Effect of Promoter Region Polymorphisms in the IP-10 Gene on HBsAg Loss in Chronic Hepatitis B Patients

Although hepatitis B virus (HBV) infection is preventable through vaccination, it continues to be a major cause of liver-related morbidity and mortality worldwide (1). According to a 2022 report by the World Health Organization (WHO), approximately 254 million people live with chronic HBV infection, which leads to around 1.1 million deaths annually (2). In Turkey, the seroprevalence of hepatitis B is reported to range from 2% to 8% (3). A crucial marker of a positive prognosis in chronic HBV infection is the loss of hepatitis B surface antigen (HBsAg) (4). However, HBsAg clearance is infrequent, primarily due to the persistence of covalently closed circular deoxyribonucleic acid (cccDNA) within hepatocytes. Spontaneous HBsAg loss is estimated to occur at an annual rate of approximately 0.5 - 1% (5). Recent studies have focused on identifying important clinical and viral factors that may help predict the spontaneous clearance of HBsAg. These factors encompass host-related variables such as gender, age, alanine aminotransferase (ALT) levels, presence of fatty liver or cirrhosis, as well as viral features including hepatitis B e antigen (HBeAg) status, HBV deoxyribonucleic acid (DNA) viral load, serum HBsAg levels, HBV genotype, and co-infection with hepatitis C virus (HCV) (6-11). Although these factors offer valuable information, accurately predicting HBsAg loss continues to be challenging. The diverse immune responses seen in chronic HBV patients are thought to be partly due to genetic variability among individuals (12).

Tremendous efforts have identified various host genetic variants influencing HBV infection susceptibility and outcomes, including polymorphisms in human leukocyte antigen (HLA) genes, cytokines, toll-like receptors (TLRs), microRNAs, the sodium taurocholate cotransporting polypeptide (NTCP, the HBV receptor), and vitamin D-related genes. A significant portion of genetic research has concentrated on cytokines and chemokines involved in modulating the immune response against HBV (13). Among these molecules, CXCL10 — also known as interferon gamma (IFN-γ)-inducible protein-10 (IP-10) — has gained attention for its involvement in HBV disease mechanisms. Research indicates that IP-10 facilitates the recruitment of leukocytes from circulation to the liver and stimulates natural killer (NK) cells to produce IFN-γ, contributing to the non-cytolytic clearance of intrahepatic cccDNA (14, 15). Additionally, the G-201A polymorphism located in the promoter region of the IP-10 gene has been associated with disease progression in patients with chronic HBV, likely through its effect on regulating IP-10 expression (16). The polymorphism known as G-201A in Human Genome 19 is specified as -135G/A in Human Genome 38 [(NM_001565.4) c.-135C>T]. Previous studies have examined the relationship between the c.-135C>T polymorphism and disease progression in hepatitis B and C (17-20); however, its direct association with HBsAg loss has not yet been explored.

To our knowledge, this is the first study to directly investigate the relationship between IP-10 promoter polymorphisms and spontaneous HBsAg clearance, aiming to elucidate a potential genetic mechanism underlying functional cure in chronic HBV infection. Based on the existing data, our study hypothesizes that polymorphisms within the IP-10 promoter region could influence the immune response and HBsAg clearance by modulating IP-10 expression. In addition to this polymorphism, we also aimed to investigate other variants in the IP-10 promoter region potentially associated with HBsAg clearance through sequencing analysis.

As one of the main causes of cirrhosis, hepatocellular carcinoma, and liver-related mortality globally, chronic HBV is still a significant infectious illness (21). It is evident that the host immune system plays a pivotal role in determining the development of persistence or clearance during the follow-up period of HBV infection (22). One of the most significant chemokines involved in the proinflammatory response is the IP-10 molecule. Several studies have demonstrated a link between serum IP-10 levels and HBsAg loss. For example, Jaroszewicz et al., Wong et al., and Yuan et al. have all reported that IP-10 levels — either elevated or reduced depending on the disease stage and sampling time — are significantly associated with the likelihood of HBsAg clearance (23-25). In our study, we examined the association between HBsAg loss and polymorphisms in the promoter, exon 1, intron 1, and exon 2 regions of the IP-10 gene in patients with chronic hepatitis B infection. While our study did not demonstrate a statistically significant association between the c.-135C>T, c.85C>T, and c.83T>G polymorphisms in the IP-10 gene and HBsAg loss, this finding should not be interpreted as conclusive evidence against a role for IP-10 in HBV immunity. Rather, our results suggest that these specific variants may have limited predictive value within the context of our cohort. The IP-10 is known to play a complex role in antiviral immune regulation, and its expression and function are likely influenced by a broader network of gene-environment interactions, epigenetic mechanisms, and immune signaling pathways. The lack of association observed in our study may reflect the multifactorial nature of HBsAg clearance, which cannot be explained by individual single nucleotide polymorphisms (SNPs) alone.

The rates of HBsAg clearance differ depending on the cohort studied and the inclusion criteria applied. For example, Buechter et al. followed 371 chronic HBV patients for approximately 12 years and reported an HBsAg loss rate of 7.8% (26). Similarly, Habersetzer et al. conducted a six-year follow-up study involving 315 chronic HBV patients in France, observing an HBsAg clearance rate of 9.2% (27). On the other hand, Chien et al. tracked 235 HBeAg-negative chronic HBV patients over seven years and found a lower HBsAg loss rate of 3.4% (28). In our own study, among 1,950 patients monitored from 2005 to 2022, 5.33% experienced HBsAg clearance. Consistent with previous research, these differences are likely influenced by multiple factors such as patient demographics, ethnic background, disease stage, duration of follow-up, and use of antiviral therapies. Studies have shown that HBsAg clearance is associated with older age and longer follow-up. Chu and Liaw demonstrated that clearance rates rise over time (6), and Buechter et al. reported a mean seroconversion period of 12 years (26). In line with these findings, our study observed a median HBsAg loss time of 15 years, with most cases occurring after 10 years. We also found that clearance was more frequent in individuals over 40. A large cohort study and a separate study from South Korea both reported higher rates of HBsAg seroclearance in patients above 40 compared to younger individuals (29, 30). In our cohort, the average age at the time of HBsAg loss was 53.2 years, and 85% of the cases occurred in patients older than 40. These results highlight age and follow-up duration as important factors in predicting HBsAg clearance.

Prior investigations suggest that certain SNPs in the IP-10 gene promoter are linked to the severity of infectious diseases like pulmonary tuberculosis and cerebral malaria (17). The c.-135C>T (also referred to as G-201A in earlier genome builds) variant has been shown in multiple studies to influence IP-10 expression levels, supported by findings from GWAS (31), protein QTL (32), and its association with HBV and HCV infections. While Talaat et al. reported no correlation between IP-10 polymorphisms and disease progression in chronic HCV patients, Thanapirom et al. highlighted the importance of the -135G/A polymorphism in achieving a sustained virologic response to pegylated IFN therapy (17, 18). The A allele of the G-201A polymorphism in the promoter region of the IP-10 gene has been associated with increased disease severity and progression. In a large-scale study conducted in China involving 2,400 individuals with chronic HBV infection, Deng et al. reported a higher frequency of disease progression among male carriers of the A allele (19). Similarly, Xu et al. found that this polymorphism was linked to liver disease development in HBV-infected patients, with the A allele being more prevalent in those with advanced disease (20). A separate study from Thailand demonstrated an association between the G-201A variant and both reduced HBsAg levels and improved virologic response to pegylated IFN therapy. Patients carrying the GG genotype exhibited lower HBsAg levels and higher response rates; however, no significant difference in HBsAg clearance rates was observed between GG and non-GG genotypes (16). Collectively, these studies suggest that the c.-135C>T polymorphism has previously been examined in the context of disease progression in HCV and HBV, as well as in relation to HBsAg level changes during IFN-based therapy. In contrast, our study directly investigated the association between this polymorphism and HBsAg clearance, finding no statistically significant correlation. Interestingly, we also identified a previously unreported CA genotype in one individual from the control group. Further genetic and functional analyses are needed to determine the clinical significance and potential biological implications of this novel variant. Although c.85C>T has not been linked to HBV, its potential role has been investigated in GWAS and protein QTL studies (31, 33). A study investigating the influence of genetic and lifestyle factors on circulating biomarkers related to inflammation and cancer identified the c.85C>T polymorphism in the IP-10 gene (34). In our research, neither the c.85C>T nor the c.83T>G polymorphism showed a significant association with HBsAg loss. The c.83T>G variant is a novel variant not reported in gnomAD v4.1, TOPMed, or the Turkish Variome, and we considered it potentially significant based on our observations. Therefore, we believe that our study is the first to evaluate the potential association of the c.-135C>T, c.85C>T, and c.83T>G polymorphisms with HBsAg loss in patients with chronic HBV infection. Given the absence of significant associations between the studied IP-10 polymorphisms and HBsAg loss, several methodological and biological factors should be considered. One key limitation is the relatively small sample size, which may have limited the statistical power to detect associations, particularly for low-frequency variants. The SNPs with low minor allele frequencies often require larger cohorts to reach adequate power, especially when investigating complex traits such as viral clearance. Additionally, population-specific genetic backgrounds may have influenced our results.

For instance, the allele frequency of the c.-135C>T polymorphism is reported as 0.402% in the Turkish population according to gnomAD v4.1, whereas it was 18.3% in our cohort. Similarly, the c.85C>T variant has a reported allele frequency of 10.737% in gnomAD, compared to 1.75% in our study population. These discrepancies likely reflect differences between the general population and disease-specific cohorts and may also point to underlying ethnic substructure within the Turkish population (35, 36). Moreover, previous studies that reported associations between IP-10 polymorphisms and HBV-related outcomes were primarily conducted in East Asian populations, where allele distributions and linkage disequilibrium patterns differ substantially from those in our cohort (19, 20). Therefore, population-specific factors must be taken into account when interpreting genetic association studies across diverse ethnic backgrounds. Beyond sample size and population variability, it is also important to consider the biological complexity of HBsAg clearance when interpreting these findings. The investigated polymorphisms may exert only a modest or indirect influence on IP-10 function or expression. HBsAg clearance is a multifactorial event involving a complex interplay between host genetics, immune response, viral factors, and treatment exposure. It is plausible that these individual variants alone are not sufficient to predict seroclearance without considering gene-gene and gene-environment interactions (37).

In addition to genetic and population-specific factors, treatment-related variables may have influenced our findings. Although we observed a significantly lower frequency and shorter duration of antiviral therapy among patients with HBsAg loss, treatment effects were not fully controlled for in the analysis. Given that antiviral therapy is a major modulator of immune-mediated viral clearance, such differences in treatment exposure may have confounded the genetic associations under investigation (38). Another important limitation of our study is the lack of functional analysis to support the observed genetic findings. Our investigation focused solely on genotyping, without evaluating the potential functional consequences of the identified polymorphisms — such as their effects on IP-10 mRNA or protein expression. However, functional validation through transcriptomic, proteomic, or promoter activity assays is essential to establish a mechanistic link between genotype and phenotype. In the absence of such data, the biological relevance of these variants remains speculative (20). Furthermore, the identification of a previously unreported genotype (CA at c.-135) and a novel SNP (c.83T>G) indicates additional genetic diversity within the IP-10 locus that has not been functionally characterized. These rare or population-specific variants may exert unique immunological effects that warrant further investigation in larger, well-characterized cohorts.

Overall, this study provides valuable negative data by demonstrating that the investigated polymorphisms are not reliable predictive markers for clinical outcomes in chronic hepatitis B. The lack of a clear association underscores the complex interplay of host genetic factors in immune-mediated viral clearance, which likely involves multiple genes and signaling pathways beyond IP-10. These findings further highlight the importance of investigating other genetic and immunological determinants — such as cytokine gene polymorphisms, HLA alleles, and components of the innate immune response — to better elucidate the mechanisms driving both spontaneous and treatment-induced HBsAg loss.