Genetic Association of Interleukin-6 Polymorphism (rs1800796) with Chronic Hepatitis B Virus Infection in Chinese Han Population

Abstract

Hepatitis B virus (HBV) infection is a global health care burden that can lead to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. We conducted this study to identify the association between interleukin-6 (IL-6) gene rs1800796 (−572G/C) polymorphism and the risk of chronic HBV infection in adults. A total of 1,048 participants including 518 cases and 530 controls were recruited for this study. The Mass Array time-of-flight mass spectrometer was applied for single-nucleotide polymorphism rs1800796 genotyping. There was a significant correlation between genotype CG in rs1800796 and chronic HBV infection in the Chinese Han population (odds ratio [OR] = 0.759, 95% confidence interval [CI]: 0.586–0.983, p = 0.04), which was also observed at allele G (OR = 0.800, 95% CI: 0.657–0.975, p = 0.02). Furthermore, significant differences in the ≤45 years old group (CC vs. CG+GG, OR = 0.616, 95% CI: 0.413–0.918, p = 0.02) and in the male group (CC vs. CG+GG, OR = 0.666, 95% CI: 0.483–0.920, p = 0.01) were found in the subgroups analysis. Our data revealed a significant association of IL-6 rs1800796 with the risk of chronic HBV infection in the Chinese Han population; meanwhile, age and gender are two coordinative risk factors, which provides new clues for the study of susceptibility of chronic HBV infection in adults.

Introduction

Hepatitis B virus (HBV) infection is a global health care burden that can lead to chronic hepatitis, liver cirrhosis (LC), and hepatocellular carcinoma (HCC) (7,28,33). Recently, the estimated prevalence of HBV surface antigen (HBsAg) in 2016 was ∼3.9% and 291 million individuals with HBV infection (from 4% to 6% of the global population), which continues to be a major public health challenge in the world (22). After HBV exposure, only 5–10% adults will develop chronic hepatitis B (CHB) and 20–30% of them will further progress to LC and/or HCC, which can lead to an estimated 887,000 deaths in 2015 (35). Despite an effective prophylactic vaccine, China still has the greatest number of HBsAg-positive patients, with >80 million adults and 37,000 children who are chronic carriers (22).

The mechanism for HBV infection can be influenced by various factors, including immunological factors, viral factors, environmental factors, and host genetic factors (15,37). Host genetic factors play an important role in the chronicity of HBV infection and have received more and more attention. In addition, numerous studies have explored the associations between polymorphisms in candidate genes and HBV infection, including human leukocyte antigen, cytokines, and chemokines (6,13,14,31,32). Cytokines play a fundamental role in regulating the hepatocyte functions and immune response (2). Among these cytokines, increasing importance has been attached to the involvement of polymorphisms in the cytokine genes (1).

Interleukin-6 (IL-6), located in chromosome 7p21 in humans, is a typical anti-inflammatory and proinflammatory cytokine that is composed of 184 amino acids (23). It is produced by a variety of cells and functions as a key regulator in the modification of the immune response, inflammation, and hematopoiesis (12,20), which is critical to the pathogenesis of HBV infection. IL-6, through interaction with its receptor (IL-6R and gp130), exerts its biological functions (11,21,36). As a key growth-promoting and antiapoptotic factor, the genetic variants of IL-6 have involved in a wide range of infectious diseases, including chronic HCV infection, persistent chlamydia pneumonia infection, respiratory tract infection, and tuberculosis (4,17,25,39).

There are three essential variations located in the promoter of IL-6 involving −174G/C (rs1800795), −572G/C (rs1800796), and −597G/A (rs1800797). Recently, several studies have extensively explored the genetic polymorphisms of IL-6 (8,26,36,38,41). However, no general conclusions have been identified, which may be attributed to small sample size and limited statistical power of each study. In view of the report that no polymorphisms at positions rs1800795 and rs1800797 was detected in Chinese HBV-infected patients (5), we designed this analytical observational case–control study to identify the association between IL-6 gene rs1800796 (−572G/C) polymorphism and the risk of chronic HBV infection.

Subjects and Methods

Subjects

A total of 1,048 adults derived from Chongqing City (in southwest China) were enrolled between January 2014 and December 2017 at the Second Affiliated Hospital and the Children's Hospital of Chongqing Medical University. Cases were defined as both HBsAg and hepatitis B core antibody (HBcAb) positive for at least 6 months (n = 518), and the controls were defined as negative for HBsAg and positive for both HBsAb and HBcAb (n = 530).

The diagnosis of chronic HBV infection was based on HBsAg seropositivity for >6 months and HBV DNA detectable according to the “Guidelines for the Prevention and Treatment of Chronic Hepatitis B” revised by the Chinese Medical Association Hepatology Branch in 2019 (30). The diagnosis of controls was age- and gender-matched subjects. Subjects with other liver diseases or autoimmune diseases were excluded. All participants gave written informed consent, and the study was performed with the approval of the ethics committee of Chongqing Medical University in Chongqing, China.

DNA extraction

Blood samples from each participant (∼3 mL taken from arm venipuncture) were collected into ethylenediamine tetra-acetic tubes without any anticoagulants. The human genomic DNA was extracted using BioTeke DP6101 kit (BiotTeke Corporation, Beijing, China) according to the manufacturer's instructions, and stored at −80°C for further experimentation. The concentration was measured using Nanodrop2000c spectrophotometer (Thermo Scientific, Delaware).

Serological assays

HBV markers (HBsAg, HBsAb, HBeAg, HBeAb, and HBcAb) were measured by enzyme-linked immunosorbent assay (ELISA). Diagnostic kits were purchased from the Shanghai Kehua Bioengineering Technology Company, Limited (Shanghai, China).

Single-nucleotide polymorphism genotyping

Single-nucleotide polymorphism (SNP) genotyping was performed using the Mass Array time-of-flight mass spectrometer (Sequenom Company, San Diego, CA). Polymerase chain reaction (PCR) and extension primers were designed according to Mass ARRAY Assay Design 3.1 software (Sequenom Company). The genotyping procedures were carried out using the manufacturer's iPLEX Application Guide (Sequenom Company). The PCR amplification conditions included an initial precycle incubation at 94°C for 15 min, followed by 45 cycles × (denaturation at 94°C for 20 s, annealing at 56°C for 30 s, and extension at 72°C for 60 s), and finally at 72°C for 5 min. All the genotyping reactions were performed in 384-well plates. Each plate included four randomly selected duplicates and six negative controls with the use of double-distilled water. The average concordance rate for the genotype was 99.5%.

Statistical analysis

All of the data were double inputted in the computer using Epidata 3.02 software, and the statistical analysis was carried out with SPSS (version 20.0; SPSS, Inc., Chicago, IL). The Hardy–Weinberg equilibrium (HWE) was examined to test the deviation of genotype distribution using SNPStats (http://bioinfo.iconcologia.net/snpstats/start.htm). The continuous variables were expressed as mean ± standard deviation (SD), and the discrete variables as percentage (%). The comparison of age and gender between the two groups was conducted by using Student's-t test and χ ² test or Fisher's exact test. One-way analysis of variance and χ ² test were used to evaluate the association of the genotypes and clinical biomarkers, including HBV DNA, HBeAg, spartate aminotransferase (AST), and alanine aminotransferase (ALT) levels. Log transformation of HBV DNA, HBeAg, AST, and ALT levels was used before the test. The analysis of the association between genotypes and HBV infection was performed by computing the odds ratios (ORs), 95% confidence intervals (CIs), and p values from the logistic regression with adjustments for age and gender, which was also used for the genetic model analysis. Each component of the model was as follows: allele C as the major allele, allele G as the minor allele, dominant model (CG + GG vs. CC), recessive model (GG vs. CG + CC), codominant model (CC vs. CG vs. GG), and overdominant model (CC + GG vs. CG). A value of p < 0.05 was considered as statistically significant.

Results

Subjects' characteristics

Baseline characteristics of subjects are presented in Table 1. This study included 518 cases (311 males and 207 females) with the mean age of 49.72 years (SD 13.55) and 530 controls (310 males and 220 females) with the mean age of 50.12 years (SD 14.91). No statistically significant difference was found in age or gender between the two groups (p = 0.66, p = 0.61).

Table 1.

Baseline Characteristics of Adult Subjects in the Case–Control Study

Variables	Total (n = 1,048)	Categorized by group		p
Variables	Total (n = 1,048)	Cases (n = 518)	Controls (n = 530)	p
Age (mean ± SD)	49.92 ± 14.25	49.72 ± 13.55	50.12 ± 14.91	0.66
Gender
Male		311 (60%)	310 (58%)	0.61
Female		207 (40%)	220 (42%)

SD, standard deviation.

Association between IL-6 rs1800796 and chronic HBV infection

The genotype and allelic frequencies of IL-6 gene rs1800796 polymorphism were in accordance with the HWE in both CHB patients and controls (p = 0.62, p = 0.75). As represented in Table 2, the rs1800796 SNP was associated with the risk of HBV infection after adjusting for gender and age (p = 0.04), and the most prevalent genotype in both groups is the CC genotype. Compared with the rs1800796 CC genotype, the CG genotype had a lower HBV infection risk, which might be considered to be a protective factor for HBV infection (OR = 0.759, 95% CI: 0.586–0.983, p = 0.04). Although the GG genotype was not significantly different in cases and controls after adjustment (OR = 0.706, 95% CI: 0.429–1.162, p = 0.17), a coincident trend can be observed that a high frequency of the GG genotype was found in the controls, which revealed that subjects with the G allele seemed to have a lower susceptibility to HBV infection than those with C allele. Finally, the comparison of allele frequency validated the result that the significant difference was also found at allele G with OR of 0.796 (95% CI: 0.654–0.970, p = 0.02).

Table 2.

Association Between Interleukin-6 rs1800796 and Risk of Chronic Hepatitis B Infection in Adults

Cytokine gene	Cases (%)	Controls (%)	Crude OR (95% CI)	p	Adjusted OR (95% CI)^a	p^a
IL-6 (−572) G/C (rs1800796)
Genotype frequency (n, %)
CC	297 (58.5)	269 (51.2)	1		1
CG	180 (35.4)	216 (41.1)	0.755 (0.583–0.977)	0.03	0.759 (0.586–0.983)	0.04
GG	31 (6.1)	40 (7.6)	0.702 (0.427–1.154)	0.16	0.706 (0.429–1.162)	0.17
Allele frequency (n, %)
C	774 (76.2)	754 (71.8)	1
G	242 (23.8)	296 (28.2)	0.796 (0.654–0.970)	0.02

Bold values represent significant values.

Adjusting for gender and age.

CI, confidence interval; IL-6, interleukin-6; OR, odds ratio.

We next conducted a genetic model analysis in the rs1800796 SNP and the result is given in Table 3. Four genetic models were applied in this study and were analyzed after adjusting for gender and age. The dominant model revealed that the (CG+GG) genotype was found to be a protective factor for chronic HBV infection (OR = 0.751, 95% CI: 0.587–0.961, p = 0.02). In the codominant model, individuals with the CG genotype showed a decreased HBV infection risk (OR = 0.759, 95% CI: 0.586–0.983, p = 0.04) compared with the CC genotype.

Table 3.

Association Between rs1800796 and Chronic Hepatitis B Infection in Genetic Models

Gene	SNP	Model	Genotype	Crude OR (95% CI)	p	Adjusted OR (95% CI)^a	p^a
IL-6	rs1800796	Dominant	CC	1		1
		Dominant	GG+CG	0.747 (0.584–0.955)	0.02	0.751 (0.587–0.961)	0.02
		Recessive	CC+CG	1		1
		Recessive	GG	0.788 (0.485–1.281)	0.34	0.791 (0.486–1.286)	0.35
		Codominant	CC	1		1
			CG	0.755 (0.583–0.977)	0.03	0.759 (0.586–0.983)	0.04
			GG	0.702 (0.427–1.154)	0.16	0.706 (0.429–1.162)	0.17
		Overdominant	CG	1		1
		Overdominant	CC+GG	1.274 (0.991–1.638)	0.06	1.267 (0.985–1.630)	0.07

Bold values represent significant values.

Adjusting for gender and age.

SNP, single-nucleotide polymorphism.

All the above indicated that the rs1800796G allele was associated with decreased risk of chronic HBV infection in the Chinese Han population.

Stratified analysis between rs1800796 polymorphism and chronic HBV infection

We further compared the genotype frequencies of rs1800796 polymorphism after segregation on the basis of age and gender. Considering the low frequency of the GG genotype, the CG and GG genotypes were combined for stratified analysis. Interestingly, as given in Table 4, in male subjects, there was a significant correlation in the genotype frequencies of rs1800796 SNP between the two groups. The rs1800796 (CG+GG) genotypes were associated with a significantly decreased risk of HBV infection compared with the CC genotype after adjusting for age (OR = 0.666, 95% CI: 0.483–0.920, p = 0.01); however, the association was not significant in female subjects (p = 0.45). In addition, in the lower age group (≤45), the comparison of the (CG+GG) genotypes and the CC genotype was significantly different in the cases and controls (OR = 0.616, 95% CI: 0.413–0.918, p = 0.02), but the difference did not come up to statistical standard in the higher age group (>45) (p = 0.24, Table 4).

Table 4.

Relationship of rs1800796 Genotypes Distribution with Age, Gender, and Clinical Biomarkers

Variables	Cases (%)		Controls (%)		p^a	Adjust OR (95% CI)^a
Variables	CC	CG+GG	CC	CG+GG	p^a	Adjust OR (95% CI)^a
Age (years)
≤45	133 (62.1)	81 (37.9)	98 (50.8)	95 (49.2)	0.02	0.616 (0.413–0.918)
>45	164 (55.8)	130 (44.2)	171 (51.5)	161 (48.5)	0.24	0.828 (0.603–1.137)
Gender
Male	188 (61.4)	118 (38.6)	158 (51.6)	148 (48.4)	0.01	0.666 (0.483–0.920)
Female	109 (54.0)	93 (46.0)	111 (50.7)	108 (49.3)	0.45	0.863 (0.587–1.269)
Clinical biomarkers	Cases (mean ± SD)
HBV DNA	2.95 ± 3.13	2.94 ± 3.20			0.61
ALT	2.25 ± 0.70	2.18 ± 0.69			0.50

Bold values represent significant values.

Adjusting for another excluded variable.

ALT, alanine aminotransferase; HBV, hepatitis B virus.

The rs1800796 polymorphism is not associated with both serum HBV DNA and ALT levels in cases

Serum HBV DNA is a highly specific marker for HBV infection, reflecting the interaction between host and HBV. The ALT levels are sensitive indicators of hepatic inflammation and liver injury. Increased serum HBV DNA, ALT levels have been considered to be important risk predictors for HBV replication and infection. Therefore, we analyzed the correlation between serum HBV DNA, ALT levels, and rs1800796 polymorphism. However, no significant difference in both of the two clinical markers was observed among cases carrying different rs1800796 genotypes (p = 0.61, p = 0.50, Table 4).

Discussion

HBV infection can lead to a diversity of clinical outcomes, such as acute self-limited hepatitis, chronic hepatitis, liver failure, LC, and HCC (30). CHB remains a challenging public health problem in China and worldwide. In 2016, China has been recognized as an area of intermediate HBV prevalence comprising ∼6.1% of the population (22). Owing to the key role of IL-6 gene in the immunoregulatory response against HBV infection, we performed this case–control study to compare the rs1800796 genotype in IL-6 promoter region with chronic HBV infection in the Chinese Han population.

In this study, our findings suggested that the IL-6 gene SNP rs1800796 was associated with the risk of chronic HBV infection in a Chinese Han population. Patients with G allele, CG, or CG+GG genotypes of rs1800796 had higher distribution in controls. Therefore, G allele and CG genotype of rs1800796 may be critical protective markers for the HBV infection. Our results are consistent with the results of Zhang et al. that reported a relationship between IL-6 rs1800796 polymorphism and HBV infection (38). Similarly, Lu et al. found that controls have significantly higher allele G frequencies for IL-6 rs1800796 than CHB patients through a case–control study with a total of 431 subjects (16). However, El-Maadawy et al. have shown that IL-6 rs1800796 CG genotype has higher distribution in CHB patients (85.2%) than in controls in Egyptians (8). In accordance with this result, the study of Saxena et al. observed that the CG genotype was more frequent in CHB patients, which may be due to the difference between the ethnicity and the study subjects (27). Several other studies also came to the same conclusions (3,24,27,34,40).

Polymorphisms in the promoter region of the IL-6 gene including −174G/C (rs1800795), −572G/C (rs1800796), and −597G/A (rs1800797) have been reported in association with the HBV infection (8,10,27,41). However, Dai et al. have shown that no polymorphism at positions −174 and −597 was detected in the Chinese population, but significant difference was found in the polymorphism at position −572 between the cases and controls (5). Besides, several previous studies have shown a lack of association between rs1800795, rs1800797, and the susceptibility to CHB infection (2,19), and the allele frequencies of the two loci can be observed in a different genetic background of ethnic diversity (18). Data from the HapMap show that the allele frequencies of the two loci are extremely rare in the Asian population in comparison with the European population (rs1800795C allele: Asian, 0.00; European, 0.352; rs1800797A allele: Asian, 0.00; European, 0.352). In contrast, the C allele frequency of rs1800796 differs from 0.86 in Asian population to 0.043 in American population. Therefore, rs1800795 and rs1800797 are unlikely to contribute significantly to CHB susceptibility in the Chinese Han population.

Recently, G allele of rs1800796 was shown to be associated with spontaneous clearance of HBV infection (16). In addition, Tang et al. have found that male subjects with the GG and CG genotypes have higher risk of HBV-related HCC than those with CC genotype (29); however, according to our study, the rs1800796 G allele decreased the susceptibility of HBV infection. A meta-analysis in 2019 also found that the rs1800796G allele may be a significant protective marker for the HBV infection, especially in the Asian population (34), which was consistent with our results. More importantly, after stratification of all subjects based on gender and age, we found that male subjects with the (CG+GG) genotypes had lower susceptibility to CHB infection than those with the CC genotype; in the patients ≤45 years, we observed that the risk of CHB infection was significantly lower among patients with the (CG+GG) genotypes. Taken together, the rs1800796 G allele could be recognized as a protective factor of HBV infection, especially in the ≤45 years old and in males. In addition, we found that there was no significant difference in ALT or HBV DNA levels among CHB patients. However, Zhang et al. observed an inconsistent conclusion that the susceptibility allele rs1800796 C was related to elevated ALT levels, but not to HBV viral load in patients with HBV infection (38).

IL-6 belongs to the interleukin family. To our knowledge, the association between IL-6 gene and HBV infection has been demonstrated in previous studies. As a candidate gene, IL-6 might influence the development of chronic HBV infection. Recently, a large body of evidence confirmed that transcription factors such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and AP-1 (Fos/Jun), combined with the corresponding sequences in the promoter region of IL-6, can highly regulate the expression of IL-6 (38). Therefore, the polymorphisms in the promoter region of IL-6 might contribute to the difference between gene transcription and expression, affecting the susceptibility to HBV infection.

Rs1800796 in the IL-6 promoter region has been reported to affect IL-6 expression and production. In the analysis of the influence of rs1800796 on IL-6 production, Zhang et al. measured the plasma IL-6 level and found that individuals with GG genotype have lower plasma IL-6 levels than those with CC genotype (38). Consistently, two studies revealed that possessing an IL-6 re1800796 GG genotype associated with the reduction in IL-6 levels (9,40). Zhang et al. also evaluated the effect of rs1800796 on promoter activity of the IL-6 by reporter luciferase activity. Consistent with the result of IL-6 production assay, rs1800796 polymorphism had a direct effect on transcription activity of IL-6 promoter (38). Thus, IL-6 gene polymorphism might be associated with CHB infection.

There were several limitations in this case–control study. First, the frequencies of IL-6 rs1800796 GG genotype were relatively small and thus statistical power may be insufficient to estimate the correlation of the IL-6 gene polymorphism with chronic HBV infection. Second, we did not conduct studies with regard to functional insights into the role of IL-6 in HBV infection. Thus, functional studies and the analysis of IL-6 expression level are required to further investigate the association between IL-6 gene and HBV infection.

In conclusion, association was identified between polymorphic variant of the wild-type allele of rs1800796 of IL-6 and protection from chronic HBV infection among a Chinese Han population. IL-6 rs1800796 CG genotype and G allele might be considered as protective factors for chronic HBV infection. Thus, determination of the polymorphism the promoter rs1800796 genotypes may be useful for the study of prevention and treatment of chronic HBV infection. Although this study allows for only preliminary conclusions due to limited analysis and should be validated by more functional studies to provide wider insight into the role of IL-6 in chronic HBV infection.

Data Availability Statement

The data used to support the findings of this study are available from the corresponding author upon request.

Ethical Approval

This study was approved by the ethics and research committee of Chongqing Medical University in Chongqing, China.

Footnotes

Acknowledgments

We are particularly grateful to all volunteers for participating in this study.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by the National Natural Science Foundation of China under Grant number 81773519.

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