Interleukin-6 plays an important role in chronic inflammation as well as tumor growth and progression. Here, a case-control study was undertaken to investigate the association of rs1800796 polymorphism of IL-6 gene and serum levels with disease progression of chronic HBV infection. Rs1800796 polymorphism was genotyped in 641 Chinese Han patients with chronic HBV infection, including 23 IT, 25 IC, 292 CHB, 153 LC, and 148 HCC patients and 265 healthy controls. Serum IL-6 levels were measured in 23 IT, 25 IC, 47 CHB, 41 LC, and 49 HCC patients and 45 healthy controls, and the classifications of HCC were accorded to BCLC staging system. We found no significant association between rs1800796 polymorphism and disease progression of chronic HBV infection; however, serum IL-6 levels showed significant statistical differences between patients with CHB, LC, and HCC. Moreover, statistical differences can be observed in patients with terminal stage HCC compared with those of early to intermediate or advanced stage HCC. Our findings suggest that rs1800796 polymorphism unlikely contribute significantly to affect the progression of chronic HBV infection, and serum IL-6 levels can act as a useful indicator for disease progression and severity of chronic HBV infection.
Hepatitis B virus (HBV) infection is a global public health problem. It is estimated that over 2 billion people have had contact with the HBV, and that 350–400 million people are chronically infected [
Recently, compelling evidence has shown that inflammation has an important role in initiation, promotion, and progression of HBV infection [
Several single nucleotide polymorphisms (SNPs) within the noncoding promoter region of IL-6 gene, including −174G > C (rs1800795), −572C > G (rs1800796), and −596G > A (rs1800797), have been reported to be related to a range of diseases including cancer [
In light of the important role of IL-6 in HBV infection, we hypothesized that genetic polymorphisms of IL-6 and serum levels of the cytokine were associated with disease progression of chronic HBV infection. To test this hypothesis, we performed a case-control study to investigate an eventual correlation between the allelic variations, the circulating levels of the cytokine, and the risk of disease progression.
A total of 641 subjects with chronic HBV infection, including 23 patients in the immune tolerant (IT) stage, 25 patients in the HBeAg-negative inactive carrier (IC) stage, 292 patients with CHB, 153 patients with liver cirrhosis (LC), 148 patients with HCC, and 265 healthy controls, were periodically enrolled between January 2008 and December 2012 at the Zhongnan Hospital of Wuhan University. The subjects were exclusively unrelated Han Chinese and recruited without restriction on gender and age. Patients with HBV infection were confirmed to be HBsAg (hepatitis B virus surface antigen) positive, HBcAb (hepatitis B virus core antibody) positive, and HBeAg (hepatitis B virus e antigen) or HBeAb (hepatitis B virus e antibody) positive for at least 6 months. IT was defined as being HBsAg-positive, HBeAg-positive, HBV-DNA levels >1 × 107 copies/mL, and having normal ALT levels on three or more occasions during at least 1 year of followup. IC was defined as being HBsAg-positive on two occasions at least 6 months apart, HBeAg-negative, HBeAb-positive with persistently normal ALT levels, and HBV-DNA levels <1 × 104 copies/mL. CHB was diagnosed by elevation of alanine aminotransferase (ALT) (≥2 times the upper limit of normal) at least once during the follow-up period, as well as positivity for HBV-DNA. LC was diagnosed pathologically or based on the clinical evidence of portal hypertension such as visible collateral vessels on the abdominal wall, esophageal varices on esophagogastroscopy, palpable splenomegaly, and sonographically definite findings of cirrhotic liver or ascites. HBV-related HCC was diagnosed based on (i) positive findings on cytological or pathological examination; and/or (ii) positive results on computed tomography (CT), magnetic resonance imaging (MRI), or ultrasonography combined; and/or (iii)
Genomic DNA was extracted from peripheral whole blood using the QIAamp DNA Blood Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. Samples were stored at −80°C until genetic polymorphism analyses were performed. Rs1800796 polymorphism genotyping was performed using the TaqMan 5′ allelic discrimination assay technology in a 7500 Real-Time PCR System according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA, USA). Genotyping was performed without knowing the subjects’ case or control status; more than 10% of samples were randomly selected for repeat analysis (which yielded 100% concordance). Finally, 10% of samples were analyzed using an ABI 3730 DNA sequencer (Applied Biosystems, Foster City, CA, USA) to confirm the accuracy of this method.
Venous blood samples were obtained from 23 IT, 25 IC, 47 CHB, 41 LC, 49 HCC patients and 45 healthy controls, respectively. After centrifugation, the serum sample was stored at −80 degree and IL-6 concentrations checked within 2 weeks using commercially available enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, San Diego, CA, USA). Results were expressed in picograms per millilitre (pg/mL) by reference to a standard curve obtained with recombinant IL-6.
Continuous variables were expressed as mean ± SD. Deviation from Hardy-Weinberg equilibrium was tested by using the
General characteristics of the subjects are summarized in Table
General characteristics of subjects.
HC ( | IT ( | CHB ( | IC ( | LC ( | HCC ( | |
---|---|---|---|---|---|---|
Age (y) (mean ± SD) | ||||||
Male, | 193 (72.8) | 16 (69.6) | 212 (72.7) | 17 (68.0) | 111 (72.5) | 110 (74.3) |
HC: healthy control; IT: immune tolerant; IC: inactive carrier; CHB: chronic hepatitis B; LC: liver cirrhosis; HCC: hepatocellular carcinoma.
Genotype and allelic frequencies of rs1800796 polymorphism of the subjects are presented in Table
Genotype distribution of rs1800796 in different groups.
rs1800796 | HC | IT | CHB | IC | LC | HCC | LC versus CHB | HCC versus CHB | HCC versus LC | |||
---|---|---|---|---|---|---|---|---|---|---|---|---|
( | ( | ( | ( | ( | ( | OR (95% CI) | OR (95% CI) | OR (95% CI) | ||||
Genotype, | ||||||||||||
CC | 176 (66.5) | 15 (65.2) | 194 (66.4) | 17 (68.0) | 101 (66.0) | 90 (60.8) | 1.00 | 1.00 | 1.00 | |||
CG | 78 (29.4) | 7 (30.4) | 87 (29.8) | 7 (28.0) | 46 (30.1) | 51 (34.5) | 1.02 (0.66–1.56) | 0.944 | 1.26 (0.83–1.94) | 0.282 | 1.24 (0.76–2.03) | 0.381 |
GG | 11 (4.1) | 1(4.4) | 11 (3.8) | 1 (4.0) | 6 (3.9) | 7 (4.7) | 1.05 (0.38–2.92) | 0.929 | 1.37 (0.52–3.66) | 0.526 | 1.31 (0.42–4.04) | 0.638 |
Dominant model | ||||||||||||
CC | 176 (66.5) | 15 (65.2) | 194 (66.4) | 17 (68.0) | 101 (66.0) | 90 (60.8) | 1.00 | 1.00 | 1.00 | |||
CG + GG | 89 (33.5) | 8 (34.8) | 98 (33.6) | 8 (32.0) | 52 (34.0) | 58 (39.2) | 1.02 (0.67–1.54) | 0.928 | 1.28 (0.85–1.92) | 0.244 | 1.25 (0.78–2.00) | 0.349 |
Hardy-Weinberg | 0.529 | 0.874 | 0.749 | 0.797 | 0.791 | 0.947 |
Note:
As shown in Figure
Serum IL-6 concentrations in HC, IT, CHB, IC, LC, and HCC groups according to different clinical-pathologic stages. NS: no significance; IT: immune tolerant; IC: inactive carrier; HC: healthy control; CHB: chronic hepatitis B; LC: liver cirrhosis; HCC: hepatocellular carcinoma.
Forty nine patients with HCC were classified according to BCLC staging system. IL-6 showed higher expression and significantly statistical differences in patients with terminal stage HCC (stage D,
Serum IL-6 concentrations in different-stage HCC patients according to the classification of BCLC staging system. BCLC-Barcelona Clinic Liver Cancer; HCC: hepatocellular carcinoma.
Cytokine mediated immunity linking innate and adaptive immunities in host may play a crucial role in determining the outcome of HBV infection [
Previous association studies have revealed that host genetic variants are related to the susceptibility to HBV clearance or persistence [
It has already been described that elevated IL-6 levels were detected in chronic liver disease [
As a proinflammatory cytokine, IL-6 is involved in the fibrotic response. Liver cirrhosis is associated with increased intrahepatic mRNA expression of IL-6 [
Our study has some limitations. First, it is a case-control study and a selection bias could not be completely excluded for the group of patients with chronic HBV infection. Second, although the highly significant association between IL-6 and susceptibility to disease progression of chronic HBV infection derives from a prior hypothesis with substantially biological basis, our initial findings should be independently verified in large size of the Chinese population and other different ancestry. Finally, only one polymorphism has been determined at this time. It is likely that with a more refined technology, such as genome-wide association studies, additional polymorphisms will be identified.
From the current study we conclude that −572C > G polymorphism (rs1800796) of IL-6 gene is unlikely to contribute significantly to affect the progression of chronic HBV infection in Chinese population. Moreover, our findings lend support to the notion that serum IL-6 levels can act as a useful indicator for disease progression and severity of chronic HBV infection, which may assist clinicians in selecting high-risk patients for HCC surveillance program.
Alanine aminotransferase
Barcelona Clinic Liver Cancer
Chronic hepatitis B
Confidence interval
Computed tomography
Enzyme-linked immunosorbent assay
Hepatitis B virus core antibody
Hepatitis B virus e antibody
Hepatitis B virus e antigen
Hepatitis B virus surface antibody
Hepatitis B virus surface antigen
Hepatitis B virus
Hepatocellular carcinoma
Hepatitis C virus
Hepatitis D virus
Human immunodeficiency virus
Immune tolerant
Inactive carrier
Interleukin-6
Magnetic resonance imaging
Odds ratio
Single nucleotide polymorphism.
All authors have no conflict of interests to declare.
The work was supported by the National Natural Science Foundation of China (nos. 81172349 and 81272692) and Science and Technology Project of Wuhan (no. 2013060501010153). The authors thank Haitao Wang, Weiliang Shen, and Mancheng Yu (Zhongnan Hospital of Wuhan University, Wuhan, China) for assistance in sample collection.