|
|
|||
|
| |||
|
| |||
|
| |||
|
|
|
|
|
|
|
|
|
|
|
|
|
||
|
|
Nattiya Hirankarn, Immunology Unit, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, ThailandIngorn Kimkong, Pittaya Kummee, Inter-Department of Medical Microbiology, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand Pisit Tangkijvanich, Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand Yong Poovorawan, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand Supported by the Thailand Research Fund, RSA4680021 Correspondence to: Dr Nattiya Hirankarn, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Rama IV Road, Bangkok 10330, Thailand. fmednpt@md.chula.ac.th Telephone: +66-2-256-4000-3667 Fax: +66-2-252-5952 Received: 2005-05-10 Accepted: 2005-08-26
Abstract AIM: To examine the effect of interleukin-1-beta (IL-1β) promoter region C-511T and IL-1 receptor antagonist (IL-1RN) polymorphism among the patients with chronic hepatitis B virus (HBV) infection (HCC and non-HCC).
METHODS: Genomic DNA from 136 Thai patients with chronic HBV infection (HCC = 46 and non-HCC = 90) and 152 healthy individuals was genotyped for IL-1β gene polymorphism (-511) using polymerase chain reaction with sequence specific primers (PCR-SSP). The variable number of tandem repeats (VNTR) of IL-1RN gene was assessed by a PCR-based assay. The association between these genes and status of the disease was evaluated by χ2 test.
RESULTS: IL-1B-511 genotype C/C was found to be significantly different in patients with HCC when compared with healthy individuals (P = 0.036, OR = 2.29, 95%CI = 1.05-4.97) and patients without HCC (P=0.036, OR = 2.52, 95%CI=1.05-6.04). Analysis of allele frequencies of IL-1B-511 showed that IL-1B-511 C allele was also significantly increased in patients with HCC, compared to that in healthy control (P = 0.033, OR = 1.72, 95%CI=1.04-2.84). However, no significant association in IL-1RN gene was found between the two groups.
CONCLUSION: IL-1B-511C allele, which may be associated with high IL-1B production in the liver, is a genetic marker for the development of HCC in chronic hepatitis B patients in Thai population.
© 2006 The WJG Press. All rights reserved.
Key words: Interleukin-1 beta gene; Polymorphism; Hepatocellular carcinoma; Hepatitis B
Hirankarn N, Kimkong I, Kummee P, Tangkijvanich P, Poovorawan Y. Interleukin-1β gene polymorphism associated with hepatocellular carcinoma in hepatitis B virus infection. World J Gastroenterol 2006; 12(5): 776-779 http://www.wjgnet.com/1007-9327/12/776.asp
INTRODUCTION Hepatitis B virus (HBV) is the most common cause of acute and chronic liver disease worldwide, especially in Asia and Africa. It has been estimated that more than 350 million people worldwide, representing more than 5% of world population are carriers of HBV infection[1,2]. Approximately 250 000 deaths occur each year resulting from fulminant hepatic failure, cirrhosis, and hepatocellular carcinoma (HCC)[3]. Family studies in China provide some evidence that the host genetic factors influence viral persistence, as a higher concordance rate has been found for HBeAg persistence in identical twins compared with non-identical twins[4]. Thus, it is conceivable that genetic difference may affect the different outcomes of patients with HBV infection. Several genetic studies on the hosts have reported that human leukocyte antigen (HLA) genes[5-7] and various cytokine genes (TNF-α, IFN-γ, IL-12, IL-18, TGF-β, IL-10, IL-4) are associated with HBV susceptibility and/or HBV persistence or disease severity[8-11]. Interleukin-1 (IL-1) is a proinflammatory cytokine with multiple biological effects[12]. The IL-1 gene family (including IL-1A, IL-1B, and IL-1RN) on chromosome 2q13-21 encodes three proteins, which comprise the agonists IL-1α, IL-1β, and their naturally occurring inhibitor, IL-1 receptor antagonist (IL-1RN)[12,13]. Recently, allele 2 of IL-1RN intron 2 has been reported as a resistant marker of HBV infection, suggesting the role of IL-1 polymorphisms in the pathogenesis of developing chronic hepatitis B[14]. This allele is associated with enhanced IL-1β production in vitro[15] and in vivo[16]. Therefore, Zhang et al [14] hypothesized that high production of IL-1β may help increase the production of other cytokines such as IL-2, IL-6, and TNF-α and trigger the complex immunological processes to eliminate the virus. Interestingly, besides its major role as a proinflammatory cytokine, IL-1β has been implicated as an important factor for tumor growth[17-19]. Several independent lines of evidence have also suggested that genetic polymorphisms within IL-1β gene are associated with gastric cancer and HCC in HCV infection[20-22]. The aim of the present study was to determine the genotype and allele frequencies of IL-1B-511 and IL-1RN VNTR polymorphisms among the Thai patients with chronic HBV infection (HCC and non-HCC) and healthy individuals to assess whether these genes are involved in chronic HBV susceptibility and/or HCC development.
MATERIALS AND METHODS Subjects One hundred and thirty-six Thai patients with chronic HBV infection were recruited into this study from Chulalongkorn Memorial Hospital. The diagnosis of chronic hepatitis B was established by seropositivity for HBsAg over a 6-month period and did not have any other type of liver diseases such as chronic hepatitis C or alcoholic liver disease. In addition, all the patients had elevated serum ALT and AST levels. Patients with chronic HBV infection were further divided into two groups: without (n = 90) and with HCC (n = 46) according to the absence or presence of concurrent HCC. Diagnosis of HCC was based on histopathology and/or a combination of mass lesion in the liver from hepatic imaging and serum alpha fetoprotein level > 400 ng/mL. Moreover, 152 ethnically and geographically matched controls from healthy blood donors of the Thai Red Cross Society were recruited.
Genotyping for SNPs of IL-1B and IL-1RN genes Molecular genetic analysis was performed on genomic DNA obtained from peripheral blood leukocytes using standard salting-out method as previously described[23]. SNP at position -511 of IL-1B was genotyped by polymerase chain reaction (PCR) with sequence specific primer (PCR-SSP) (F-5’ CTCATCTGGCATTGATCTGG-3’ and R-5’ GGTGCTGTTCTCTGCCTCGA-3’)[24]. The PCR conditions were established as previously described[25]. The VNTR of IL-1RN gene was assessed by a PCR-based assay. Oligonucleotides F-5’ CTCAGCAACACTCCTAT-3’ and R-5’ TCCTGGTCTGCAGGTAA-3’ flanking this region were used as primers[24]. The PCR conditions were an initial denaturation at 94 °C for 2 min, followed by 35 cycles at 94 °C for 20, at 59 °C for 50 s, at 72 °C for 20 s, and a final extension at 72 °C for 7 min. Each allele was identified according to its size[24].
Statistical analysis The association between these genes and disease status was evaluated by the statcalc from Epi info version 6 program[26] to calculate the odds ratio (OR) and 95% confidence interval (CI), Yates’ corrected χ2 and associated P values. P < 0.05 was considered statistically significant. The groups were tested for conformity to the Hardy-Weinberg equilibrium by 2 × 2 χ2 test comparing observed and expected numbers.
RESULTS The genotype and allele frequencies of IL-1B and IL-1RN in healthy control subjects and patients with chronic hepatitis B, including patients with and without HCC are shown in Table 1. All the three groups were in Hardy-Weinberg equilibrium with no significant χ2 values compared to the observed and expected genotype frequencies of each of the tested polymorphisms. The heterozygous C/T of IL-1B was the most common genotype in all the three groups (51.97% in healthy controls, 45.65% in patients with HCC and 56.67% in patients without HCC). The homozygous T/T was the second most common genotype in healthy controls (27.63%) and patients without HCC (24.44%), followed by the C/C genotype that was found in 20.39% of healthy controls and in 18.89% of patients without HCC, respectively. In contrast to these two groups, the C/C was the second most common genotype in patients with HCC (36.96%), followed by the T/T genotype (17.39%). Comparison of IL-1B-511 genotype revealed that IL-1B-511 C/C genotype was significantly increased in patients with HCC compared to that in healthy controls (P = 0.036, OR = 2.29, 95%CI = 1.05-4.97) and patients without HCC (P = 0.036, OR = 2.52, 95%CI = 1.05-6.04). Analysis of allele frequencies of IL-1B-511 showed that IL-1B-511 C allele was also significantly increased in patients with HCC, compared to that in the healthy controls (P = 0.033, OR = 1.72, 95%CI = 1.04-2.84). The effect of IL-1B-511 C allele was similar to autosomal recessive mode of inheritance. The presence of two C alleles (CC) was required to increase the likelihood of HCC development.
Table 1 IL-1β and IL-1RN polymorphisms in patients with HBV and healthy controls
Four genotypes of IL-1RN (1/1, 1/2, 2/2, 1/4) were found in this study. The IL-1RN 1/1 genotype was the most common genotype in all the three groups (79.61% in healthy controls, 82.61% in patients with HCC and 82.22% in patients without HCC), followed by 1/2 genotype that was 19.08% in healthy controls, 17.39% in patients with HCC and 16.67% in patients without HCC, respectively. The 2/2 genotype was 0.66% in healthy control and 1.11% in patients without HCC, whereas the 1/4 genotype was 0.66% in healthy controls. There were no significant differences in genotype or allele frequencies of IL-1RN between patients with chronic hepatitis B and healthy controls.
DISCUSSION The association between the development of chronic hepatitis B and the polymorphisms of IL-1B gene (-511C/T) and IL-1RN gene (VNTR at intron 2) was not observed in this study. However, the -511C allele of IL-1B gene was identified as a genetic marker for the development of HCC in patients with chronic HBV infection. The hypothesis regarding IL-1 genetic polymorphism and hepatocarcinogenesis is based on the assumption that carriers of these genotypes are associated with increased levels of IL-1B in the liver in response to HBV infection and hepatocyte damage that may finally lead to the development of HCC. This hypothesis is supported by the observation that IL-1B level is increased in the liver tissue surrounding the tumor tissue[27]. IL-1B is a proinflammatory cytokine as well as a tumor growth factor. There are several lines of evidence that support its role in tumor growth development. First, IL-1B can increase the production of prostaglandin E2 and hepatocyte growth factor[17]. Second, IL-1B can induce angiogenesis which is an important step in promoting tumor growth by either upregulating COX-2 or inducing nitric oxide[19] and vascular endothelial growth factor (VEGF)[28]. Third, IL-1B can also attenuate interferon-induced antiviral activity and STAT1 activation in the liver[29]. However, the IL-1B polymorphism in the promoter area in association with cancer remains controversial. Briefly, a number of studies mostly in the Caucasian population support that -511T in linkage disequilibrium with -31C is a risk haplotype for the development of gastric cancer[30-34]. There are more conflicting data regarding the effect of IL-1B-511/-31 haplotype on the risk of gastric cancer and HCV-related HCC in Asian population, while some studies support the result from the Caucasian group[16,21,35] and a number of studies reported that -511C/-31T is a risk haplotype for the development of cancer[22,36-40]. Interestingly, functional studies of IL-1B genotype seem to support that -511C/-31T haplotype is associated with high-production of IL-1B. First, the IL-1B -31 polymorphism involves a TATA sequence in the promoter and the -31T allele is associated with a five-fold elevated binding activity with the transcription initiation factor[30,31]. Second, mucosal IL-1B level is higher than IL-1B-31T level in H pylori-infected gastric cancer patients[40]. Although IL-1B -511T/-31C is associated with high level of IL-1B in the plasma[41], it is likely that gene expression in each organ is differently regulated and the assessment of IL-1B level in targeted organ is more reliable. The functional study in liver tissue is also required for the better understanding of the role of IL-1B genotype in chronic hepatitis and HCC development. In conclusion, IL-1B-511C allele which may be associated with the high IL-1B production in the liver is a genetic marker for the development of HCC in chronic hepatitis B patients in Thai population.
ACKNOWLEDGMENTS The authors greatly appreciate the participants and all the staff members who participated in chronic HBV study at King Chulalongkorn Memorial Hospital. Also, we would like to thank the National Blood Center for the recruitment of healthy controls and collection of research materials.
References 1 Lee WM. Hepatitis B virus infection. N Engl J Med 1997; 337: 1733-1745 2 Andre FE, Zuckerman AJ. Review: protective efficacy of hepatitis B vaccines in neonates. J Med Virol 1994; 44: 144-151 3 Perrillo RP. How will we use the new antiviral agents for hepatitis B? Curr Gastroenterol Rep 2002; 4: 63-71
4 Lin TM, Chen CJ, Wu MM, Yang CS, Chen
JS, Lin CC, Kwang TY, Hsu ST, Lin SY, Hsu LC. Hepatitis B virus
markers in 5 van Hattum J, Schreuder GM, Schalm SW. HLA antigens in patients with various courses after hepatitis B virus infection. Hepatology 1987; 7: 11-14 6 Almarri A, Batchelor JR. HLA and hepatitis B infection. Lancet 1994; 344: 1194-1195 7 Ahn SH, Han KH, Park JY, Lee CK, Kang SW, Chon CY, Kim YS, Park K, Kim DK, Moon YM. Association between hepatitis B virus infection and HLA-DR type in Korea. Hepatology 2000; 31: 1371-1373 8 Hohler T, Kruger A, Gerken G, Schneider PM, Meyer zum Buschenefelde KH, Rittner C. A tumor necrosis factor-alpha (TNF-alpha) promoter polymorphism is associated with chronic hepatitis B infection. Clin Exp Immunol 1998; 111: 579-582
9 Kim YJ, Lee HS, Yoon JH, Kim CY, Park MH,
Kim LH, Park BL, Shin HD. Association of TNF-alpha promoter
polymorphisms
10 Ben-Ari Z, Mor E, Papo O, Kfir B,
Sulkes J, Tambur AR, Tur-Kaspa R, Klein T. Cytokine gene
polymorphisms in patients
11 Nieters A, Yuan JM, Sun CL, Zhang ZQ,
Stoehlmacher J, Govindarajan S, Yu MC. Effect of cytokine genotypes
on the 12 Dinarello CA. Biologic basis for interleukin-1 in disease. Blood 1996; 87: 2095-2147 13 Nicklin MJ, Weith A, Duff GW. A physical map of the region encompassing the human interleukin-1 alpha, interleukin-1 beta, and interleukin-1 receptor antagonist genes. Genomics 1994; 19: 382-384 14 Zhang PA, Li Y, Xu P, Wu JM. Polymorphisms of interleukin-1B and interleukin-1 receptor antagonist genes in patients with chronic hepatitis B. World J Gastroenterol 2004; 10: 1826-1829 15 Santtila S, Savinainen K, Hurme M. Presence of the IL-1RA allele 2 (IL1RN*2) is associated with enhanced IL-1beta production in vitro. Scand J Immunol 1998; 47: 195-198 16 Hwang IR, Kodama T, Kikuchi S, Sakai K, Peterson LE, Graham DY, Yamaoka Y. Effect of interleukin 1 polymorphisms on gastric mucosal interleukin 1beta production in Helicobacter pylori infection. Gastroenterology 2002; 123: 1793-1803 17 Bamba H, Ota S, Kato A, Matsuzaki F. Nonsteroidal anti-inflammatory drugs may delay the repair of gastric mucosa by suppressing prostaglandin-mediated increase of hepatocyte growth factor production. Biochem Biophys Res Commun 1998; 245: 567-571 18 Roshak AK, Jackson JR, McGough K, Chabot-Fletcher M, Mochan E, Marshall LA. Manipulation of distinct NFkappaB proteins alters interleukin-1beta-induced human rheumatoid synovial fibroblast prostaglandin E2 formation. J Biol Chem 1996; 271: 31496-31501 19 Rahman MA, Dhar DK, Yamaguchi E, Maruyama S, Sato T, Hayashi H, Ono T, Yamanoi A, Kohno H, Nagasue N. Coexpression of inducible nitric oxide synthase and COX-2 in hepatocellular carcinoma and surrounding liver: possible involvement of COX-2 in the angiogenesis of hepatitis C virus-positive cases. Clin Cancer Res 2001; 7: 1325-1332 20 Lee KA, Ki CS, Kim HJ, Sohn KM, Kim JW, Kang WK, Rhee JC, Song SY, Sohn TS. Novel interleukin 1beta polymorphism increased the risk of gastric cancer in a Korean population. J Gastroenterol 2004; 39: 429-433 21 Tanaka Y, Furuta T, Suzuki S, Orito E, Yeo AE, Hirashima N, Sugauchi F, Ueda R, Mizokami M. Impact of interleukin-1beta genetic polymorphisms on the development of hepatitis C virus-related hepatocellular carcinoma in Japan. J Infect Dis 2003; 187: 1822-1825 22 Wang Y, Kato N, Hoshida Y, Yoshida H, Taniguchi H, Goto T, Moriyama M, Otsuka M, Shiina S, Shiratori Y, Ito Y, Omata M. Interleukin-1beta gene polymorphisms associated with hepatocellular carcinoma in hepatitis C virus infection. Hepatology 2003; 37: 65-71 23 Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988; 16: 1215 24 Hutyrova B, Pantelidis P, Drabek J, Zurkova M, Kolek V, Lenhart K, Welsh KI, Du Bois RM, Petrek M. Interleukin-1 gene cluster polymorphisms in sarcoidosis and idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2002; 165: 148-151 25 Bunce M, O’Neill CM, Barnardo MC, Krausa P, Browning MJ, Morris PJ, Welsh KI. Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP). Tissue Antigens 1995; 46: 355-367 26 Center for Disease Control and Prevention. Epi info version 6 Program, online, 1994, cited 2004-08-15. Available from: URL: http//www.cdc.gov/epiinfo/EI6dnjp.htm 27 Bortolami M, Venturi C, Giacomelli L, Scalerta R, Bacchetti S, Marino F, Floreani A, Lise M, Naccarato R, Farinati F. Cytokine, infiltrating macrophage and T cell-mediated response to development of primary and secondary human liver cancer. Dig Liver Dis 2002; 34: 794-801 28 Ben-Av P, Crofford LJ, Wilder RL, Hla T. Induction of vascular endothelial growth factor expression in synovial fibroblasts by prostaglandin E and interleukin-1: a potential mechanism for inflammatory angiogenesis. FEBS Lett 1995; 372: 83-87 29 Tian Z, Shen X, Feng H, Gao B. IL-1 beta attenuates IFN-alpha beta-induced antiviral activity and STAT1 activation in the liver: involvement of proteasome-dependent pathway. J Immunol 2000; 165: 3959-3965 30 El-Omar EM, Carrington M, Chow WH, McColl KE, Bream JH, Young HA, Herrera J, Lissowska J, Yuan CC, Rothman N, Lanyon G, Martin M, Fraumeni JF Jr, Rabkin CS. Interleukin-1 polymorphisms associated with increased risk of gastric cancer. Nature 2000; 404: 398-402 31 El-Omar EM, Carrington M, Chow WH, McColl KE, Bream JH, Young HA, Herrera J, Lissowska J, Yuan CC, Rothman N, Lanyon G, Martin M, Fraumeni JF Jr, Rabkin CS. The role of interleukin-1 polymorphisms in the pathogenesis of gastric cancer. Nature 2001; 412: 99 32 Figueiredo C, Machado JC, Pharoah P, Seruca R, Sousa S, Carvalho R, Capelinha AF, Quint W, Caldas C, van Doorn LJ, Carneiro F, Sobrinho-Simoes M. Helicobacter pylori and interleukin 1 genotyping: an opportunity to identify high-risk individuals for gastric carcinoma. J Natl Cancer Inst 2002; 94: 1680-1687 33 Machado JC, Pharoah P, Sousa S, Carvalho R, Oliveira C, Figueiredo C, Amorim A, Seruca R, Caldas C, Carneiro F, Sobrinho-Simoes M. Interleukin 1B and interleukin 1RN polymorphisms are associated with increased risk of gastric carcinoma. Gastroenterology 2001; 121: 823-829 34 Machado JC, Figueiredo C, Canedo P, Pharoah P, Carvalho R, Nabais S, Castro Alves C, Campos ML, Van Doorn LJ, Caldas C, Seruca R, Carneiro F, Sobrinho-Simoes M. A proinflammatory genetic profile increases the risk for chronic atrophic gastritis and gastric carcinoma. Gastroenterology 2003; 125: 364-371 35 Furuta T, El-Omar EM, Xiao F, Shirai N, Takashima M, Sugimura H. Interleukin 1beta polymorphisms increase risk of hypochlorhydria and atrophic gastritis and reduce risk of duodenal ulcer recurrence in Japan. Gastroenterology 2002; 123: 92-105 36 Kato S, Onda M, Yamada S, Matsuda N, Tokunaga A, Matsukura N. Association of the interleukin-1 beta genetic polymorphism and gastric cancer risk in Japanese. J Gastroenterol 2001; 36: 696-699 37 Hamajima N, Matsuo K, Saito T, Tajima K, Okuma K, Yamao K, Tominaga S. Interleukin 1 polymorphisms, lifestyle factors, and Helicobacter pylori infection. Jpn J Cancer Res 2001; 92: 383-389 38 Matsukura N, Yamada S, Kato S, Tomtitchong P, Tajiri T, Miki M, Matsuhisa T, Yamada N. Genetic differences in interleukin-1 betapolymorphisms among four Asian populations: an analysis of the Asian paradox between H. pylori infection and gastric cancer incidence. J Exp Clin Cancer Res 2003; 22: 47-55 39 Yang J, Hu Z, Xu Y, Shen J, Niu J, Hu X, Guo J, Wei Q, Wang X, Shen H. Interleukin-1B gene promoter variants are associated with an increased risk of gastric cancer in a Chinese population. Cancer Lett 2004; 215: 191-198 40 Chang YW, Jang JY, Kim NH, Lee JW, Lee HJ, Jung WW, Dong SH, Kim HJ, Kim BH, Lee JI, Chang R. Interleukin-1B (IL-1B) polymorphisms and gastric mucosal levels of IL-1beta cytokine in Korean patients with gastric cancer. Int J Cancer 2005; 114: 465-471 41 Hulkkonen J, Laippala P, Hurme M. A rare allele combination of the interleukin-1 gene complex is associated with high interleukin-1 beta plasma levels in healthy individuals. Eur Cytokine Netw 2000; 11: 251-255
|
|
|
|
|
PubMed