TNFA -308G>A and IL10 -1082A>G Polymorphisms as Predictive Biomarkers of Chronic HCV Infection

Genetic changes may induce dysregulated cytokine production and affect the progression of the chronic disease caused by the hepacivirus C (HCV) because the balance of pro- and anti-inammatory cytokines determines the outcome of infection. This study evaluated the TNFA − 308G > A and IL10 -1082A > G polymorphisms in the susceptibility and progress of chronic hepatitis C. The study included 101 samples from patients with chronic hepatitis C and 300 samples from healthy donors. Polymorphisms were typed by real-time PCR and were analyzed for associations with histopathological parameters (according to METAVIR classication) and HCV viral load. The polymorphic genotype for the TNFA − 308G > A variant was not present in the group of patients with chronic hepatitis C and was associated with protection against HCV infection (p = 0.0477). Patients with the polymorphic genotype of the IL10 -1082A > G polymorphism had higher HCV viral load than wild-type patients (p = 0.0428). Neither polymorphism was associated with different levels of necroinammatory activity or brosis scores. The polymorphic genotype at TNFA − 308G > A protected against chronic HCV infection, and the polymorphic genotype at the IL10 -1082A > G variant was associated with viral persistence.


Introduction
Hepacivirus C (HCV) is the causative agent of hepatitis C and is considered the main cause of liver cancer. It is estimated that over 71 million people are chronically infected by the virus and approximately 399 000 people died from hepatitis C, mostly from cirrhosis and hepatocellular carcinoma [1]. Persistent HCV infection leads to chronic hepatitis, which is mainly due to the inability of the immune system to eliminate the virus [2]. Dysregulated cytokine production is related to the chronicity of hepatitis C; however, no pro le of cytokines involved in the development of liver injury has been identi ed [3].
Tumor necrosis factor (TNF-α) is a pro-in ammatory cytokine that acts both as a mediator of innate immunity and in the cellular immune response. Abnormal TNF-α levels have been associated with chronic HCV infection [4]. Some polymorphisms in the TNFA gene are associated with the regulation of cytokine production and coincide with the binding regions of transcription factors [5]. The TNFA − 308G > A polymorphism has higher transcriptional activity than the wild-type allele [6] and has been associated with different infectious diseases [7][8][9][10][11].
Interleukin (IL)-10 is a potent suppressor of the effector function of T cells, natural killer (NK) cells and, mainly, activated macrophages [8]. Several functional polymorphisms have been described in the promoter region of the IL10 gene 10 (12), among which the IL10 -1082A > G polymorphism promotes changes in cytokine levels, with the A allele being related to lower levels and the G allele with higher levels of IL-10 [12]. This polymorphism has been associated with chronic and infectious diseases [13,14].
Because the liver is a highly immunotolerant organ, an imbalance of the components related to its suppressor and effector functions may contribute to the persistence of HCV and the progression of chronic cases of hepatitis C. On this background, the present study investigated the in uence of the TNFA − 308G > A and IL10 -1082A > G polymorphisms on the susceptibility to chronic HCV infection, the progression to different disease stages, and viral persistence. The ndings in this study may help to understand the physiology of the biomarkers analyzed and their response to chronic HCV infection as well as the progression to the hepatic diseases.

Study population
The study included patients, both sexes, treated at the liver disease outpatient clinics of the Santa Casa de Misericórdia do Pará Foundation and the João de Barros Barreto University Hospital. Consecutive patients with chronic HCV were included. The HCV group consisted of 101 patients with chronic hepatitis C, characterized by clinical changes, abnormal liver tests and HCV RNA positivity. For diagram of patient ow chart see Fig. 1.
The inclusion criteria adopted for the individuals were as follows: age 18 or older, and positivity for HBsAg for more than 6 months or positivity for HCV RNA, as criteria for chronic HCV infection, and without antiviral therapy. Individuals coinfected with hepatitis B virus (HBV), hepatitis delta virus, or human immunode ciency virus (HIV) and patients who used or were using speci c antiviral therapy against HCV were excluded from the study. Genotyping TNF − 308G > A (rs1800629) e IL10 − 1082A > G (rs1800896) Polymorphisms were genotyped by quantitative real-time polymerase chain reaction in the StepOne PLUS Sequence Detector (Applied Biosystems, Foster City, CA, USA). The assay used for each polymorphism contained a pair of primers and a pair of VIC-and FAM-labeled probes for the respective alleles. For both polymorphisms, predesigned and customized TaqMan® SNP Genotyping Assays were used: C_7514879_10 for TNFA − 308G > A and C_1747360_10 for IL10 -1082A > G (Thermo Fisher, Carlsbad, California, USA). For each reaction, 2X TaqMan® Universal PCR Master Mix, 1X TaqMan® Assay (diluted from 20X), and 20 ng of DNA was used in a nal reaction volume of 10 µL. The following temperature cycle was used for the ampli cation: 60°C for 30 seconds, 95°C for 10 minutes, and 50 cycles of 92°C for 30 seconds and 60°C for 1 minute and 30 seconds.

Complementary exams
All selected patients were clinically evaluated and subjected to a complimentary investigation consisting of biochemical (liver enzyme levels: alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT), serological (HBV surface antigen (HBsAg), HBV e antigen (HBeAg), anti-HBeAg, total anti-HBc and anti-HCV), virological (HBV DNA and hepacivirus C RNA), ultrasound, endoscopic tests and liver biopsies. These data were transcribed from the medical records into a form developed speci cally for this study.

Histopathological procedures
Liver biopsy specimens were obtained only from patients with medical indications for the investigation of liver parenchyma changes, in compliance with the clinical care protocol. The liver biopsies were performed by a medical professional from one of the study hospitals using a Tru-Cut needle under ultrasound guidance. The sample was sent to the Department of Pathological Anatomy of Federal University of Pará, where they were examined following the department's routines, which included hematoxylin-eosin (HE), chromotrope aniline blue (CAB), Gomori's reticulin, and Shikata's orcein staining.
The histopathological diagnosis followed the METAVIR classi cation [15], which classi es the activity of the portal and periportal in ammatory in ltrate from 0 to 3 (A0-A3), A0-A1 indicating absent to mild in ammation and A2-A3 indicating moderate to severe in ammation. The structural changes in the liver parenchyma (degree of brosis) were classi ed from 0 to 4 (F0-F4), F0-F1 indicating absent to mild liver brosis, F2 indicating moderate liver brosis, and F3-F4 indicating liver brosis that has progressed to cirrhosis. All data regarding the histopathological pro le were obtained from the patients' medical records.

Statistical analysis
Hardy-Weinberg equilibrium analysis was performed in all samples through the chi-squared test. Comparative analyses of the allelic and genotypic frequencies were done using the G-test and chisquared test. Comparisons of viral load levels (HCV RNA) were done using the Kruskal-Wallis test and the Mann-Whitney test. Statistical analyses were done with BioEstat software version 5.3, adopting a signi cance level of p < 0.05.

Results
Most patients with chronic HCV were male (n = 52; 51%). The median alanine aminotransferase, aspartate aminotransferase, and gamma-glutamyltransferase concentrations were 77.9 UI/L, 69.9 UI/L, and 99.6 UI/L, respectively. The mean viral load of 5.4 log (Table 1). Table 1 Characterization of patients with chronic HCV. According to the METAVIR classi cation (Table 1), most patients with HCV had absent to moderate brosis, F0-F2 (n = 67; 66.3%). In ammatory activity was evaluated only in 90 patients because 11 were diagnosed with liver cirrhosis by imaging tests and therefore did not meet the medical indications for biopsy. The majority of the evaluated patients presented with absent or mild in ammatory activity, A0-A1 (n = 54; 60%).
All the genotype frequencies were in Hardy-Weinberg equilibrium. Evaluation of the TNFA − 308G > A polymorphism (rs1800629) showed that no patient with chronic HCV had the homozygous polymorphic genotype (AA). The comparison of genotypic frequencies showed a signi cant difference between the HCV group and control group (p = 0.0477). However, no signi cant differences were found in allelic frequencies ( Table 2). At the IL10 -1082A > G polymorphism (rs1800896), the wild-type genotype and allele (AA and A, respectively) were the most frequent in the HCV and control groups, with no signi cant differences between the groups ( Table 2).
The evaluation of the frequencies of genotypes and alleles of TNFA − 308G > A and IL10 -1082A > G in relation to histopathological markers of in ammatory activity and brosis score showed no signi cant differences (Table 3). The analysis of viral load showed no signi cant difference between patients with different TNFA − 308G > A genotypes. However, patients with the polymorphic genotype of IL10 -1082A > G had higher HCV viral load than those with the wild-type genotype (p = 0.0428; Fig. 2).

Discussion
The course and outcome of HCV infection are determined by its virological characteristics and the immune responses triggered by the virus [16]. HCV is a hepatotropic virus that induces the development of acute and chronic necroin ammatory disease, escaping the immune system in up to 85% of cases [3]. Several cytokines play dual roles in viral infection and are responsible for viral clearance and tissue damage [17].
TNF-α is an important cytokine in the immune response, mediating the in ammatory process through innate immunity pathways and activation of the cellular response, which induces apoptosis or necrosis [18]. Thus, genetic variations in the TNFA gene that alter cytokine production levels may contribute to the progression of HCV infection.
In the present study, the polymorphic genotype for the TNFA − 308G > A variant was not present in the group of patients with HCV. This genotype is correlated with increased expression of the cytokine [19]. In this case, the presence of the homozygous allele may contribute to better immune control, preventing the progression of HCV infection. High levels of TNF-α increase the expression of vascular endothelial adhesion molecules and increase the stimulation of endothelial cells and macrophages, which may lead to better infection resolution [17].
The high frequency of the wild-type allele (G) in the group of patients with chronic HCV infection suggests that in addition to having a higher risk of developing the infection, these patients seem to have a greater chance of developing the chronic form of the disease. The inadequate production of TNF-α by dendritic cells favors the differentiation of CD4 + T cells into IL-10-and non-IFN-γ-producing cells [20]. As the IL-10 cytokine is not effective in resolving the infection, the infection progressed to the chronic form.
Studies on the TNFA − 308G > A polymorphism performed in other ethnic groups also observed different frequencies of the polymorphic genotype in patients with HCV than without, showing that although the presence of the homozygous polymorphism was not observed in patients from France [21], in India the prevalence of the polymorphism was higher in the group of patients with HCV [22]. As the population evaluated in this study is trihybrid, formed from the genetic contributions of whites, blacks, and indigenous people [23], the association of the polymorphism with the prevention or risk of HCV infection needs to be better investigated in other ethnic groups.
IL-10 is an anti-in ammatory cytokine produced by Th2 cells that inhibits the activity of Th1, NK, and macrophage cells, the main cells responsible for pathogen elimination. The cytokine acts by limiting the marked pro-in ammatory response and damage caused by in ammation [8]. In infectious processes, there is a direct correlation between lower IL-10 production and greater disease severity [24].
The IL10 -1082A > G polymorphism is associated with changes in IL-10 level, the wild-type genotype being associated with lower levels [12]. In the present study, no differences in genotype frequencies were found between the groups with and without HCV infection. The evaluation of this polymorphism in HCV infection by other studies has shown different results. Although the present results were similar to those of another study, which also did not nd an association between the frequency of the IL10 -1082A > G polymorphism and susceptibility to HCV infection [25], Ramos et al. [26] observed that the GG (polymorphic) genotype was associated with increased chances of viral infection resolution. The combined analysis of these results shows that the polymorphism does not in uence the protection from or susceptibility to HCV infection but can in uence the disease resolution, reducing the chances of progression to the chronic form among those who develop hepatitis C.
The polymorphic genotype (GG) of the IL10 -1082A > G variant was associated with higher HCV viral load than the wild-type genotype. Most studies that investigated this polymorphism in HCV infection did not assess viral load levels. In the study by Abbas et al. [27], no difference in viral load was observed between genotypes in patients from Pakistan. Viral load has been associated with the frequency of the homozygous genotype (AA) and that of the wild-type (A) allele [28]. The divergence between the results of that study and the present study may be related to the type of analysis performed, Gao et al. [28] evaluated the frequencies of genotypes in relation to the presence or absence of HCV RNA, while the present study evaluated the absolute plasma levels, which were converted to their base-10 logarithm. In addition, the differences may also be related to the ethnicity of the populations assessed between the different studies. The population evaluated in this work is originally from the Brazilian Amazon and has a genetic contribution from Europeans, native Indians and Africans [23], which could contribute to the result found. Some studies have shown that the genetic in uence of ethnicity is associated with variations in genes related to the individual's response to diseases [29,30].
The polymorphic genotype (GG) for IL10 -1082A > G is associated with higher IL-10 expression. This cytokine inhibits the activation of Th1, NK, and macrophage cells, which are the main cells responsible for the elimination of HCV; higher levels of IL-10 reduce in ammatory activity at the infection site, favoring the persistence of the virus in the tissue, the main characteristic of chronic infection [2]. In this sense, our ndings raise the hypothesis that the polymorphic genotype may favor the persistence of HCV in the liver tissue. Follow-up studies are needed to con rm this hypothesis.
The TNFA − 308G > A and IL10 -1082A > G polymorphisms were not associated with different levels of necroin ammatory activity or with brosis score. Several studies have also found no relationship between these polymorphisms in the TNF and IL10 genes and different stages of the disease [21,25,28,31,32). Thus, these polymorphisms seem not to in uence the progression of the histopathological processes of chronic HCV infection because in this disease, in addition to the host immunological factors, others factors inherent to the virus act directly on the in ammatory process, causing tissue damage.
In conclusion, the polymorphic genotype at TNFA − 308G > A was not present in the group of patients with chronic hepatitis C, but we do not know if it could represent a protective action of this SNP against HCV infection. In the same way, considering that this was a cross-sectional study, the polymorphic genotype for variant IL10 -1082A > G need to be better analyzed in a follow-up study in order to con rm its association with viral persistence. Figure 1 Diagram illustrating the ow of participating patients during the study.