Insights into the Correlation between Toll-Like Receptor 2 Polymorphism and HBV-Related Disease Progression and Occurrence of Hepatocellular Carcinoma: A Case-Control Study in Egyptian Patients

Methods In total, 170 chronic HBV patients and 50 healthy controls of comparable age and gender were included in this case-control study. Clinical, laboratory, and imaging evaluations were conducted. ELISA was used to determine serum IL-6 levels, and TLR2 (rs3804099) genotyping allelic discrimination assay was performed using real-time PCR. Results IL-6 values were significantly higher in the HCC group, followed by the cirrhotic group, than those in chronic hepatitis and control groups (p < 0.001), with a significant correlation with disease activity and progression parameters. TRL2 homozygous TT was the most frequent in the control group, but the CC genotype was significantly more prevalent in the HCC group than that in the other groups. Furthermore, the CC genetic variant was associated with higher levels of IL-6 and viral load in all HBV patients, whereas the TT genotype was associated with larger tumor size. Multivariate regression analysis demonstrated that in chronic HBV patients, viral load and TRL2 polymorphism are independent risk factors associated with the progression from chronic hepatitis to liver cirrhosis and to HCC. Similarly, the HBV viral load (p=0.03, OR = 2.45, and 95% CI: 1.69–3.65), IL-6 levels (p=0.04, OR = 3.45, and 95% CI: 2.01–6.9), and TRL2 variants (p=0.01, OR = 4.25, and 95% CI: 2.14–13.5) are independent risk factors associated with disease progression from cirrhosis to HCC. Conclusion In chronic HBV patients, TRL2 polymorphism and higher IL-6 levels were positively correlated with a higher likelihood of HCC and chronic hepatitis B disease activity and progression.


Introduction
Hepatitis B infection is a common infectious disease of the liver caused by a hepatotropic virus known as the hepatitis B virus (HBV) [1].Over two billion individuals have been infected with HBV globally, with over 290 million developing chronic HBV infections [2].HBV infection is known to result in clinical manifestations that guarantee acute, fulminant, chronic, and occult forms.In the chronic form of HBV infection, the immune system fails to clear the virus from hepatocytes and sera [1].Furthermore, prolonged HBV infections could induce liver cirrhosis and may alter cell conditions, causing hepatocellular carcinoma (HCC) [3].
Chronic hepatitis B (CHB) progression is substantially afected by host, viral, and environmental factors [4].Te interaction between viral and host genetic characteristics triggers viral infection susceptibility and disease progression, additionally infuenced by metabolic, physiological, and environmental factors [5].Han et al. discovered that HBV mutations signifcantly impact the efect of miRNA single nucleotide polymorphisms (SNPs) on vulnerability to HBVassociated hepatocellular carcinoma.Consequently, infections by a particular HBV genotype could be linked to host genetic polymorphisms.Furthermore, individual genetic predisposition may infuence HCC development risk [6].
TLRs (toll-like receptors) are critical pattern recognition receptors.Tey have crucial functions in immune and infammatory control, including antigen presentation, innate immune response and acquired immune response, and, most importantly, cytokine gene expression.TLR2 is indeed an outer membrane receptor that responds primarily to bacterial surface-associated pathogen-associated molecular patterns (PAMPs) in humans [7].TLRs are thought to play a crucial role in the pathophysiology of a diversity of liver diseases because they are expressed in all types of liver cells, which comprise hepatic stellate cells (HSCs), Kupfer cells (KCs), and hepatocytes, as well as immune cells such as hepatic dendritic cells [8].TLR-mediated infammatory signaling pathways are linked to a variety of liver diseases, which include alcoholic and nonalcoholic liver disease, ischemia/reperfusion injury, hepatitis, liver fbrosis, cirrhosis, regeneration of the liver, and HCC [9].
Since TLRs are considered major molecules in immune responses to HBV infection, it was hypothesized that they also have essential functions in either eradicating HBV or inducing virus complications such as liver cirrhosis and HCC [10].Tus, more research into the causal relationship between TLR genetic polymorphisms and the progression of HBV infection is required.
Variations in various cytokine activities have been observed during the course of HBV infections; however, an imbalance in proinfammatory and anti-infammatory cytokine release impacts their immunopathogenesis [11].Interleukin-6 (IL-6) is the primary mediator of infammation and acute phase responses in the liver; it may also moderate chronic disease progression by safeguarding T cells from apoptosis during the infammation process [12].Additionally, IL-6 has pivotal roles in the malignant transformation of HCC progenitor cells and the growth of HCC [13].
Terefore, the purpose of the current research is to investigate the role of IL-6 and TLR2 gene polymorphism in chronic active hepatitis B and how they afect the progression of HBV-associated liver disease (liver cirrhosis and HCC).Chronic hepatitis B was identifed by the detection of HBsAg for more than six months, and liver cirrhosis was confrmed by clinical evaluations, laboratory investigations, and pelvi-abdominal ultrasonography.According to the WHO guidelines on the treatment of patients with CHB, the cutof values for FibroScan were 7 to 8.5 kPa for the diagnosis of signifcant liver fbrosis and 11 to 14 kPa for the diagnosis of cirrhosis, respectively [14].Moreover, triphasic CT abdomen was done to confrm HCC diagnosis (arterial enhancement with delayed washout).Patients with hepatic focal lesions other than HCC, those who had liver diseases other than HBV, such as chronic hepatitis C, autoimmune hepatitis, and metabolic hepatitis, and those with a history of hepatotoxic drug use or alcohol consumption were excluded from this study.

Materials and Methods
Patients and controls were subjected to a thorough history, clinical evaluation, and laboratory tests such as complete blood count, liver function tests, renal function tests, alpha-fetoprotein (AFP), and serological tests for viral markers by ELISA HBsAg, HIV, and HCV antibodies.Realtime PCR was conducted to assess the HBV viral load.Radiological evaluation was carried out for all participants using pelvi-abdominal ultrasonography, FibroScan for staging of fbrosis, and triphasic abdominal CT to confrm HCC diagnosis.Baseline computed tomography of the chest, abdomen, and pelvis and bone scans were performed to assess distant metastases in HCC patients.Moreover, the Child-Turcotte-Pugh (CTP) score was assessed, and esophagogastroduodenoscopy was done to determine the severity of liver disease and presence of gastroesophageal varices, respectively, in patients with liver cirrhosis and HCC.

Ethical Consideration
Following the development of the research questions and objectives, each participant was informed about the study and asked to provide informed consent before participating.Te research was approved by the Research Ethics Committee, Faculty of Medicine, Menoufa University, Egypt (IRB: 1/2023TROP2), and was conducted in conformity with the Declaration of Helsinki.

Laboratory Investigations
In total, 10 mL of whole blood was collected from all participants from the cubital vein by venipuncture, 4 ml were collected into 2 EDTA tubes, one for immediate CBC measurement, then was centrifuged and plasma was preserved for IL6 assessment and the second was preserved at −80 till TLR2 SNP analysis.2 mL was collected into sodium citrate tube for INR.Te remaining 4 mL were collected into plain tube and centrifuged at high speed and the resultant serum was used to measure LFTs and AFP.

IL-6
Assay.IL-6 plasma level assessment: According to the manufacturer's instructions, the Human IL-6 ELISA Kit (enzyme-linked immunosorbent assay, Sigma-Aldrich, Germany, product number RAB0306) was used for measuring plasma IL-6 levels.Within thirty minutes, the results were read at 450 nm.Te IL-6 concentrations in each sample were calculated after a standard curve was constructed.

DNA Extraction.
As per the manufacturer's instructions (Termo Scientifc, Lithuania, Gene jet whole blood genomic DNA purifcation Mini kit).Te human genomic DNA has been extracted from whole venous blood collected in tubes containing EDTA by a spin column technique.Each qPCR reaction amplifcation volume is twenty μL, composed of: 0.5 μl of genotyping (primer/probe mix) assay, 10 μL of genotyping qPCR Master Mix (2×) (Termo Fisher Scientifc, MA, USA), 3.5 μL of DNAse-free water and 6 μl (0.1 μg/ μL) of genomic DNA.6 μL of DNAse-free water has been added to the negative control reaction.

Real-Time PCR.
Te PCR and genotyping have been carried out.Te following cycling parameters were set: for 1 minute; holding stage at 60 °C, at 95 °C for 2 minutes' initial denaturation of genomic DNA occurred, 35 repeats of following: for 20 seconds denature at 94 °C, for 20 seconds denature at 94 °C, and for 20 seconds annealing at 55 °C, for 30 seconds extension at 72 °C.Furthermore, at 72 °C for 2 minutes' fnal extension occurred and eventually holding stage for 1 minute at 60 °C.

SNP Analysis of TLR2 Gene.
Te TLR-2 SNP identifcation: Te TaqMan allelic discrimination assay was performed for rs3804099 genotyping.Using TaqMan 5′ nuclease assay chemistry for amplifcation and detection of specifc SNP alleles variants in purifed genomic DNA samples for the SNP Genotyping Assays.Every predesigned TaqMan SNP genotyping assay comprises 2 allele-specifc TaqMan MGB probes that contains unique fuorescent dye as well as a PCR primer pair for identifying specifc targets of SNP utilizing 96 well 7500 real-time fast PCR instrument (Termo Fisher Scientifc Inc., Life Technologies TM, CA, USA).To ofer unmatched specifcity for the allele of interest, these TaqMan probe and primer sets (assays) align in a unique way with the genome.Samples that contain both alleles 1 and 2 represented the heterozygotes.Meanwhile, samples containing the same allele (either allele 1 or allele 2) demonstrated the homozygotes.

Statistical Analysis.
Te data were collected and analyzed by SPSS (Statistical Package for Social Science) version 20.0 on an IBM-compatible computer (SPSS Inc., Chicago, IL, USA).Qualitative data were described as frequency and percentage and analyzed using the chi-square test, while quantitative data was represented as mean, standard deviation, median, and range and was analyzed between the four studied groups using the ANOVA test when the data were normally distributed.Kruskal-Wallis test was used to compare multiple groups with normally distributed data, and the post hoc test was employed to compare the groups in pairs.Spearman correlation was performed to correlate IL-6 and other quantitative parameters.Te crude OR measured the risk of exposure to mutant genotypes.Regression analysis is a statistical process for estimating independent risk for progression of liver disease.Te P value was considered signifcant when it was less than 0.05.

Results
Te demographic, clinical, imaging, and upper endoscopy characteristics of the studied participants are summarized in Table 1.In total, 220 participants were included in this casecontrol study.Tere were 50 age-and gender-matched healthy controls (group I), 58 patients with chronic hepatitis B (group II) with a mean age of 54.57 ± 9.56 years, 62 patients with HBV-related liver cirrhosis (group III) with a mean age of 54.92 ± 10.88 years, and 50 patients with HBVrelated HCC (group VI) with a mean age of 56.8 ± 8.87 years.A nonsignifcant diference was observed between the studied groups in terms of age and gender (p value � 0.68 and 0.16, respectively).According to the clinical evaluation of studied patients, there were no signifcant diferences observed between the cirrhotic and HCC groups in terms of the presence of jaundice, history of hepatic encephalopathy, ascites grade, and presence of esophageal varices in upper endoscopy (p value � 0.86, 0.76, 0.79, and 0.35, respectively).
Regarding radiological evaluation of the studied patients, fbrosis staging by FibroScan difered signifcantly between the three patient groups (p < 0.001), where, in chronic hepatitis B groups, F1, F2, and F3 were 15.5%, 55.2%, and 29.1%, respectively, and all cirrhotic patients (100%) were F4.However, in the HCC group, six (12%) patients were F3 and Canadian Journal of Infectious Diseases and Medical Microbiology the rest (88%) were F4.Te ultrasonography and triphasic CT fndings in patients' groups are displayed in Table 1.
Concerning laboratory investigations, all parameters of complete blood count showed signifcant diferences between the studied groups, with lower platelet counts were observed in the cirrhotic and HCC groups.Moreover, liver function tests and alpha-fetoprotein were signifcantly different among the four groups (p < 0.001 for all).HBV-DNA copies were higher in the cirrhotic group than those in other groups (p � 0.002).IL-6 was found to have higher levels in   1).Additionally, the T allele was the most prevalent in control, chronic hepatitis, and cirrhotic groups, while the C allele represents the most prevalent one in the HCC group.
Table 6 demonstrates a signifcant association between TLR-2 genetic variants with parameters of disease activity and progression in chronic hepatitis and cirrhotic groups.For both groups, patients carrying the homogenous CC genetic variant had higher values of ALT, IL-6 (Figure 2), and viral load than the other variants.Additionally, as a marker of disease progression, the presence of the homogenous CC genetic variant is associated with higher mean values of total bilirubin and INR (P � 0.02, 0.07), and with lower mean values of serum albumin (P � 0.01).
According to Table 7, patients carrying the homogenous CC genetic variant of TLR-2 had higher values of IL-6 (Figure 2) and viral load (P � 0.047 and 0.04, respectively).Additionally, those with the CT and CC variants had larger tumor sizes (P � 0.03).

Discussion
Liver cancer is the third leading cause of cancer-related death and ranks sixth as the most commonly diagnosed cancer.It accounts for 8.3% of cancer deaths overall and 4.7% of newly diagnosed cases.In 2021, liver cancer was responsible for approximately 30,000 deaths [15,16].Being the most common histological type of liver cancer, HCC accounts for more than 75% of all primary liver cancers worldwide [17], indicating its signifcant burden.
Because of the disease's poor prognosis, the incidence and mortality rates for HCC are roughly equal.Te signifcant variation in HCC incidence and mortality rates throughout geographical areas could be explained by variations in environmental exposure, timing, and the prevalence of chronic viral hepatitis, access to healthcare resources, and the ability to detect and treat cancer early [18].Te majority of HCC cases are reported in individuals with underlying liver disease, the majority of which is caused by chronic hepatitis B and C virus infection or excessive consumption of alcohol.In addition to the viral and environmental risk factors, individual genetic predisposition may infuence the risk of HCC development.Considerations of heritability and familial aggregations supported the infuence of genetic factors [19][20][21].Despite the immunization program's great success, a large number of patients have chronic HBV infection and, as a result, are at increased risk of cancer.Host immunity is indeed essential in the progression and HBV infection outcome [22].In chronic HBV infection, IL-6 is a potent pleiotropic multifunctional infammatory cytokine that is a crucial driver of hepatocyte repair and replication [23].TLRs are a type of receptor that serves as the frst line of defense against microbes.Tey are capable of recognizing both invading pathogens as well as endogenously dangerous molecules produced by damaged tissues and dying cells.Being expressed on DCs that link innate and adaptive immunity, TLRs serve as bridging molecules [24].
We investigated serum IL-6 and TRL2 rs3804099 gene distribution in healthy controls and HBV-infected patients.Furthermore, we explored the association between them and disease progression, liver function parameters, and risk of HCC development, as well as the tumor characteristics in HBV patients.
In the current study, IL-6 levels were considerably higher in the HCC group, followed by the cirrhotic group, than those in the chronic hepatitis and control groups.IL-6 exhibited a signifcant positive association with ALT, AST, and HBV-DNA copies in chronic hepatitis and HCC groups, as well as a signifcant positive correlation with serum creatinine in cirrhosis and HCC groups.Additionally, serum IL-6 was found to be signifcantly associated with AFP in HCC patients.
It has been reported that IL-6 is highly expressed in the early stages of both acute and chronic liver injury [25].As an infammatory mediator, IL-6 may contribute to the progression of HBV-associated liver cirrhosis [26].Furthermore, Xia et al. previously demonstrated that the levels of IL-6 rise with the expression of hepatitis B Virus X protein (HBx) in the hepatocytes, and this could be explained by MyD88-(myeloid diferentiation factor 88) dependent manner production of HBx-induced IL-6 [27].In this research, we noticed that higher values of IL-6 were linked with higher levels of ALT and AST, pointing out the IL-6's role as an indicator of liver cell necrosis.Furthermore, IL-6 is positively associated with total bilirubin and INR while being negatively associated with serum albumin and platelets, implying that IL-6 might potentially be used as a marker for the progression of liver disease.Tese fndings are consistent with those of previous studies that revealed a link between IL-6 levels and biochemical markers of liver disease [28,29].
A meta-analysis study found that IL-6 concentrations were considerably higher in the HCC group than in healthy participants and patients with liver cirrhosis [30].A fourmarker model incorporating IGF2, IL-6, AFP, and platelet count could discriminate HCC from the liver with an AUC of 0.97 [31].El-Folly et al. reported that high serum IL-6 levels could predict HCC development in chronic hepatitis B patients and that the diagnostic value of IL-6 improved when combined with AFP measurement.Additionally, they stated that the gathering of IL-6 and AFP values may provide new hope for earlier detection of HCC in HBV patients [32].It was reported that IL-6 trans-signaling is required to promote the development of HCC by preventing DNA-damageinduced hepatocyte apoptosis and inducing endothelial cell proliferation, which in turn promotes tumor angiogenesis [33].
When HBV patients were compared to control subjects in terms of TRL2 rs3804099 gene distribution, we found that the CC homozygote and CT heterozygote gene variants were the least frequent in the controls.However, in HBV patients, both CC and CT gene variants are more prevalent, especially in HCC patients.Similarly, the T allele was the most common in controls, while the C allele was more prevalent in cirrhotic and HCC groups.Elbrolosy et al. found TRL2 polymorphism to be related to disease progression in chronic HBV infection in Egyptian patients, which is consistent with our fndings.Tey reported that chronic HBV-infected patients with the mutant TT genotypes or heterozygous CT of TLR2 rs3804099 experienced less disease activity compared to those with the CC variant [29].
TLRs are a type of pattern recognition receptor family that serves as the foundation of the innate immune response [7].Toll was discovered as a gene that controls the dorsalventral polarity of the Drosophila embryo; nevertheless, later studies revealed that it is implicated in antifungal immunity [34].Te discovery of these receptors increased the importance of the innate immune system.Tey can recognize both the external PAMPs and the internal damageassociated molecular patterns (DAMPs) [24].
TLRs can identify various viral components, such as nucleocapsids, envelope peptides, and nucleic acids, activate signaling pathways, and intensify the release of proinfammatory cytokines and interferons [35].Tere is mounting evidence that cytokine-mediated immune responses have a substantial impact on the clinical outcomes of HBV infections [36].Hepatitis B virus-mediated hepatic TLR2 signaling may postpone, but not preclude, HBV spread and persistent replication in hepatocytes.HBV infection can be detected and responded to by both the circulating and intrahepatic innate immune systems.Regrettably, the strong response is also responsible for hepatic necroinfammation, resulting severe liver damage [37].
Tis study identifed substantial diferences in genotype frequencies and allelic distribution for TLR rs3804099 among the studied groups.We discovered that the CC genotype and C allele were signifcantly more prevalent in the HCC group when compared to chronic hepatitis and cirrhotic groups, implying a role in HCC development.Tis is supported by multivariate regression analysis, revealing that TRL2 gene variants and HBV viral load are independent risk factors for the development of HCC in HBV-related chronic hepatitis and liver cirrhosis.8 Canadian Journal of Infectious Diseases and Medical Microbiology TLR2 has been linked to a variety of cancers.TLR2 (Delta22) polymorphisms were linked to an increased risk of gallbladder cancer [38], while TLR2 microsatellite GT polymorphism has been linked to sporadic colorectal cancer in Croatians [39].Additionally, a link between TLR2 (−196 to −174 del) and cervical cancer susceptibility [40], as well as HCC development in HCV patients, was reported [41].
TLR2 rs3804099 is a synonymous SNP.For a long time, it was assumed that synonymous SNPs were insignifcant because they did not change the polypeptide structure.Notwithstanding, since synonymous mutations were proven to be involved in diseases in numerous studies over the past decade, this concept has changed.Te following mechanisms may be involved in the efect on gene function: mRNA structure, mRNA stability, mRNA splicing changes, and protein folding [42].
Although TLRs are important components of the innate immune system and play an important role in the hostdefensive mechanism against microbes, overactivation of TLRs could disrupt immune homeostasis, resulting in excessive proinfammatory cytokine production, which is undoubtedly a contributing factor to the pathogenesis of several infammatory and autoimmune diseases [42].Tereby, TLR signaling pathway inhibition is expected to be Canadian Journal of Infectious Diseases and Medical Microbiology a benefcial therapeutic strategy for suppressing undesired, disease-linked infammatory responses, which can be achieved using two main methods: blocking TLR ligand binding to the receptor or interrupting with intracellular signaling pathways to halt signal transduction [43].Furthermore, some clinical trials indicated that therapeutic manipulation of TLR pathways could serve as a novel strategy for reversing chronic liver diseases [44].
Tis study has some limitations, as follows.First, this is a cross-sectional study, which does not allow us to follow up on the same patients at diferent stages of HBV infection.Terefore, a longitudinal study could aid in more precise detection of the actual impact of IL-6 levels and TLR2 rs3804099 SNP variation on liver disease progression and outcome in HBV patients.Second, the study has small sample size; thus, larger studies are warranted to validate our results.

Conclusion
Te current study revealed that TLR2 rs3804099 polymorphism and IL-6 were positively correlated with CHB disease activity and progression.Terefore, they may be performed practically to assess the disease progression in CHB patients and those who are more susceptible to developing HCC.
2.1.Study Design and Participants.Tis case-control study was performed at Menoufa University's Departments of Tropical Medicine, Internal Medicine, Clinical Oncology, Radiology, and Clinical Pathology in collaboration with the Clinical Biochemistry and Molecular Diagnostics Department, National Liver Institute.In total, 220 participants were included in this study.Tey were chosen from Tropical Medicine, Internal Medicine, and Clinical Oncology departments' inpatient and outpatient clinics between January 2023 and May 2023.Tese participants were categorized into 58 HBV-linked chronic hepatitis patients, 62 patients with HBV-related cirrhosis, 50 patients with HCC on top of HBV infection, and 50 healthy controls of comparable age and gender.

Table 1 :
Comparison between the studied groups regarding clinical data, ultrasound, CT scanning, and upper endoscopy.
the HCC group, followed by the cirrhotic group, than that in chronic hepatitis and control groups (p < 0.001), as shown in

Table 2 .
Table3lists the genotype frequencies and allelic distribution of the TLR2 polymorphism (rs3804099) in the studied groups.When HBV patients were compared to

Table 2 :
Comparison between the studied groups regarding laboratory parameters and serum IL-6 level.

Table 3 :
Genotypes frequencies and allelic distribution for TLR-2 polymorphism among the studied groups.

Table 4 :
Risk assessment (odds ratio) of TLR-II polymorphism between the HCC group and both chronic hepatitis and cirrhotic groups.

Table 5 :
Correlation between IL-6 profle and disease activity parameters among CHBV groups.

Table 6 :
Correlation between rs3804099 genotypes of TLR2 and disease activity parameters and IL-6 profle among Chronic Hepatitis B and cirrhosis groups.

Table 7 :
Correlation between rs3804099 genotypes of TLR2 and disease activity parameters and IL-6 profle in HCC group.