Abstract
We conducted a case-control study to investigate the association between three common SNPs in IL-10 gene (rs1800896, rs1800871 and rs1800872) and the development of liver cirrhosis in a Chinese population. Between January 2013 and December 2014, a total of 318 patients with liver cirrhosis and 318 health control subjects were enrolled into our study. The IL-10 rs1800896, rs1800871 and rs1800872 polymorphisms were analyzed using polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP). By multivariate logistic regression analysis, we found that individuals with the AA genotype and GA+AA genotype of IL-10 rs1800896 were more likely to have an increased risk of liver cirrhosis when compared with the GG genotype, and the ORs (95% CI) for the AA genotype and GA+AA genotype were 2.04 (1.20-3.50) and 1.41 (1.02-1.96), respectively. We found that the GA+AA genotype of IL-10 rs1800896 had higher risk of liver cirrhosis in individuals with chronic hepatitis B when compared with the GG genotype (OR = 1.95, 95% CI = 1.01-3.59). In conclusion, we found that IL-10 rs1800896 polymorphism was correlated with an increased risk of liver cirrhosis, especially in individuals with chronic hepatitis B.
Keywords: Interleukin-10, polymorphism, liver cirrhosis
Introduction
Liver cirrhosis is a severe public health problem worldwide, which is correlated with higher morbidity and mortality worldwide [1-3]. It is reported that about 2 billion population are infected with HBV, and 3/4 of them are in Asia-Pacific region [4]. It is well known that long-term infection with HBV is associated with the development of liver cirrhosis, and other lifestyle factors may influence the susceptibility to liver cirrhosis, such as long-term heavy alcohol drinking [5,6]. However, not all of the individuals who exposed to long-term infection with HBV and heavy alcohol drinking would suffer from liver cirrhosis, which suggests that molecular and cellular may contribute to the development of liver cirrhosis.
It is reported that cytokines play a fundamental role in the immunopathogenesis of HBV related diseases [7-9]. Previous studies have reported that cytokines gene polymorphisms, such as Interleukin-1β (IL-1β), IL-28B, IL-17 and IL-35, are associated with the development of HBV-related liver cirrhosis [10-12]. Interleukin-10 (IL-10) is an immunoregulatory cytokine, which is produced by Th2 cells, regulatory T cells, and monocytes/macrophages. The encoding gene of IL-10 is located on chromosome 1 (1q31-1q32). IL-10 is an anti-inflammatory cytokine, which could inhibit the synthesis of cytokines such as IL-6, IL-1β, IL-1α and TNF-α in activated macrophage and IFNγ by T cells [13]. Few studies investigated the role of IL-10 gene polymorphisms in the susceptibility to liver cirrhosis, and the results were inconsistent [14-17]. Therefore, we conducted a case-control study to investigate the association between three common SNPs in IL-10 gene (rs1800896, rs1800871 and rs1800872) and the development of liver cirrhosis in a Chinese population.
Materials and methods
Study subjects
A case-control study was conducted in our study. Between January 2013 and December 2014, a total of 352 patients with liver cirrhosis were enrolled into our study. Liver cirrhosis was diagnosed either by histopathologically diagnosis, ultrasound, computer tomography (CT) or magnetic resonance imaging (MRI). Patients who had organ transplantation were excluded from this study. Finally, 318 patients were enrolled in our study, and the participation rate was 90.35%.
For the frequency-matched controls on sex and age, 318 health control subjects were randomly selected from individuals who came to our hospital for health check-up. Controls that had a history of liver cirrhosis and other HBV-related diseases were excluded from our study. All patients with liver cirrhosis and control subjects signed written informed consents. The signed written informed consents were obtained from patients with liver cirrhosis and control subjects. Our study was approved by the ethics committee of our hospital.
Genotyping assays
Each subject was asked to provide 5 ml peripheral blood samples, and the blood samples were kept in -80°C before DNA extraction. Genomic DNA was selected from 5 mL peripheral blood of each sample, using the TIANamp Blood DNA Kit (Tiangen Biotech, Beijing, China). Therefore, the DNA was stored at -80°C for following genotype analysis. DNA was extracted from peripheral blood samples 100 collected from patients and controls using the TIANamp Blood DNA Kit (Tiangen, Beijing, China), according to the manufacturer’s instructions. After extraction, genomic DNA was diluted to a final concentration of 15-20 ng/µl for the genotyping assays. The IL-10 rs1800896, rs1800871 and rs1800872 polymorphisms were analyzed using polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP). Primers for amplification and extension reactions were designed using Sequenom Assay Design 3.1 software (Sequenom, San Diego, CA, USA). The Primers for IL-10 rs1800896, rs1800871 and rs1800872 were as follows: for IL-10 rs1800896, 5’-CTACTAAGGCTTCTTTGGGAG-3’ (forward) and 5’-ACTACTAAGGCTTCTTTGGGAA-3’ (reverse); for rs1800871, 5’-TCATTCTATGTGCTGGAGATGG-3’ (forward) and 5’-TGGGGGAAGTGGGTAAGAGT-3’ (reverse); for rs1800872, 5’-GGTGAGCACTACCTGACTAGC-3’ (forward) and 5’-CCTAGGTCACAGTGACGTGG-3’ (reverse). The restriction enzymes for IL-10 rs1800896, rs1800871 and rs1800872 were BseRI, MsII and RsaI, respectively. The amplification conditions were 95°C for 5 min, then 30 cycles of 94°C for 0.5 min, 60°C for 0.5 min and 72°C for 1 min, at last 72°C for 10 min. Digestion products were separated by electrophoresis on ethidium bromide stained agarose gel and visualized under UV light.
Statistical methods
The statistical difference between cases and controls was analyzed by A Chi-squared test and t test. The Hardy-Weinberg equilibrium (HWE) was tested by Fisher’s exact test for each SNP in controls. The odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using logistic regression models adjusted for confounding factors for genotype analysis. Additional regression models were designed where subjects were stratified on drinking status, chronic hepatitis B status and genetic common type and polymorphism carriers of the studied variations. A P-value of less than 0.05 was considered to be statistically significant. All statistical analyses were analyzed using the SPSS statistical package software, version 17.0 (SPSS Inc, Chicago, IL, USA).
Results
Population characteristics
The demographic and clinical characteristics of patients with liver cirrhosis and controls were shown in Table 1. There were no significant differences between patients and controls in terms of age and sex (P > 0.05). By Chi-squared test, we found that patients with liver cirrhosis were more likely to be infected with chronic hepatitis B and chronic hepatitis C, and have a habit of alcohol drinking. Of 318 patients, 163 (51.26%) were at A of Child-Pugh score, 128 (40.25%) were at B and 27 (8.49%) were at C.
Table 1.
Characteristics of included patients with liver cirrhosis and controls
| Variables | Patients | % | Controls | % | t or χ2 | P value |
|---|---|---|---|---|---|---|
| Age, years | ||||||
| < 55 | 150 | 47.17 | 157 | 49.37 | ||
| ≥ 55 | 168 | 52.83 | 161 | 50.63 | 0.31 | 0.58 |
| Sex | ||||||
| Females | 100 | 31.45 | 100 | 31.45 | ||
| Males | 218 | 68.55 | 218 | 68.55 | 0.00 | 1.00 |
| Chronic hepatitis B | ||||||
| No | 148 | 46.54 | 291 | 91.51 | ||
| Yes | 170 | 53.46 | 27 | 8.49 | 118.71 | < 0.001 |
| Chronic hepatitis C | ||||||
| No | 293 | 92.14 | 315 | 99.06 | ||
| Yes | 25 | 7.86 | 3 | 0.94 | 18.09 | < 0.001 |
| Alcohol status | ||||||
| No | 132 | 41.51 | 253 | 79.56 | ||
| Yes | 186 | 58.49 | 65 | 20.44 | 96.36 | < 0.001 |
| Child-Pugh score | ||||||
| A | 163 | 51.26 | ||||
| B | 128 | 40.25 | ||||
| C | 27 | 8.49 |
The genotype distributions of IL-10 rs1800896, rs1800871 and rs1800872 were in line with HWE, and the P values for HWE were 0.90, 0.23 and 0.65, respectively (Table 2). The genotype distribution of IL-10 rs1800896 showed significant difference between patients with liver cirrhosis and controls (χ2 = 8.08, P value = 0.02). By multivariate logistic regression analysis, we found that individuals with the AA genotype and GA+AA genotype of IL-10 rs1800896 were more likely to have an increased risk of liver cirrhosis when compared with the GG genotype, and the ORs (95% CI) for the AA genotype and GA+AA genotype were 2.04 (1.20-3.50) and 1.41 (1.02-1.96), respectively. Moreover, there was no significant association between IL-10 rs1800871 and rs1800872 polymorphisms and development of liver cirrhosis.
Table 2.
Association between IL-10 rs1800896, rs1800871 and rs1800872 polymorphisms and risk of liver cirrhosis
| SNPs | Patients | % | Controls | % | HWE | OR (95% CI) | P value |
|---|---|---|---|---|---|---|---|
| rs1800896 | |||||||
| GG | 125 | 39.31 | 152 | 47.80 | 0.90 | 1.0 (Ref.) | - |
| GA | 141 | 44.34 | 135 | 42.45 | 1.27 (0.90-1.80) | 0.16 | |
| AA | 52 | 16.35 | 31 | 9.75 | 2.04 (1.20-3.50) | 0.005 | |
| GA+AA | 193 | 60.69 | 166 | 52.20 | 1.41 (1.02-1.96) | 0.03 | |
| rs1800871 | |||||||
| TT | 128 | 40.25 | 139 | 43.71 | 0.23 | 1.0 (Ref.) | - |
| CT | 139 | 43.71 | 135 | 42.45 | 1.12 (0.79-1.59) | 0.52 | |
| CC | 51 | 16.04 | 44 | 13.84 | 1.26 (0.77-2.07) | 0.34 | |
| CT+CC | 190 | 59.75 | 179 | 56.29 | 1.15 (0.83-1.60) | 0.38 | |
| rs1800872 | |||||||
| AA | 110 | 34.59 | 122 | 38.36 | 0.65 | 1.0 (Ref.) | - |
| AC | 159 | 40.25 | 153 | 48.11 | 1.15 (0.81-1.64) | 0.41 | |
| CC | 49 | 25.16 | 43 | 13.52 | 1.26 (0.76-2.11) | 0.34 | |
| AC+CC | 208 | 65.41 | 196 | 61.64 | 1.18 (0.84-1.65) | 0.32 |
We conducted analysis on the association between IL-10 rs1800896 polymorphism and risk of liver cirrhosis stratified by drinking and chronic hepatitis B status (Table 3). We found that the GA+AA genotype of IL-10 rs1800896 had higher risk of liver cirrhosis in individuals with chronic hepatitis B when compared with the GG genotype (OR = 1.95, 95% CI = 1.01-3.59). However, no significant interaction was found between IL-10 rs1800896 polymorphism and alcohol drinking in the risk of liver cirrhosis.
Table 3.
Association between IL-10 rs1800896 polymorphism and risk of liver cirrhosis stratified by drinking and chronic hepatitis B status
| Variables | GG | GA+AA | OR (95% CI) | P value | ||
|---|---|---|---|---|---|---|
|
| ||||||
| Patients | Controls | Patients | Controls | |||
| Chronic hepatitis B | 69 | 137 | 79 | 154 | 1.02 (0.67-1.55) | 0.93 |
| No | 56 | 15 | 114 | 12 | 2.54 (1.03-6.36) | 0.02 |
| Yes | ||||||
| Alcohol drinking | ||||||
| No | 52 | 118 | 80 | 135 | 1.34 (0.86-2.11) | 0.17 |
| Yes | 73 | 34 | 113 | 31 | 1.70 (0.92-3.12) | 0.07 |
Discussion
Genetic susceptibility to cancers has obtained a growing attention to investigate the gene polymorphisms in the development of several diseases. Inflammation and related cytokines play an important role in activating stellate cells and causing liver fibrosis [17], and the inflammatory responses of immune cells and following cytokine expression in the liver could contribute to the development of liver cirrhosis. In our study, we found that IL-10 rs1800896 polymorphism was correlated with an increased risk of liver cirrhosis, and had interaction with chronic hepatitis B in the pathogenesis of liver cirrhosis.
Previous studies have reported that IL-10 gene polymorphisms are associated with the development of hepatocellular carcinoma, and could influence the long-term infection of chronic hepatitis B and C [18-21]. Gao et al. assessed the relationship between the polymorphisms of IL-10 gene at position rs1800896 and rs1800872 and long-term infection of HBV and/or HCV, and they reported that IL-10 rs1800896 polymorphism could influence the chronic infection of HBV and HCV replication [18]. Gong et al. conducted a study in a Chinese population, and they indicated that IL-10-producing regulatory B cells were associated with the development of impaired anti-HBV immunity and the pathogenesis of chronic hepatitis B [19]. Ren et al. conducted a meta-analysis with 24 studies, and they reported that IL-10 rs1800872 was associated with an increased susceptibility to HBV infection, but IL-10 rs1800896 and rs1800871 were not [20]. Saxena et al. reported that IL-10 rs1800871 and rs1800872 gene polymorphisms were associated with the progression of HBV infection related disease, and they could promote the inactive carrier state to malignancy in an Indian population [21]. In our study, we found that IL-10 rs1800896 polymorphism had interaction with chronic hepatitis B in the pathogenesis of liver cirrhosis, which is in line with previous studies.
In our study, we found that IL-10 rs1800896 polymorphism was correlated with an increased risk of liver cirrhosis. Several studies reported their association and the results are inconsistent [14,16,17,20]. Yang et al. conducted a study in a Taiwanese population, and they reported that the IL-10 promoter haplotype is correlated with alcoholic liver cirrhosis [17]. Jin et al. suggested that IL-10 rs1800872 gene polymorphism would promote the risk for liver cirrhosis, and this gene has interaction with chronic hepatitis B infection in the development of liver cirrhosis [16]. Liu et al. reported the association between IL-10 rs1800896, rs1800871 and rs1800872 polymorphisms and risk of liver cirrhosis, and they found that IL-10 rs1800896 polymorphism slightly enhanced the risk of liver cirrhosis [14]. The discrepancies of the above mentioned results could be explained by differences in ethnicities, study design and disease status as well as sample size.
There were several limitations in our study. First, selection bias may exist in our study due to the hospital-based subjects in our study, but the matched on age and sex could reduce the bias in this study. Second, the sample size of our study is relatively small, which may limit the statistical power to find difference between groups. Therefore, further large-scale studies in different ethnic groups are greatly needed to confirm our results.
In conclusion, we found that IL-10 rs1800896 polymorphism was correlated with an increased risk of liver cirrhosis; especially in individuals with chronic hepatitis B. Future studies with larger sample size may contribute to elucidate the impact of IL-10 polymorphisms on the risk of liver cirrhosis.
Disclosure of conflict of interest
None.
References
- 1.Hsiang JC, Bai WW, Raos Z, Stableforth W, Upton A, Selvaratnam S, Gane EJ, Gerred SJ. Epidemiology, disease burden and outcomes of cirrhosis in a large secondary care hospital in South Auckland, New Zealand. Intern Med J. 2015;45:160–9. doi: 10.1111/imj.12624. [DOI] [PubMed] [Google Scholar]
- 2.Scaglione S, Kliethermes S, Cao G, Shoham D, Durazo R, Luke A, Volk ML. The Epidemiology of Cirrhosis in the United States: A Population-based Study. J Clin Gastroenterol. 2015;49:690–6. doi: 10.1097/MCG.0000000000000208. [DOI] [PubMed] [Google Scholar]
- 3.Damgaard Sandahl T. Alcoholic hepatitis. Dan Med J. 2014;61:B4755. [PubMed] [Google Scholar]
- 4.Liaw YF, Chu CM. Hepatitis B virus infection. Lancet. 2009;373:582–92. doi: 10.1016/S0140-6736(09)60207-5. [DOI] [PubMed] [Google Scholar]
- 5.Ansari N, Makvandi M, Samarbaf-Zadeh AR. Hepatitis B virus Genotyping Among patients with cirrhosis. Jundishapur J Microbiol. 2015;8:e14571. doi: 10.5812/jjm.14571. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Lin CW, Lin CC, Mo LR, Chang CY, Perng DS, Hsu CC, Lo GH, Chen YS, Yen YC, Hu JT, Yu ML, Lee PH, Lin JT, Yang SS. Heavy alcohol consumption increases the incidence of hepatocellular carcinoma in hepatitis B virus-related cirrhosis. J Hepatol. 2013;58:730–5. doi: 10.1016/j.jhep.2012.11.045. [DOI] [PubMed] [Google Scholar]
- 7.Yao JY, Chao K, Li MR, Wu YQ, Zhong BH. Interleukin-21 gene polymorphisms and chronic hepatitis B infection in a Chinese population. World J Gastroenterol. 2015;21:4232–9. doi: 10.3748/wjg.v21.i14.4232. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Shi X, Chi X, Pan Y, Gao Y, Li W, Yang C, Zhong J, Xu D, Zhang M, Minuk G, Jiang J, Niu J. IL28B is associated with outcomes of chronic HBV infection. Yonsei Med J. 2015;56:625–33. doi: 10.3349/ymj.2015.56.3.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Romani S, Hosseini SM, Mohebbi SR, Kazemian S, Derakhshani S, Khanyaghma M, Azimzadeh P, Sharifian A, Zali MR. Interleukin-16 gene polymorphisms are considerable host genetic factors for patients’ susceptibility to chronic hepatitis B infection. Hepat Res Treat. 2014;2014:790753. doi: 10.1155/2014/790753. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Shi M, Wei J, Dong J, Meng W, Ma J, Wang T, Wang N, Wang Y. Function of interleukin-17 and -35 in the blood of patients with hepatitis B-related liver cirrhosis. Mol Med Rep. 2015;11:121–6. doi: 10.3892/mmr.2014.2681. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Kil H, Jeong SH, Kim JW, Byoun YS, Min BY, Woo BH, Lee YJ, Kim YS. Role of interleukin-28B genetic polymorphisms in Korean patients with hepatitis C virus infection. Gut Liver. 2014;8:70–8. doi: 10.5009/gnl.2014.8.1.70. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Biswas A, Panigrahi R, Pal M, De BK, Chakrabarti S, Ghosh MK, Chandra Seth BC, Roychowdhury S, Chakravarty R. Association of Interleukin-1β and Gene Polymorphisms with Liver Pathogenesis in Hepatitis B Virus Infection among Eastern Indian Population. J Clin Exp Hepatol. 2013;3:281–7. doi: 10.1016/j.jceh.2013.11.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.D’Andrea A, Aste-Amezaga M, Valiante NM, Ma X, Kubin M, Trinchieri G. Interleukin 10 (IL-10) inhibits human lymphocyte interferon gamma-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J Exp Med. 1993;178:1041–8. doi: 10.1084/jem.178.3.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Liu Y, Yu MC, Zhang AQ, Wang YB, Jiang K, Dong JH. Interleukin-10 gene promoter polymorphism and risk of liver cirrhosis. Genet Mol Res. 2015;14:1229–34. doi: 10.4238/2015.February.13.1. [DOI] [PubMed] [Google Scholar]
- 15.Guo PF, Jin J, Sun X. Influence of IL10 gene polymorphisms on the severity of liver fibrosis and susceptibility to liver cirrhosis in HBV/HCV-infected patients. Infect Genet Evol. 2015;30:89–95. doi: 10.1016/j.meegid.2014.12.011. [DOI] [PubMed] [Google Scholar]
- 16.Jin XY, Wang YQ, Yan T, Wang J, Qing S, Ding N, Tian H, Zhao P. Interleukin-10 gene promoter polymorphism and susceptibility to liver cirrhosis. Hepatogastroenterology. 2014;61:442–6. [PubMed] [Google Scholar]
- 17.Yang AM, Wen LL, Yang CS, Wang SC, Chen CS, Bair MJ. Interleukin 10 promoter haplotype is associated with alcoholic liver cirrhosis in Taiwanese patients. Kaohsiung J Med Sci. 2014;30:291–8. doi: 10.1016/j.kjms.2014.02.016. [DOI] [PubMed] [Google Scholar]
- 18.Gao Q, Zhang S, Wu L, Jia M, Mi Y, Liu D. Association of IL-10-1082 and IL-10-592 polymorphisms with chronic hepatitis B and/or hepatitis C virus infection among plasma donors in a rural area of Hebei Province, China. Wei Sheng Yan Jiu. 2011;40:709–13. [PubMed] [Google Scholar]
- 19.Gong Y, Zhao C, Zhao P, Wang M, Zhou G, Han F, Cui Y, Qian J, Zhang H, Xiong H, Sheng J, Jiang T. Role of IL-10-Producing Regulatory B Cells in Chronic Hepatitis B Virus Infection. Dig Dis Sci. 2015;60:1308–14. doi: 10.1007/s10620-014-3358-1. [DOI] [PubMed] [Google Scholar]
- 20.Ren H, Zhang TT, Hu WL. A -819 C/T polymorphism in the interleukin-10 promoter is associated with persistent HBV infection, but -1082 A/G and -592A/C polymorphisms are not: a meta-analysis. Arch Virol. 2015;160:747–56. doi: 10.1007/s00705-014-2317-7. [DOI] [PubMed] [Google Scholar]
- 21.Saxena R, Chawla YK, Verma I, Kaur J. Association of interleukin-10 with hepatitis B virus (HBV) mediated disease progression in Indian population. Indian J Med Res. 2014;139:737–45. [PMC free article] [PubMed] [Google Scholar]