Genetic polymorphisms of interleukin-6 influence the development of hepatitis B virus-related liver cirrhosis

Background Interleukin-6 (IL-6) plays an important role in chronic inflammation. Thus, we aimed to investigate the effects of IL-6 polymorphisms in predicting the progression of hepatitis B virus (HBV) -related liver cirrhosis. Methods A cross-sectional study was conducted to analysis IL-6 polymorphisms and serum levels of IL-6 in HBV-infected patients of different clinical phases and in healthy controls. IL-6 polymorphisms were detected by Taqman PCR method and plasma IL-6 levels were assessed by ELISA. Results Our analysis included 182 chronic hepatitis B (CHB) patients, 190 HBV-infected liver cirrhosis cases, 125 inactive HBsAg carriers, and 246 healthy controls. Seven SNPs in IL-6 including rs10499563, rs17147230, rs1800796, rs2069837, rs1524107, rs2066992, rs2069852 were analyzed. In haplotype analysis between HBV-infected liver cirrhosis cases with CHB patients, inactive HBV-carriers or healthy controls, haplotype CT in block 1 and haplotype GGCGG in block 2 were associated with liver cirrhosis (P<0.05). What’s more, the genotype or allele frequencies were significantly different in IL-6 rs10499563 and rs2069837 when HBV-infected liver cirrhosis patients compared with CHB patients, inactive HBV-carriers or healthy controls. A further study found that compared with the controls or CHB patients, plasma IL-6 was elevated in HBV-infected liver cirrhosis patients (P<0.05).


Background
Interleukin-6 (IL-6) plays an important role in chronic inflammation.Thus, we aimed to investigate the effects of IL-6 polymorphisms in predicting the progression of hepatitis B virus (HBV) -related liver cirrhosis.

Methods
A cross-sectional study was conducted to analysis IL-6 polymorphisms and serum levels of IL-6 in HBV-infected patients of different clinical phases and in healthy controls.IL-6 polymorphisms were detected by Taqman PCR method and plasma IL-6 levels were assessed by ELISA.

Results
Our analysis included 182 chronic hepatitis B (CHB) patients, 190 HBV-infected liver cirrhosis cases, 125 inactive HBsAg carriers, and 246 healthy controls.Seven SNPs in IL-6 including rs10499563, rs17147230, rs1800796, rs2069837, rs1524107, rs2066992, rs2069852 were analyzed.In haplotype analysis between HBV-infected liver cirrhosis cases with CHB patients, inactive HBV-carriers or healthy controls, haplotype CT in block 1 and haplotype GGCGG in block 2 were associated with liver cirrhosis (P<0.05).What's more, the genotype or allele frequencies were significantly different in IL-6 rs10499563 and rs2069837 when HBV-infected liver cirrhosis patients compared with CHB patients, inactive HBV-carriers or healthy controls.A further study found that compared with the controls or CHB patients, plasma IL-6 was elevated in HBV-infected liver cirrhosis patients (P<0.05).

Conclusion
In conclusion, the polymorphisms of the IL-6 rs10499563 and rs2069837 are associated with the susceptibility of liver cirrhosis may through their effects on IL-6 expressions and these two single nucleotide polymorphisms can be used as potential predicting markers for prognosis of HBV-infected liver cirrhosis.

Background
HBV infection is a global public health issue.What's more, it is the principal reason for hepatitis, liver cirrhosis, and even hepatocellular carcinoma [1].Natural outcome of this infection varies greatly between individuals, and both viral and host factors contribute to the diversity [2].Specifically, single nucleotide polymorphisms (SNPs) of a great many genes have been reported to take part in the variety of HBV clinical course [3,4].Liver cirrhosis, as a common ending of most HBV infection, is increasingly becoming a global health burden [5].Thus, early diagnosis of HBV-infected liver cirrhosis becomes necessary.
Cytokines are thought to be the pivotal mediators which regulate many inflammatory immune responses and play an important part in determining the outcome of HBV infection [6].IL-6, one of primary immunomodulatory cytokines, has been documented to act a pivotal part in regulating the biological processes of many cells including hepatocytes [7].The gene of IL-6 lies at chromosome 7p21 and it contains 184 amino acids [8].IL-6 is a multifunctional cytokine produced by diverse cell types and it plays an important role in various biological responses [9].Many studies have proved that cytokine genetic polymorphisms such as IL-6 are related with HBV infection outcomes [10].
However, the literature is little concerning about IL-6 polymorphisms in HBV-infected liver cirrhosis in China [11].In this study, we explored the relationships about IL-6 polymorphisms and HBV-infected liver cirrhosis.What's more, compared with other studies, We paid attention to functional SNPs in whole genome of IL-6 gene besides those at the promoter region.

Study population
We recruited at the First Affiliated Hospital of Zhejiang University (Hangzhou, China) and 246 healthy controls and 497 Han Chinese with HBV infection (125 inactive HBsAg carriers, 182 CHB patients, 190 HBV-infected liver cirrhosis subjects) were enrolled in our study.All participants gave the written informed consents.Individuals with concurrent hepatitis C virus, hepatitis D virus or human immunodeficiency virus infections, alcoholic liver diseases or autoimmune liver diseases were excluded.Chronic hepatitis B patients should fulfill the criteria that elevated alanine aminotransferase (ALT) and positive for HBsAg and HBV DNA.Patients with inactive HBsAg carriers fulfilled the diagnostic criteria: HBsAg-positive, HbeAg-negative and undetectable serum HBV DNA levels with persistently normal ALT.HBV-infected liver cirrhosis patients met the criterions that a histology or clinical evidence of liver cirrhosis and a clear etiology of HBV infections.Meanwhile, controls were negative for HBsAg.

DNA isolation and genotyping
We followed the manufacturer's instructions to extract the genomic DNA from the whole blood of all subjects with a DNA extraction kit (QIAamp DNA Blood Mini Kit, USA).After extracting, we stored the DNA samples at a concentration of 100 ng/μl at -80 °C.All seven IL6 SNPs (rs10499563, rs17147230, rs1800796, rs2069837, rs1524107, rs2066992 and rs2069852) were genotyped with TaqMan probes (Applied Biosystems, Foster City, CA, USA).We used 5 % random samples to verify accuracy and repeatability of our experiment.

Detection for IL6
Plasma IL-6 was measured by a commercial ELISA kit (MultiSciences, China).We followed the kit protocol to perform the procedures.

Statistical analysis
All the statistical analyses were performed by SPSS software version 19.0 (SPSS, Inc., Chicago, IL).
Chi-square test was used to analyze Hardy-Weinberg (HW) equilibrium.The genotype and allele frequencies were calculated by direct gene counting method and compared by Chi-square test.
Student's t-test was used to analyze continuous variables among the groups.Odds ratios (ORs) and 95% confidence intervals (95% CI) were described.Haploview 4.2 software was used for linkage disequilibrium (LD) and haplotype analysis.It was statistically significant when p-value ≤0.05.

Hardy-Weinberg Equilibrium
General characteristics of the subjects are summarized in Table 1.Among the groups, there was no difference in age (P>0.05).The genotype distributions of IL-6 polymorphisms showed no significant difference from Hardy-Weinberg equilibrium in each group (P>0.05)(Table 2) and we could see the relative positions of the seven SNPs in Table 2.

Haplotype Block Structure and Linkage Disequilibrium Analysis
First, we conduct the LD analysis in HBV-infected liver cirrhosis cases with CHB patients (Fig. 1 A), inactive HBV-carriers (Fig. 1 B) and healthy controls (Fig. 1 C).As shown in Fig. 1, LD analysis showed that there were two blocks in the IL-6 gene.Rs10499563 and rs17147230 were in LD block 1 and rs1800796, rs2069837, rs1524107, rs2066992, and rs2069852 were located in LD block 2. In Table 3, we could see that in haplotype analysis between liver cirrhosis cases and CHB patients, haplotype CT (P=0.0022) in block 1 and haplotype GGCGG (P=0.0235) in block 2 were associated with liver cirrhosis.In haplotype analysis between liver cirrhosis cases and HBV-carriers, haplotype CT in block 1 (P=9.757E-6)and haplotype GGCGG in block 2 (P=0.0029)were associated with liver cirrhosis.In haplotype analysis between liver cirrhosis cases and healthy controls, haplotype CT in block 1 (P=0.0228)and haplotype GGCGG in block 2 (P=0.0127)were associated with liver cirrhosis.

The Polymorphisms of IL-6 and The Development of HBV-Related Liver Cirrhosis
Based on the haplotype analysis, we further did the genotype and allele analyses.In Table 4, we could find that the allele frequencies were different in rs10499563 and rs2069837 (P=0.0031,P=0.0240) between HBV-infected liver cirrhosis cases and CHB patients.And TC genotype of rs10499563 had a significant protective effect on liver cirrhosis patients (P=0.0246)(The Bonferroni correction was applied.P<0.025).In HBV-infected liver cirrhosis patients and inactive HBV-carriers, TC genotype of rs10499563 had a significant protective effect on liver cirrhosis patients (P=7.0000E-6) (The Bonferroni correction was applied.P<0.025).GG genotype of rs2069837 also had protective effects on liver cirrhosis patients (P=0.0239)(The Bonferroni correction was applied.P<0.025).And the allele frequencies were also different in rs10499563 and rs2069837 (P=1.4000E-5,P=0.0026).In addition, the allele frequencies were different between HBV-infected liver cirrhosis patients and control group in rs10499563 and rs2069837 (P=0.0245,P=0.0062).

Comparison of IL-6 Expressions in Different Groups
For the sake of researching whether there are any differences of IL-6 expressions between controls and HBV-cirrhotic group, we detected the IL-6 expressions in control, CHB and HBV-cirrhotic groups.

Discussion
In the study, we tested our hypothesis that the IL-6 polymorphisms may associate with the progression of HBV-infected liver cirrhosis.Our result showed that the polymorphisms of the IL-6 rs10499563 and rs2069837 were associated with the susceptibility of liver cirrhosis may through their effects on IL-6 expressions and these two SNPs could be used as potential predicting markers for prognosis of hepatitis B development.
As we all know that the interaction between host immunity and viral replication influences the clinical outcome of HBV infection, and nowadays more attentions are paid to the genetic background influence [12,13].Gene polymorphisms such as SNP may change the corresponding protein's structure or biological function.An SNP in the promoter region could induce decreased or increased productions of the corresponding protein.Thus, a person may be more resistant or susceptible to a certain disease [14].Consequently, the impact of IL-6 gene polymorphisms on disease outcomes is attracting more and more attention.It was stated that IL-6 rs10499563 was a functional polymorphism site in the promoter region of IL-6 and could transcriptionally regulate the expression of IL-6 [15].Recently, several researchers found that IL-6 rs10499563 polymorphism was linked to gastric cancer risk [16,17].However, the relationship of IL-6 rs10499563 and the progression of HBVrelated liver cirrhosis has not been studied.Based on these, our research explored the relationship between IL-6 rs10499563 and the liver cirrhosis risk in China.Compared with TT wild-type carriers, we discovered that subjects with TC and CC genotype were related with decreased risk of HBV-related liver cirrhosis.It was reported that in acute inflammatory states, TT wild-type carriers had a higher level of IL-6 while CC genotype was associated with decreased IL-6 concentrations, which was confirmed by in vitro luciferase assay [15].These results were in line with our IL-6 expression levels in patients.That is, the IL-6 rs10499563 polymorphism with wild-type genotype could produce a higher level of IL-6 and might be related with increased risk of HBV-related liver cirrhosis; the IL-6 rs10499563 polymorphism with mutant-type genotype could produce a lower level of IL-6 and might be related with decreased risk of HBV-related liver cirrhosis.The IL6 rs2069837 SNP was located at the intron region.It has been studied in many diseases, such as nephropathy [18], chronic periodontitis [19], cervical cancer [20] and chronic hepatitis B virus infection [21].One research predicted that IL6 rs2069837 SNP might participate in IL6 gene regulatory networks [22].Chen et al. deduced that carriers of rs2069837 A allele had higher plasma levels of IL-6 than in others [23].
However, it needs to be decided by further functional studies whether the IL6 rs2069837 SNP could regulate the expressions of IL-6, which may reveal the exact mechanisms underlying the observed association with the progression of HBV-related liver cirrhosis.
Recently, IL-6 polymorphisms in liver diseases such as hepatitis C and anti-tuberculosis drug-induced hepatitis (ATDH) were focused.Motawi et al. [24] have explored the influences of IL-6,α1-antitrypsin (A1AT) polymorphisms in HCV-infected liver cirrhosis patients.In their study, 85 chronic HCV-infected patients and 65 cirrhotic patients together with 100 healthy control subjects were genotyped for IL-6 rs1800795, A1AT Z mutation rs28929474 and A1AT S mutation rs17580.However, the results showed that IL-6 rs1800795 was not associated with cirrhotic patients but the A1AT gene polymorphisms were related with the progression of chronic hepatitis C. In addition, Wang et al. investigated the distribution of the IL-6, HSPA1L and STAT3 SNPs in patients with ATDH in Chinese Han population [25].
A total of 356 healthy controls and 89 ATDH patients were enrolled in their study.However, they found that the three selected IL-6 SNPs (rs2066992, rs2069837, rs1524107) did not contribute to ATDH susceptibility and STAT3 polymorphisms were associated with ATDH.The association between IL-6 SNPs and the outcome of HBV infection could be found in earlier studies.Some researchers found that there was no significant relationship between IL-6 rs1800796 polymorphisms and the outcome of chronic hepatitis B disease [26].However, after adjusting all subjects based on gender, males with the G allele had a higher chance of HCC than those with the CC genotype [10].Furthermore, Lu et al.
reported that IL-6 rs1800796 allele G may be helpful for spontaneous clearance of HBV [27].They found that compared to CHB patients, controls with spontaneous clearance of HBV had higher GG genotype and allele G frequencies in IL-6 rs1800796 site.Hence, they drawn the conclusion that IL-6 rs1800796 was related with spontaneous clearance of HBV.
In our study, the haplotype analysis showed that the polymorphisms of the IL-6 rs10499563, rs17147230, rs1800796, rs2069837, rs1524107, rs2066992 and rs2069852 were associated with the susceptibility of liver cirrhosis.This discovery is in accordance with the concept that IL-6 haplotypes are more functionally correlative to some illnesses than single polymorphisms [28].What's more, based on the genotype and allele analyses, there were definitive evidences pointing to the relationships about IL-6 rs10499563 and rs2069852 in HBV-infected liver cirrhosis cases with CHB patients, inactive HBV-carriers or healthy controls.

Conclusions
In conclusion, it was found that the polymorphisms of the IL-6 rs10499563 and rs2069837 were associated with the susceptibility of liver cirrhosis.Moreover, we found that plasma IL-6 levels were remarkably higher in HBV-cirrhotic groups than in CHB group and in control group.We assume that the genetic influences of the IL-6 SNPs on susceptibility to liver cirrhosis may through their effects on IL-6 expressions.Our works find a pivotal role of IL-6 SNP in predicting the progression of HBV-related liver cirrhosis in a Chinese population, which is helpful of the existing prognostic and therapeutic strategies in the HBV-related liver cirrhosis disease.Until now, our research is the first one to explore the distribution of functional SNPs in whole genome of IL-6 gene in a Chinese population about the HBV-related liver cirrhosis disease.Nevertheless, larger comprehensive population-based studies with different races are needed to further validate these preliminary observations.What's more, our next study will focus on the functional research whether the IL6 SNP could regulate the expressions of IL-6, which may reveal the exact mechanisms underlying the observed association with the progression of HBV-related liver cirrhosis.

Abbreviations
a: Controls mean CHB patients; b: Controls mean inactive HBV-carriers; c: Controls mean healthy controls.Cases all mean HBV-infected liver cirrhosis patients.d: The Bonferroni correction was applied for multiple comparisons.

Figures
Figures

Figure 1 LD
Figure 1 LD (D') plot of IL-6 gene.Two blocks were in the IL-6 gene.D' means the degree of LD between two SNPs.(A) The LD analysis in HBV-infected liver cirrhosis cases with CHB patients.(B) The LD analysis in HBV-infected liver cirrhosis cases with inactive HBV-carriers.(C)The LD analysis in HBV-infected liver cirrhosis cases with healthy controls.

Figure 2 Plasma
Figure 2 Plasma IL-6 levels in control, CHB and HBV-cirrhotic groups.a: compared with healthy control group P<0.05 b: compared with CHB group P<0.01.

Table 4
Comparison of genotype and allele distributions among HBV-infected liver cirrhosis group and three other groups (CHB patients, inactive HBV-carriers; and healthy controls)