Association between Signal Transducer and Activator of Transcription 4 Genetic Polymorphisms and the Spontaneous Clearance of Hepatitis B Surface Antigen: A Large Population Case Control Study in China

How to cite this article: Qi X, Jiang Q, Lv Y, Yang S, Li J, Huang Y, Chen L, Zhang J. Association between Signal Transducer and Activator of Transcription 4 Genetic Polymorphisms and the Spontaneous Clearance of Hepatitis B Surface Antigen: A Large Population Case Control Study in China. J Biomed Res Environ Sci. 2020 Oct 31; 1(6): 256-262. doi: 10.37871/jbres1152, Article ID: JBRES1152 ORIGINAL ARTICLE


INTRODUCTION
Hepatitis B Virus (HBV) infection is a major global health problem, though the eff ective HBV vaccine is widely used. HBV infects about 2.5 billion people worldwide and 300 million of those are chronically infected [1,2]. China has one of the highest HBV carrier prevalences in the world. It is estimated that there are approximately HEPATOLOGY | GASTROENTEROLOGY 30 million Chronic HBV (CHB) patients in China, almost 5% -6% of the entire population [3]. About 95% of adults can successfully clear HBV after being infected and 5% -10% will develop CHB; even fewer progress to liver failure [4]. Several host and viral genetic factors are known to infl uence susceptibility to HBV infection, ability to clear the virus, and maintenance of a chronic state. Among these factors, host immunogenetic background has a signifi cant role in mediating the clinical progression of viral hepatitis infections [5][6][7][8].
Signal Transducer and Activator of Transcription 4 (STAT4), a member of the STAT family, can be activated by several cytokines including IL 12, type I Interferon (IFN), and IL 23, as well as lead to T helper (Th) type 1 and 17 diff erentiation, monocyte activation, and production of IFN- [9]. In fact, more and more studies indicated that STAT4 variations are correlated with the risk of developing autoimmune diseases. The Single Nucleotide Polymorphism (SNP) rs7574865, which is in the third intron of the STAT4 gene, is associated with a higher risk of rheumatoid arthritis [10][11][12][13], Systemic Lupus Erythematosus (SLE) [14], type I diabetes [15], and systemic scleroderma [16]. Furthermore, it is reported that the G allele of rs7574865 can increase the risk of Hepatocellular Carcinoma (HCC) and decrease the effi cacy of IFN treatment in CHB patients [17][18][19].
However, few studies have assessed the relationship between STAT4 variations and Hepatitis B surface Antigen (HBsAg) clearance in adults infected with HBV, and the results of these studies have been inconsistent. Our aim was to evaluate the association between the genetic variations in STAT4 and HBsAg clearance in HBV infected adults in a large sample size population.

Patients
This case control study enrolled 1,858 of 2,398 patients with CHB infection, aged 18-70 years, who were followed up at our hospitals between March 2016 and December 2018.
These patients were divided into two groups: CHB patients, and HBsAg Clearance after Treatment (TC). There were 1,688 and 170 patients in each group, respectively. In the CHB group, the patients were categorized into four subgroups according to the natural history [20]

HEPATOLOGY | GASTROENTEROLOGY
in which all HBV markers were negative. SC subjects were defi ned as those who were negative for HBsAg, but positive for both the Hepatitis B surface Antibody (HBsAb) and Hepatitis B core Antibody (HBcAb). All control subjects had no other ailments and had normal liver function ( Figure 1).
The study was in complied with the Helsinki Declaration. Our Center's Ethics Committee reviewed and approved the study design (KY2016-196) and all patients provided written informed consent.

Study design
All data of the subjects were collected at the baseline, including history, liver function, and White Blood Cell Count (WBC), Platelet Count (PLT), and HBV markers. In CHB infected patients, HBV DNA load and HBV genotype were tested.

DNA isolation and rs7574865 genotyping
Genomic DNA was extracted from 1 ml of peripheral whole blood from each subject using Qiagen DNA kits (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The STAT4 variants were genotyped using a realtime Polymerase Chain Reaction (PCR) in a 50 μl reaction system including 20ng of genomic DNA. The thermal cycling conditions applied were initial activation for 5 min at 95°C, 40 cycles of denaturation at 94°C for 30s, annealing at 57°C for 30s, and extension at 72°C for 30s. All the samples were analyzed by direct sequencing.

Statistical methods
All data were processed using Stata 16

Baseline characteristics of the subjects
In this study, 5,823 subjects were screened, and 3,922 met the inclusion criteria and were included ( Figure 1).
The proportion of male, age, and the values of Alanine Transaminase (ALT) and Aspartate Aminotransferase (AST) were higher in the CHB and TC groups than in the HC and SC groups. However, the values of WBC and PLT in the CHB and TC groups were lower than in the HC and SC groups. In CHB patients, the major HBV genotype was the C genotype, and the percentage of Hepatitis B e Antigen (HBeAg) positive patients was 56% (Table 1).

Association between STAT4 polymorphisms and HBsAg clearance
To evaluate the relationship between rs7574865 variation and HBsAg spontaneous clearance, the SC group was compared with the CHB, TC, and HC groups. The frequency of the G allele in STAT4 rs7574865 was higher in the CHB and TC groups, compared with the SC group (CHB vs. SC: 70% vs. 67%, p = 0.018; TC vs. SC: 72% vs. 67%, p = 0.047)

Association between STAT4 polymorphisms and HBV natural history
The relationship between the rs7574865 variation and HBV natural history was also analyzed. In the recession   Lu, et al. [28] found the association between SNP rs7574865 and HBV spontaneous clearance in a study including 288 subjects. However, in two studies, Liao, et al. [29,30]  INF- production [18]. INF- is a critical cytosolic factor to control antiviral and immune response [33]. Therefore, we speculated that subjects infected with HBV with rs7574865 T may express more STAT4 genes and proteins, produce more INF-, and have a higher chance of clearing HBV naturally.
However, there are more factors infl uencing HBsAg clearance after treatment, including drug types, duration of the treatment and the host factor [18,19]. Therefore, there is no association between HBsAg clearance after treatment and STAT4 variations.
Furthermore, the relationship between CHB patients in diff erent natural history phases and SNP rs7574865 was analyzed. The results indicated that there was no signifi cant association between genetic variations in STAT4 and CHB natural history. In addition, genetic variations in STAT4 were not correlated with the ALT, load of HBV DNA, and HBeAg positive in CHB patients, which is consistent with the results reported by Lu, et al. [28].
Although the sample size of the study is large, there are still limitations. This is a case control study, which has a weaker evidence power than the prospective study due to lack of follow-up information. Moreover, there were more confounders than in the prospective study, although logistic regression analysis was used. Therefore, matched case control studies are warranted to obtain more potent evidence.
In summary, SNP rs7574865 in STAT4 is correlated with HBV spontaneous clearance. Subjects with the T allele had a higher chance of clearing HBV naturally than patients with the G allele. However, there is no relationship between genetic variations in STAT4 and CHB natural history phases.