not hepatitis B, suggesting a critical role of the aetiology underlying HCC. Both polymorphisms in the DR4 gene have already been reported to affect the risk for other types of malignancy. However, in a previous study investigating the role of the DR4 A683C polymorphism in chronic lymphocytic leukaemia, mantle cell lymphoma, prostate cancer, head and neck squamous cell carcinoma and bladder cancer an increased frequency of the CC genotype was apparently linked to malignancy . In contrast, Frank et al. observed an increased percentage of carriers with the AA genotype in patients with breast cancer . Moreover, they found an increased percentage of carriers of the 626CC genotype similar to our results. At present, it remains unclear which mechanisms underlie DR4 polymorphisms C626G and A683C to affect the risk for malignancy.
However, both amino acid exchanges are in the extracellular cysteine-rich domain of DR4. Therefore both genetic variants may lead to alterations in the TRAIL-binding domain and thus alter DR4 affinity for TRAIL. Since TRAIL signalling presumably contributes importantly to the control of HCV infection, our finding of significantly increased HCV viral loads in carriers of both DR4 risk factors suggests that TRAIL-DR4 signalling is less efficient in these patients. In line with this reasoning less efficient signalling of cell death in transformed cells and reduced susceptibility of transformed hepatocytes towards TRAIL-induced apoptosis would also facilitate HCC development. On the other hand, we cannot exclude the possibility that the increased HCC risk in HCV-infected patients carrying the C626G and A683C risk variants of TRAIL receptor I simply reflects less efficient immune control over HCV infection via TRAIL-mediated mechanisms. TRAIL is likely to affect tumour surveillance of transformed HCV-infected cells by the immune system and viral loads, once HCV infection has occurred . On the other hand our data suggest that neither of the DR4 polymorphisms affects susceptibility to HCV infection. This constellation can be explained by the fact that DR4 expression is rather low on normal liver cells while up-regulated DR4 expression following HCV infection sensitizes liver cells towards TRAIL-mediated apoptosis [14, 21]. Thus, sufficient DR4 expression on transformed liver cells appears to be a pivotal prerequisite for efficient tumour surveillance by the immune system, in line with recent clinical findings .
Therefore the role of TRAIL and DR4 polymorphisms should be further studied in patients whose risk for HCC is attributed to different aetiologies.
Here, we provide first evidence that HCV genotype 1 infected carriers of a DR4 626C allele in combination with the DR4 683AA genotype have an increased risk for HCC indicating that TRAIL and its receptors contribute importantly to control of HCV infection and tumour surveillance by the immune system. The combined presence of the two DR4 risk variants can help to identify HCV-infected patients with an increased risk for liver cancer, who may need more intensive cancer monitoring.
This work was supported by a grant from the German Federal Ministry of Education and Research (BMBF) within the network for resistance in hepatitis C, grant number (01KI0792) to JN and US. By a grant from the H.W. & J. Hector foundation, grant number (M42) to JN and by the Deutsche Krebshilfe grant number (107865) to HDN and US.
- Alter MJ: Epidemiology of hepatitis C. Hepatology. 1997, 26 (3 Suppl 1): 62S-65S.View ArticlePubMedGoogle Scholar
- Moradpour D, Blum HE: Pathogenesis of hepatocellular carcinoma. Eur J Gastroenterol Hepatol. 2005, 17 (5): 477-483. 10.1097/00042737-200505000-00002.View ArticlePubMedGoogle Scholar
- Tsukuma H, Hiyama T, Tanaka S, Nakao M, Yabuuchi T, Kitamura T, Nakanishi K, Fujimoto I, Inoue A, Yamazaki H, et al: Risk factors for hepatocellular carcinoma among patients with chronic liver disease. N Engl J Med. 1993, 328 (25): 1797-1801. 10.1056/NEJM199306243282501.View ArticlePubMedGoogle Scholar
- Altekruse SF, McGlynn KA, Reichman ME: Hepatocellular carcinoma incidence, mortality, and survival trends in the United States from 1975 to 2005. J Clin Oncol. 2009, 27 (9): 1485-1491. 10.1200/JCO.2008.20.7753.View ArticlePubMedPubMed CentralGoogle Scholar
- Schutte K, Bornschein J, Malfertheiner P: Hepatocellular carcinoma-epidemiological trends and risk factors. Dig Dis. 2009, 27 (2): 80-92. 10.1159/000218339.View ArticlePubMedGoogle Scholar
- Shepard CW, Finelli L, Alter MJ: Global epidemiology of hepatitis C virus infection. Lancet Infect Dis. 2005, 5 (9): 558-567. 10.1016/S1473-3099(05)70216-4.View ArticlePubMedGoogle Scholar
- Mellier G, Huang S, Shenoy K, Pervaiz S: TRAILing death in cancer. Mol Aspects Med. 2010, 31 (1): 93-112. 10.1016/j.mam.2009.12.002.View ArticlePubMedGoogle Scholar
- Yang A, Wilson NS, Ashkenazi A: Proapoptotic DR4 and DR5 signaling in cancer cells: toward clinical translation. Curr Opin Cell Biol. 2010, 22 (6): 837-844. 10.1016/j.ceb.2010.08.001.View ArticlePubMedGoogle Scholar
- Chen B, Liu S, Wang XL, Xu W, Li Y, Zhao WH, Wu JQ: TRAIL-R1 polymorphisms and cancer susceptibility: an evidence-based meta-analysis. Eur J Cancer. 2009, 45 (14): 2598-2605. 10.1016/j.ejca.2009.06.023.View ArticlePubMedGoogle Scholar
- Frank B, Hemminki K, Shanmugam KS, Meindl A, Klaes R, Schmutzler RK, Wappenschmidt B, Untch M, Bugert P, Bartram CR, et al: Association of death receptor 4 haplotype 626 C-683C with an increased breast cancer risk. Carcinogenesis. 2005, 26 (11): 1975-1977. 10.1093/carcin/bgi164.View ArticlePubMedGoogle Scholar
- Frank B, Shanmugam KS, Beckmann L, Hemminki K, Brenner H, Hoffmeister M, Chang-Claude J, Burwinkel B: Death receptor 4 variants and colorectal cancer risk. Cancer Epidemiol Biomarkers Prev. 2006, 15 (10): 2002-2005. 10.1158/1055-9965.EPI-06-0053.View ArticlePubMedGoogle Scholar
- Ulybina YM, Kuligina E, Mitiushkina NV, Rozanov ME, Ivantsov AO, Ponomariova DN, Togo AV, Levchenko EV, Shutkin VA, Brenister SI, et al: Coding polymorphisms in Casp5, Casp8 and DR4 genes may play a role in predisposition to lung cancer. Cancer Lett. 2009, 278 (2): 183-191. 10.1016/j.canlet.2009.01.012.View ArticlePubMedGoogle Scholar
- Wang M, Wang M, Cheng G, Zhang Z, Fu G, Zhang Z: Genetic variants in the death receptor 4 gene contribute to susceptibility to bladder cancer. Mutat Res. 2009, 661 (1-2): 85-92. 10.1016/j.mrfmmm.2008.11.009.View ArticlePubMedGoogle Scholar
- Lan L, Gorke S, Rau SJ, Zeisel MB, Hildt E, Himmelsbach K, Carvajal-Yepes M, Huber R, Wakita T, Schmitt-Graeff A, et al: Hepatitis C virus infection sensitizes human hepatocytes to TRAIL-induced apoptosis in a caspase 9-dependent manner. J Immunol. 2008, 181 (7): 4926-4935.View ArticlePubMedGoogle Scholar
- Ahlenstiel G, Edlich B, Hogdal LJ, Rotman Y, Noureddin M, Feld JJ, Holz LE, Titerence RH, Liang TJ, Rehermann B: Early Changes in Natural Killer Cell Function Indicate Virologic Response to Interferon Therapy for Hepatitis C. Gastroenterology. 2011, 141 (4): 1231-1239. 10.1053/j.gastro.2011.06.069.View ArticlePubMedPubMed CentralGoogle Scholar
- Stegmann KA, Bjorkstrom NK, Veber H, Ciesek S, Riese P, Wiegand J, Hadem J, Suneetha PV, Jaroszewicz J, Wang C, et al: Interferon-alpha-induced TRAIL on natural killer cells is associated with control of hepatitis C virus infection. Gastroenterology. 2010, 138 (5): 1885-1897. 10.1053/j.gastro.2010.01.051.View ArticlePubMedGoogle Scholar
- Kriegl L, Jung A, Engel J, Jackstadt R, Gerbes AL, Gallmeier E, Reiche JA, Hermeking H, Rizzani A, Bruns CJ, et al: Expression, cellular distribution, and prognostic relevance of TRAIL receptors in hepatocellular carcinoma. Clin Cancer Res. 2010, 16 (22): 5529-5538. 10.1158/1078-0432.CCR-09-3403.View ArticlePubMedGoogle Scholar
- Chen XP, He SQ, Wang HP, Zhao YZ, Zhang WG: Expression of TNF-related apoptosis-inducing Ligand receptors and antitumor tumor effects of TNF-related apoptosis-inducing Ligand in human hepatocellular carcinoma. World J Gastroenterol. 2003, 9 (11): 2433-2440.PubMedPubMed CentralGoogle Scholar
- He S, Chen Y, Chen X, Zhao Y, Wang H, Zhang W, Wang S: Antitumor effects of soluble TRAIL in human hepatocellular carcinoma. J Huazhong Univ Sci Technolog Med Sci. 2005, 25 (1): 51-54. 10.1007/BF02831386.View ArticlePubMedGoogle Scholar
- Wolf S, Mertens D, Pscherer A, Schroeter P, Winkler D, Grone HJ, Hofele C, Hemminki K, Kumar R, Steineck G, et al: Ala228 variant of trail receptor 1 affecting the ligand binding site is associated with chronic lymphocytic leukemia, mantle cell lymphoma, prostate cancer, head and neck squamous cell carcinoma and bladder cancer. Int J Cancer. 2006, 118 (7): 1831-1835. 10.1002/ijc.21502.View ArticlePubMedGoogle Scholar
- Zhu H, Dong H, Eksioglu E, Hemming A, Cao M, Crawford JM, Nelson DR, Liu C: Hepatitis C virus triggers apoptosis of a newly developed hepatoma cell line through antiviral defense system. Gastroenterology. 2007, 133 (5): 1649-1659. 10.1053/j.gastro.2007.09.017.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/12/85/prepub
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