Genetic polymorphisms of interleukin-6 in�uence the development of hepatitis B virus-related liver cirrhosis in the Han Chinese population

Background Interleukin-6 (IL-6) plays an important role in chronic in�ammation. Thus, we aimed to investigate the effects of IL-6 polymorphisms on predicting the progression of hepatitis B virus (HBV)-r elated liver cirrhosis.Methods A cross-sectional study was conducted to analyse IL-6 polymorphisms and serum levels of IL-6 in HBV-infected patients at different clinical phases and in healthy controls. IL-6 polymorphisms were detected by the 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, and rs2069852 were analysed. In a haplotype analysis between HBV-infected liver cirrhosis cases and 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). Moreover, the genotype or allele frequencies were signi�cantly different in IL-6 rs10499563 and rs2069837 when HBV-infected liver cirrhosis patients were compared with CHB patients, inactive HBV carriers or healthy controls. A further study found that compared with that in the healthy controls, inactive HBV carriers or CHB patients, plasma IL-6 was elevated in HBV-infected liver cirrhosis patients (P<0.05).Conclusion In conclusion, the IL-6 rs10499563 and rs2069837 polymorphisms are associated with susceptibility to liver cirrhosis may through their effects on IL-6 expression and these two single nucleotide polymorphisms can be used as potential prognostic markers of HBV-related liver cirrhosis.


Background
Hepatitis B virus (HBV) infection is a global public health issue.Moreover, it is the principal reason for hepatitis, liver cirrhosis, and even hepatocellular carcinoma [1][2][3].The natural outcome of this infection varies greatly between individuals, and both viral and host factors contribute to the diversity [4].
Speci cally, single nucleotide polymorphisms (SNPs) of great many genes have been reported to take part in the variability of the HBV clinical course [5,6].Liver cirrhosis, as a common endpoint of most HBV infections, is increasingly becoming a global health burden [7].Thus, early diagnosis of HBV-infected liver cirrhosis patients becomes necessary.
Cytokines are thought to be pivotal mediators that regulate many in ammatory immune responses and play an important role in determining the outcome of HBV infection [8,9].IL-6, a primary immunomodulatory cytokine, has been documented to play a pivotal role in regulating the biological processes of many cells including hepatocytes [10,11].The IL-6 gene is located on chromosome 7p21 and encodes 184 amino acids [12].IL-6 is a multifunctional cytokine produced by diverse cell types and plays an important role in various biological responses [13][14][15].For example, Jing et al. found that IL-6 levels were closely related to myocardial infarction-induced myocardial remodelling [16].Keshari et al. revealed that IL-6 mediated calcium phosphate-induced pruritus [17].Moreover, many studies have demonstrated that cytokine genetic polymorphisms such as those in IL-6 are related to HBV infection outcomes [18][19][20][21].
However, little is known about IL-6 polymorphisms in HBV-related liver cirrhosis in China.In this study, we explored the relationships between IL-6 polymorphisms and HBV-related liver cirrhosis.Moreover, compared with other studies, we focused on functional SNPs in the whole genomic sequence of the IL-6 gene in addition to those in the promoter region.

Study population
A total of 246 healthy controls and 497 Han Chinese with HBV infection (125 inactive HBsAg carriers, 182 CHB patients, and 190 HBV-infected liver cirrhosis subjects) were recruited and enrolled in our study at the First A liated Hospital of Zhejiang University (Hangzhou, China).All participants gave written informed consent.Individuals with concurrent hepatitis C virus, hepatitis D virus or human immunode ciency virus infections, alcoholic liver diseases or autoimmune liver diseases were excluded.Chronic hepatitis B patients had to ful l the criteria of elevated alanine aminotransferase (ALT) and HBsAg positive status for more than six months.Patients that were inactive HBsAg carriers ful lled the following diagnostic criteria: HBsAg-positive, HBeAg-negative and undetectable serum HBV DNA levels with persistently normal ALT.HBV-infected liver cirrhosis patients met the criteria of histology or clinical evidence of liver cirrhosis and a clear aetiology of HBV infection.Moreover, healthy controls were negative for HBsAg.

SNP selection
The SNPs were selected according to the following principle: (1) minor allele frequency (MAF) > 0.05; (2) r 2 > 0.8; (3) SNPs shown to be related to diseases in previous studies.Finally, seven SNPs, including rs10499563, rs17147230, rs1800796, rs2069837, rs1524107, rs2066992, and rs2069852 (Table 2), in the IL-6 gene were selected from the International HapMap Project (http://hapmap.ncbi.nlm.nih.gov/), and in the Allele column of Table 2, the rst one is the mutant allele and the next is the wild-type allele.

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 (Qiagen, USA).After extraction, we stored the DNA samples at a concentration of 100 ng/μl at -80 °C.All seven IL-6 SNPs (rs10499563, rs17147230, rs1800796, rs2069837, rs1524107, rs2066992 and rs2069852) were genotyped with TaqMan probes (Applied Biosystems, Foster City, CA, USA).We randomly selected 5% of the samples to verify the accuracy and repeatability of our experiment.

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

Statistical analysis
All statistical analyses were performed using SPSS software version 19.0 (SPSS, Inc., Chicago, IL).A chisquare test was used to analyse the Hardy-Weinberg equilibrium (HWE).The genotype and allele frequencies were calculated by the direct gene counting method and compared by the Chi-square test.The student's t test was used to analyse age among the groups.A non-parametric test (Mann-Whitney U) was used to analyse IL-6 levels among the groups.Data are presented as the means (± standard deviation, SD/standard error, SE) and counts (percentage).Odds ratios (ORs) and 95% con dence intervals (95% CIs) were determined.Haploview 4.2 software was used for linkage disequilibrium (LD) and haplotype analysis.The Bonferroni correction was applied for multiple comparisons.Differences were statistically signi cant when p-value ≤ 0.05, except for Bonferroni correction.

Clinical information of study population
The general characteristics of the subjects are summarized in Table 1.Among the groups, there were no differences in age (P>0.05), and the genotype distributions of IL-6 polymorphisms showed no signi cant departure from HWE in each group (P>0.05)(Table 2).The relative positions of the seven SNPs are shown in Table 2. Haplotype block structure and LD analysis First, we conducted the LD analysis of HBV-infected liver cirrhosis cases and 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. Table 3 shows that in the 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 the 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 the 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.Cases all mean the HBV-infected liver cirrhosis patients.

The polymorphisms of IL-6 and the development of HBV-related liver cirrhosis
Based on the haplotype analysis, we further performed genotype and allele analyses.As shown in Table 4, we found that the allele frequencies were different in rs10499563 and rs2069837 (P=0.0031,P=0.0240, respectively) between HBV-infected liver cirrhosis cases and CHB patients.The TC genotype of rs10499563 had a signi cant 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, the TC genotype of rs10499563 had a signi cant protective effect on liver cirrhosis patients (P=7.0000E-6)(the Bonferroni correction was applied; P<0.025).The GG genotype of rs2069837 also had protective effects on liver cirrhosis patients (P=0.0239)(the Bonferroni correction was applied; P<0.025).The allele frequencies were also different in rs10499563 and rs2069837 (P=1.4000E-5,P=0.0026, respectively).In addition, the allele frequencies were different between HBV-infected liver cirrhosis patients and the healthy control group in rs10499563 and rs2069837 (P=0.0245,P=0.0062, respectively).However, there were no signi cant differences in rs17147230, rs1800796, rs1524107, rs2066992 and rs2069852 between the HBV-infected liver cirrhosis group and the other three groups (see Supplementary Table 2).Cases all mean the HBV-infected liver cirrhosis patients.d: The Bonferroni correction was applied for multiple comparisons.

Comparisons of IL-6 expression in different groups
To determine whether there were any differences in IL-6 expression between the controls and the HBVcirrhotic group, we detected the IL-6 expression levels in the healthy controls, the inactive HBV-carrier group, the CHB group and the HBV-cirrhotic group.As shown in Table 5, we could not detect IL-6 levels in the plasma of the healthy control group or the inactive HBV-carrier group.There were signi cantly high IL-6 expression levels in the CHB group and HBV-cirrhotic group compared with the healthy control group and the inactive HBV-carrier group.We next evaluated the association of changes in the plasma concentrations of IL-6 with the progression of HBV-related liver cirrhosis.Plasma IL-6 levels were remarkably higher in the HBV-cirrhotic group than in the CHB group.Furthermore, we also analysed the plasma concentrations of IL-6 associated with different genotypes and alleles of rs10499563 and rs2069837 in each group (Figure 2).For rs10499563, plasma IL-6 levels in the TT genotype and T allele were higher than those in the CT and CC genotypes and C allele in the CHB group and HBV-cirrhotic group.For rs2069837, plasma concentrations of IL-6 in the AA genotype and A allele were lower than those in the AG genotype and G allele in the CHB group and HBV-cirrhotic group.However, there were no differences between each group.

Discussion
In this study, we tested our hypothesis that IL-6 polymorphisms may be associated with the progression of HBV-associated liver cirrhosis.Our results showed that the IL-6 polymorphisms rs10499563 and rs2069837 were associated with susceptibility to liver cirrhosis may through their effects on IL-6 expression, and these two SNPs could be used as potential prognostic markers of hepatitis B development.
It is well known that the interaction between host immunity and viral replication in uences the clinical outcome of HBV infection, and more attention is currently paid to the genetic background in uence [22,23].Gene polymorphisms such as SNPs may change the structure or biological function of the corresponding protein.An SNP in the promoter region could induce decreased or increased production of the corresponding protein.Thus, a person may be more resistant or susceptible to a certain disease [24].Consequently, the impact of IL-6 gene polymorphisms on disease outcomes is attracting increasing attention [25].It has been shown that IL-6 rs10499563 is a functional polymorphism site in the promoter region of IL-6 and could transcriptionally regulate the expression of IL-6 [26].Recently, several researchers have found that the IL-6 rs10499563 polymorphism is linked to gastric cancer risk [27,28].However, the relationship between IL-6 rs10499563 and the progression of HBV-related liver cirrhosis has not been studied.Based on these ndings, our research explored the relationship between IL-6 rs10499563 and liver cirrhosis risk in China.Compared with TT wild-type carriers, we discovered that subjects with the TC and CC genotypes had a decreased risk of HBV-related liver cirrhosis.It has been reported that in acute in ammatory states, TT wild-type carriers have a higher level of IL-6, while the CC genotype has been associated with decreased IL-6 concentrations, which has been con rmed by an in vitro luciferase assay [26].These results are in line with our IL-6 expression levels in patients.That is, the IL-6 rs10499563 polymorphism with the wild-type genotype could produce a higher level of IL-6 and might be related to an increased risk of HBV-related liver cirrhosis; the IL-6 rs10499563 polymorphism with the mutant-type genotype could produce a lower level of IL-6 and might be related to a decreased risk of HBV-related liver cirrhosis (Figure 2).IL-6 rs2069837 is located in the intron region.It is known that introns can increase gene expression through intron-mediated enhancement [29].For example, introns can not only in uence the rate of transcription and nuclear export but also enhance the effectiveness of mRNA translation [30].
As an SNP within the intron region, IL-6 rs2069837 has been studied in many diseases, such as nephropathy [31], chronic periodontitis [32], cervical cancer [33] and chronic hepatitis B virus infection [34].One study predicted that the IL-6 rs2069837 SNP might participate in IL-6 gene regulatory networks [35].Chen et al. deduced that carriers of the rs2069837 A allele had higher plasma IL-6 levels than others [36].However, in our study, carriers of the rs2069837 G allele had higher plasma levels of IL-6 than the A allele (Figure 2 D).Therefore, further functional studies are needed to determine whether the IL-6 rs2069837 SNP could regulate the expression 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 virus infection and anti-tuberculosis drug-induced hepatitis (ATDH) have become the focus of research studies.Motawi et al. [37] explored the in uences of IL-6 and 1-antitrypsin (A1AT) polymorphisms in HCV-infected liver cirrhosis patients in Egyptians.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 to the progression of chronic hepatitis C. Nevertheless, a study in a population from Rio de Janeiro, Brazil found that the IL-6 rs1800795 C allele was associated with protection from chronic hepatitis C and could decrease in ammation scores [38].In addition, Wang et al. investigated the distribution of IL-6, HSPA1L and STAT3 SNPs in patients with ATDH in a Chinese Han population [39].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, and rs1524107) did not contribute to ATDH susceptibility and that STAT3 polymorphisms were associated with ATDH.The association between IL-6 SNPs and the outcome of HBV infection can be found in earlier studies.Some researchers found that there was no signi cant relationship between IL-6 rs1800796 polymorphisms and the outcome of chronic hepatitis B disease [40].However, after adjusting for gender, males with the G allele were at a higher risk of HCC than those with the CC genotype [41].Furthermore, Lu et al. reported that the IL-6 rs1800796 G allele may be helpful for spontaneous clearance of HBV [42].The authors found that compared to CHB patients, controls with spontaneous clearance of HBV had higher GG genotype and allele G frequencies in the IL-6 rs1800796 site.Hence, they concluded that IL-6 rs1800796 was associated with spontaneous clearance of HBV.What's more, in a recent study in Egyptians, researchers concluded that IL-6 (rs1800795, rs1800796 and rs1800797) gene polymorphisms were associated with susceptibility to HBV infection [43].
In our study, the haplotype analysis showed that the polymorphisms of the IL-6 rs10499563, rs17147230, rs1800796, rs2069837, rs1524107, rs2066992 and rs2069852 SNPs were associated with susceptibility to liver cirrhosis.This discovery is in accordance with the concept that IL-6 haplotypes are more functionally correlative to some illnesses than single polymorphism [44].Moreover, based on the genotype and allele analyses, there was de nitive evidence indicating the relationships of IL-6 rs10499563 and rs2069852 in HBV-infected liver cirrhosis cases and CHB patients, inactive HBV carriers or healthy controls.

Conclusions
In conclusion, it was found that the IL-6 rs10499563 and rs2069837 polymorphisms were associated with susceptibility to liver cirrhosis.Moreover, we found that plasma IL-6 levels were remarkably higher in the HBV-cirrhotic group than in the CHB group, in the inactive HBV-carrier group and in the healthy control group.We assume that the genetic in uences of the IL-6 SNPs on susceptibility to liver cirrhosis may occur through their effects on IL-6 expression.Our works found a pivotal role of IL-6 SNPs in predicting the progression of HBV-related liver cirrhosis in a Han Chinese population, which is helpful for the existing prognostic and therapeutic strategies for the HBV-related liver cirrhosis.To date, our research is the rst to explore the distribution of functional SNPs in the whole genomic sequence of the IL-6 gene in a Han Chinese population and their association with HBV-related liver cirrhosis.Nevertheless, larger comprehensive population-based studies with different races are needed to further validate these preliminary observations.Furthermore, our next study will focus on functional research on whether IL-6 SNPs could regulate the expression of IL-6, which may reveal the exact mechanisms underlying the observed association with the progression of HBV-related liver cirrhosis.

Table 1
General characteristics of the subjects SNP, single nucleotide polymorphism; MAF, minor allele frequency.a: From both HapMap and dbSNP databases.b: P value for HWE test in the whole group.

Table 3
Haplotype analysis between cases and controlsHapMap block Haplotype Cases Controls Chi Square P value a: Controls mean the CHB patients; b: Controls mean the inactive HBV carriers; c: Controls mean the healthy controls.

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

Table 5
n.d., not detected; a: compared with the CHB group, P<0.05.Data are presented as mean ± SD.