chronic hepatitis b is an infectious disease caused by hepatitis b virus ( hbv ) , which results in acute or chronic liver diseases . it is estimated that more than 350 million people worldwide are infected by this virus and 75% of them are asians . several evidence indicate that the clinical appearance of infection varies among individuals and includes the following modes : spontaneously recovered infection , chronic and acute hbv , cirrhosis or hepatocellular carcinoma , and liver cancer - related death ( 1 ) . several factors such as characteristics of the virus , environmental factors , and host properties such as age , ethnicity , and genetic background can affect the susceptibility to the chronic hbv infection ( 2 ) . a study on twins in china showed that identical twins might have more incidence of hbeag compare to the non - identical twins . it means that , the incidence of infection is influenced by host genetic factors ( 3 ) . moreover , reports indicate that genetic factors might increase the chronic hbv carrier in some populations ( 4 ) . the studies about the host immune responses indicated a significant association between the t - cell functions and infectious diseases ( 5 ) . more recently , results suggest that chronic hbv infection is an immune related disease , and cellular immune activation was observed through some cytokines such as interleukin 10 ( il-10 ) ( 6 ) and interleukin 28 ( il-28 ) ( 7 , 8) , as well as macrophage migration factor ( 9 ) . in addition , the host genetic background also regulates the natural history of chronic hbv infection ( 10 ) . based on the relationship between cytokines and immune responses , the serum level of cytokines has an effect on disease outcome ( 11 ) . in this regard , single nucleotide polymorphisms ( snps ) , specially in promoter and regulatory regions of the cytokine genes , change the level of cytokine expression and host response , as well ( 12 ) . tumour necrosis factor - alpha ( tnf- ) is a potential pro - inflammatory cytokine , which increases in hbv - infected individuals and induces the clearance of hbv via reducing the replication of hbv in the hepatocytes ( 13 , 14 ) . in - vivo studies indicated that tnf- can reduce the existence of hbv antigen via the regulation of the human leucocyte antigen ( hla)-ii molecules ( 15 , 16 ) . also , tnf- levels vary in subjects with some snps in promoter regions ( 17 - 19 ) . more than 600 snps in the tnf- gene have been identified , which some of them ( -238 g / a , 244 a / g , 308 g / a , 376 a / g , 575 a / g , 857 c / t , 863 c / a , 1031 t / c , 1125 g / c , and 1196 c / t ) are located in the promoter ( 20 ) and can change the production of protein ( 21 ) . ( 22 ) investigated the association of tnf-  gene polymorphisms among iranian pulmonary tuberculosis patients as an infectious disease . they found that polymorphisms in tnf--238 gene were associated with the risk of developing pulmonary tuberculosis . studies in caucasians revealed a positive correlation between -238a allele and chronic hbv infection ( 23 ) . in the korean patients , the resolution of hbv infection also 163g/238g/308g/857c/863c/1031 t and 163g/238g/308g/857c/863a/1031c haplotypes were related to the clearance and persistence of infection , respectively ( 24 ) . ( 25 ) reported that -1031 c , -308 a , and -238 a alleles are related to the increased levels of tnf- , while the -863 a allele is related to the reduced level of tnf-. in above mentioned studies , conflicting results have been reported , hence further studies are necessary . in this case - control study , we aimed to explore whether the tnf- promoter snps ( -238 a / g , -308 a / g , -857 c / t , -863 a / c ) were in association with the outcome of hbv infection in a sample of iranian population . in this study , 100 patients with chronic hepatitis b virus ( hbv group ) ( mean age of 29.03  5.710 and age range of 18 - 43 ) , 40 spontaneously recovered subjects ( sr group ) ( mean age of 29.72  5.517 and age range of 18 - 42 ) , and 100 healthy controls ( c group ) ( mean age of 30.44  4.539 and age range of 22 - 45 ) were recruited . the participants were selected among those who referred to the blood transfusion organization clinics in zahedan , iran , between july and december 2015 . participants were selected based on the following inclusion criteria : hbv patients were positive for hbsag and antibodies against anti - hbc and had impaired liver function test so that their transaminases were twofold more than the normal level for at least six months . in hbv group , serological tests ( presence of hbsag by enzyme - linked immunosorbent assay ( elisa ) ) and clinical findings were compatible with chronic liver disease . the spontaneously recovered group was negative for hbsag and anti - hbc and positive for antibodies against anti - hbs and anti - hbc . the control group comprised hbsag , anti - hbs , and anti - hbc negative , healthy volunteers with normal values for alanine transaminase ( alt ) , without any history of hepatitis b infection . there was no difference between groups in terms of age , gender , and ethnicity . subjects with hcv , hev , hav , hiv , alcohol consumption , drug abuse , and liver diseases were excluded . the study was approved by the institutional ethics committee of zahedan university of medical sciences ( grant number : 6809 ) and carried out in infectious diseases and tropical medicine research center , zahedan , iran . the serum level of tnf- was measured using a sensitive sandwich elisa technique ( eastbiopharm , china ) , according to the manufacturer s instructions . briefly , serially diluted standards and samples were added to the 96-well plates in triplicate . enzyme - labeling reagents ( 50 ml ) were then added to each well , and plates were incubated for 30 minutes at 37c . subsequently , chromogenic substrates a and b ( 50 ml each ) were added and plates were incubated in the dark at 37c for 10 minutes to develop signals . plates were read on a microplate reader to record the optical density values at 450 nm . genomic dna was extracted from the peripheral blood leukocytes using the salting - out method . briefly , 500 l blood was transferred to 1.5-ml microfuge tubes , and 1 ml cell lysis buffer ( 10 mm tris - hcl , 11% w / v sucrose , 5 mm mgcl2 , and 11% v / v triton x-100 ) was added . the solutions were gently mixed and centrifuged for 2 minutes at 6000 rpm at room temperature . next , 400 l buffer ii ( 10 mm tris - hcl , 10 mm edta , and 10 mm sodium citrate ) and 50 l 10% sds were added , and the mixture incubated for 5 minutes at room temperature . then , 100 l saturated nacl and 600 l chloroform were added with gentle mixing , and the mixture was centrifuged for 2 minutes at 6000 rpm . the supernatant was transferred to a new microfuge tube , where 800 l cold isopropanol was added , followed by gentle mixing and centrifugation for 1 minute at 12,000 rpm for 2 minutes at 4c . the supernatant was discarded and 800 l cold 70% ethanol was added . the suspension was gently mixed and centrifuged for 1 minute at 12,000 rpm at 4c . the tnf- polymorphisms ( -238 a / g , -308 a / g , -857 c / t , and -863 a / c ) were detected by polymerase chain reaction - restriction fragment - length polymorphism ( pcr - rflp ) . the primers were designed according to the data achieved from the ncbi data bank ( http : www.ncbi.nlm.nih.gov ) for identification of snps and listed in table 1 . every reaction contained 1 l of each primer , 100 ng of template dna , and 10 l of 2x prime taq premix ( genet bio , korea ) and 7 l of ddh2o in a 20 l of total reaction volume . the pcr conditions were as follows : initial denaturation at 95c for 5 minutes followed by 35 cycles of denaturation at 95c for 30 seconds , annealing at 58.5c for -238 a / g , 59c for -308 a / g , 63c for -857 c / t , and 52c for -863 a / c polymorphisms of the tnf- for 30 seconds and extension at 72c for 30 seconds , followed by a nal extension step at 72c for 5 minutes . finally , the pcr products digested by the restriction enzymes ( fermentas , vilnius , and lithuania ) and digested products were resolved by electrophoresis in 4 % agarose gel and stained with ethidium bromide ( figure 1a , b , c , and d ) . ( a ) , the digestion pattern of msp i restriction enzyme on 4% agarose gel at -238 a / g snp ; m marker 100bp , 1 genotype aa , 2 genotype ag , 3 genotype gg ; ( b ) , the digestion pattern of ncoi restriction enzyme on 4% agarose gel at -308 a / g snp ; m marker 100bp,1 genotype aa , 2 genotype ag , 3 genotype gg ; ( c ) , the digestion pattern of hincii restriction enzyme on 4% agarose gel at -857 c / t snp , m marker 100 bp,1 genotype tt , 2 genotype ct , 3 genotype cc ; ( d ) , the digestion pattern of styi restriction enzyme on 4% agarose gel at -863 a / c snp ; m marker 100bp,1 genotype aa , 2 genotype ac , 3 genotype cc . data were reported as mean values  sd for parametric variables and as percentages for non - parametric values . the statistical analyses were performed using spss statistical package , version 17.0 for windows ( spss . pearson s  test ( for categorical variables ) and kruskal - wallis h test ( for continuous variables including age ) were used to examine differences among the three groups in the distributions of demographic characteristics and genotype frequencies . the frequency of the genotypes in patients and control groups was estimated by direct gene counting . the risk of hbv infection associated with snps was estimated using odds ratios ( ors ) and 95% confidence intervals ( 95% cis ) calculated by a multivariate logistic regression model after adjustment for sex , age , and ethnicities . a p value of less than 0.05 was considered significant at 95% confidence interval ( ci ) . in this study , 100 patients with chronic hepatitis b virus ( hbv group ) ( mean age of 29.03  5.710 and age range of 18 - 43 ) , 40 spontaneously recovered subjects ( sr group ) ( mean age of 29.72  5.517 and age range of 18 - 42 ) , and 100 healthy controls ( c group ) ( mean age of 30.44  4.539 and age range of 22 - 45 ) were recruited . the participants were selected among those who referred to the blood transfusion organization clinics in zahedan , iran , between july and december 2015 . participants were selected based on the following inclusion criteria : hbv patients were positive for hbsag and antibodies against anti - hbc and had impaired liver function test so that their transaminases were twofold more than the normal level for at least six months . in hbv group , serological tests ( presence of hbsag by enzyme - linked immunosorbent assay ( elisa ) ) and clinical findings were compatible with chronic liver disease . the spontaneously recovered group was negative for hbsag and anti - hbc and positive for antibodies against anti - hbs and anti - hbc . the control group comprised hbsag , anti - hbs , and anti - hbc negative , healthy volunteers with normal values for alanine transaminase ( alt ) , without any history of hepatitis b infection . there was no difference between groups in terms of age , gender , and ethnicity . subjects with hcv , hev , hav , hiv , alcohol consumption , drug abuse , and liver diseases were excluded . the study was approved by the institutional ethics committee of zahedan university of medical sciences ( grant number : 6809 ) and carried out in infectious diseases and tropical medicine research center , zahedan , iran . the serum level of tnf- was measured using a sensitive sandwich elisa technique ( eastbiopharm , china ) , according to the manufacturer s instructions . briefly , serially diluted standards and samples were added to the 96-well plates in triplicate . enzyme - labeling reagents ( 50 ml ) were then added to each well , and plates were incubated for 30 minutes at 37c . subsequently , chromogenic substrates a and b ( 50 ml each ) were added and plates were incubated in the dark at 37c for 10 minutes to develop signals . plates were read on a microplate reader to record the optical density values at 450 nm . genomic dna was extracted from the peripheral blood leukocytes using the salting - out method . briefly , 500 l blood was transferred to 1.5-ml microfuge tubes , and 1 ml cell lysis buffer ( 10 mm tris - hcl , 11% w / v sucrose , 5 mm mgcl2 , and 11% v / v the solutions were gently mixed and centrifuged for 2 minutes at 6000 rpm at room temperature . next , 400 l buffer ii ( 10 mm tris - hcl , 10 mm edta , and 10 mm sodium citrate ) and 50 l 10% sds were added , and the mixture incubated for 5 minutes at room temperature . then , 100 l saturated nacl and 600 l chloroform were added with gentle mixing , and the mixture was centrifuged for 2 minutes at 6000 rpm . the supernatant was transferred to a new microfuge tube , where 800 l cold isopropanol was added , followed by gentle mixing and centrifugation for 1 minute at 12,000 rpm for 2 minutes at 4c . the suspension was gently mixed and centrifuged for 1 minute at 12,000 rpm at 4c . the tnf- polymorphisms ( -238 a / g , -308 a / g , -857 c / t , and -863 a / c ) were detected by polymerase chain reaction - restriction fragment - length polymorphism ( pcr - rflp ) . the primers were designed according to the data achieved from the ncbi data bank ( http : www.ncbi.nlm.nih.gov ) for identification of snps and listed in table 1 . every reaction contained 1 l of each primer , 100 ng of template dna , and 10 l of 2x prime taq premix ( genet bio , korea ) and 7 l of ddh2o in a 20 l of total reaction volume . the pcr conditions were as follows : initial denaturation at 95c for 5 minutes followed by 35 cycles of denaturation at 95c for 30 seconds , annealing at 58.5c for -238 a / g , 59c for -308 a / g , 63c for -857 c / t , and 52c for -863 a / c polymorphisms of the tnf- for 30 seconds and extension at 72c for 30 seconds , followed by a nal extension step at 72c for 5 minutes . finally , the pcr products digested by the restriction enzymes ( fermentas , vilnius , and lithuania ) and digested products were resolved by electrophoresis in 4 % agarose gel and stained with ethidium bromide ( figure 1a , b , c , and d ) . ( a ) , the digestion pattern of msp i restriction enzyme on 4% agarose gel at -238 a / g snp ; m marker 100bp , 1 genotype aa , 2 genotype ag , 3 genotype gg ; ( b ) , the digestion pattern of ncoi restriction enzyme on 4% agarose gel at -308 a / g snp ; m marker 100bp,1 genotype aa , 2 genotype ag , 3 genotype gg ; ( c ) , the digestion pattern of hincii restriction enzyme on 4% agarose gel at -857 c / t snp , m marker 100 bp,1 genotype tt , 2 genotype ct , 3 genotype cc ; ( d ) , the digestion pattern of styi restriction enzyme on 4% agarose gel at -863 a / c snp ; m marker 100bp,1 genotype aa , 2 genotype ac , 3 genotype cc . data were reported as mean values  sd for parametric variables and as percentages for non - parametric values . the statistical analyses were performed using spss statistical package , version 17.0 for windows ( spss . pearson s  test ( for categorical variables ) and kruskal - wallis h test ( for continuous variables including age ) were used to examine differences among the three groups in the distributions of demographic characteristics and genotype frequencies . the frequency of the genotypes in patients and control groups was estimated by direct gene counting . the risk of hbv infection associated with snps was estimated using odds ratios ( ors ) and 95% confidence intervals ( 95% cis ) calculated by a multivariate logistic regression model after adjustment for sex , age , and ethnicities . a p value of less than 0.05 was considered significant at 95% confidence interval ( ci ) . study subjects were divided into three groups : patients with chronic hbv infection ( female / male : 43.0/57.0 ) , patients who spontaneously recovered from infection ( female / male : 37.5/62.5 ) , and healthy people ( female / male : 49.0/51.0 ) . the gender ratio ( female / male ) was not significantly different between the 3 groups ( p = 0.427 ) . the mean age in the study groups was not statistically different ( p = 0.125 ) . the prevalence of various ethnicities was also not different between hbv , sr , and c groups ( p = 0.292 ) . genotype results at positions -238 a / g , -308 a / g , -857 c / t , and -863 a / c in the tnf- gene are listed in tables 3 and 4 . the distribution of the polymorphisms was in hardy - weinberg equilibrium ( p > 0.05 ) . for -308 a / g , -857 c / t , and -863 a / c snps , we found that compared to the healthy group ( c + sr ) , chronic hbv patients have signicantly higher g , c , and a alleles and gg , cc / ct , and aa / ac genotypes frequency , respectively . this study showed that the existence of -308 g , -857 c , and -863 a alleles signicantly increased the risk of chronic hbv infection compared to the other alleles . also , these results showed that there is not any relationship between -308 g , -857 c , and -863 a alleles and elimination of hbv . for -238 a / g polymorphism , no signicant differences were found in allele or genotype frequencies between the groups . we conducted the -238 a / g , -308 a / g , -857 c / t , and -863 a / c haplotype association analyses and found 5 genotypes . as shown in tables 5 and 6 , the distribution frequency of haplotypes showed a signicant association with chronic hbv infection compared to the c and sr groups . in our study , ggca , ggcc , and ggta haplotypes had a higher frequency in hbv patients than c and sr groups . also , -238 a / g , -308 a / g , -857 c / t , and -863 a / c polymorphisms indicated enrichment of ggca haplotype among patients than healthy controls ( c + sr ) ( 48.0% vs. 30.7 , p = 0.005 ) . linkage disequilibrium ( ld ) analysis did not show any significant statistical relationship between any of the 4 positions . tnf- levels in hbv patients , sr subjects , and c group were 1.41  0.062 ( pg / ml ) , 1.99  0.159 ( pg / ml ) , and 2.03  0.193 ( pg / ml ) , respectively . we found a significant difference in the serum levels of tnf- between the groups ( p < 0.05 ) . the serum levels of tnf- significantly decreased in the chronic hbv patients compared to the healthy groups ( c + sr ) ( p < 0.05 ) . statistical analysis indicated positive correlations between tnf- genotypes and serum levels of the tnf- in hbv patients ( table 7 ) . chronic hbv patients with -308 gg / ag , -857 cc , and -863 aa genotypes had lower levels of tnf- compared to those with -308 aa ( p = 0.000/0.001 ) , -857 tt ( p = 0.009 ) , and -863cc ( p = 0.041 ) genotypes , respectively . the results showed that the risk of hbv infection significantly increased in subjects with at least one of -308 g , -857c , and -863a alleles that were effective in reducing the levels of tnf- , compared to the other alleles ( table 8) . genotype results at positions -238 a / g , -308 a / g , -857 c / t , and -863 a / c in the tnf- gene are listed in tables 3 and 4 . the distribution of the polymorphisms was in hardy - weinberg equilibrium ( p > 0.05 ) . for -308 a / g , -857 c / t , and -863 a / c snps , we found that compared to the healthy group ( c + sr ) , chronic hbv patients have signicantly higher g , c , and a alleles and gg , cc / ct , and aa / ac genotypes frequency , respectively . this study showed that the existence of -308 g , -857 c , and -863 a alleles signicantly increased the risk of chronic hbv infection compared to the other alleles . also , these results showed that there is not any relationship between -308 g , -857 c , and -863 a alleles and elimination of hbv . for -238 a / g polymorphism , we conducted the -238 a / g , -308 a / g , -857 c / t , and -863 a / c haplotype association analyses and found 5 genotypes . as shown in tables 5 and 6 , the distribution frequency of haplotypes showed a signicant association with chronic hbv infection compared to the c and sr groups . in our study , ggca , ggcc , and ggta haplotypes had a higher frequency in hbv patients than c and sr groups . also , -238 a / g , -308 a / g , -857 c / t , and -863 a / c polymorphisms indicated enrichment of ggca haplotype among patients than healthy controls ( c + sr ) ( 48.0% vs. 30.7 , p = 0.005 ) . linkage disequilibrium ( ld ) analysis did not show any significant statistical relationship between any of the 4 positions . tnf- levels in hbv patients , sr subjects , and c group were 1.41  0.062 ( pg / ml ) , 1.99  0.159 ( pg / ml ) , and 2.03  0.193 ( pg / ml ) , respectively . we found a significant difference in the serum levels of tnf- between the groups ( p < 0.05 ) . the serum levels of tnf- significantly decreased in the chronic hbv patients compared to the healthy groups ( c + sr ) ( p < 0.05 ) . statistical analysis indicated positive correlations between tnf- genotypes and serum levels of the tnf- in hbv patients ( table 7 ) . chronic hbv patients with -308 gg / ag , -857 cc , and -863 aa genotypes had lower levels of tnf- compared to those with -308 aa ( p = 0.000/0.001 ) , -857 tt ( p = 0.009 ) , and -863cc ( p = 0.041 ) genotypes , respectively . the results showed that the risk of hbv infection significantly increased in subjects with at least one of -308 g , -857c , and -863a alleles that were effective in reducing the levels of tnf- , compared to the other alleles ( table 8) . the current study showed a significant association between tnf- polymorphism and susceptibility to chronic hbv infection , which support some previous studies in other countries and emphasizes the critical role of tnf- in chronic hbv infection . our results showed that -308 gg , -857 cc / ct , and -863aa / ac genotypes were associated with the susceptibility to the hbv infection , and -308 g , -857 c , and -863 a alleles were clearly related to the disease . in addition , the ggca haplotype was more frequent in hbv patients , confirming that tnf- polymorphism can be a risk factor for the infection . it has been recognized that host genetic background same as virus profile , can affect the outcome of hbv infection ( 26 ) . a study on twins showed an increased rate of hbv infection in identical twins in comparison with non - identical twins , which indicates the effect of host genetic on the disease ( 3 ) . tnf- , as an important pro - inflammatory cytokine , can also regulate transcription of other immune regulatory proteins , which amplify immune responses against infections ( 3 ) . in vitro studies have indicated inhibitory effects of tnf- on the hbv virus replication and increasing hbv mrna destruction ( 27 ) . also , serum level of tnf- may be associated with the progression of the disease ( 28 ) . it has been revealed that the amount of tnf- was significantly different in hbv patients compared to the simultaneously recovered subjects from hbv ( 29 ) . an in vivo study found that tnf- reduced infection by destabilizing hbv nucleocapsids and reducing the cccdna ( 30 ) .   in this regard , snps in promoter region might change the expression of protein at the transcriptional and post - transcriptional levels . some studies have identified tnf- promoter snps and their association with the susceptibility to hbv infection , but the results are conflicting because of the different inclusion criteria , various genotypes of the virus , and insufficient sample size . in our study , the results revealed a signicantly increased risk of hbv infection because of the presence of the tnf- -308 g , -857 c , and -863 a alleles in the iranian population . the tnf- gene is linked to a cluster of genes such as lymphotoxin - a ( lta ) that relates to the disease progression ( 31 ) . tnf- can bind to the tnf receptors 1 and 2 on parenchymal cells ( such as hepatocytes ) and leucocytes and then mediate immune responses against various infectious pathogens in human ( 32 ) . in addition , tnf- polymorphisms regulate the rna polymerase ii - binding and allele - specic lta transcription in the host immune system ( 33 ) . the current study did not demonstrate a significant association between -238 a / g polymorphism and hbv infection . however , the expression of tnf- is affected by snps , but we did not find any difference among -238 a / g genotypes with regard to the serum levels of tnf-. in accordance with our findings , du et al . the current study indicated a relative increase in the frequency of -238 gg genotype and g allele in hbv infected patients ; but it was not significant . the lack of signicance is probably attributed to the complication of the human s immune response and also may be due to the low sample size and other genetic or environmental factors . by the way , the analysis of this polymorphism in hbv infection has shown conflicting results . it has been determined that the binding capabilities of allele - specic binding elements with trans factors mediated the relationship between polymorphisms and protein expression . tnf- -238 a / g is located in the regulatory sequences of the promoter named y - box which can bind to the dna - binding proteins . therefore , genetic variation in the y - box will change the functional activity of the promoter ( 36 ) . the results showed that the frequency of -308 gg genotype in the chronic hbv patients was higher than that in the sr and c groups . also , in hbv patients the amount of tnf- decreased , which could be explained by the existence of the -308 g allele in a way that -308 g can inhibit the production of the tnf- and then intensify the disease . they highlighted the key role of -308 a / g polymorphism both in susceptibility to hbv infection and increased serum tnf- level in hbv patients and emphasized that the presence of g at the position -308 increased the risk of hbv infection . however , our findings were different from the results of some studies ( 41 - 43 ) claiming that the a allele may have a role in hbv aggravation . in addition , somi et al . ( 44 ) found that tnf- promoter polymorphism -308 was common in iranian population , but they did not find any association between this snp and development of chronic hbv infection . their failure to find an association may be related to the type ii error , and larger studies may provide more helpful results . also , it seems that ethnic differences have an effect on the distribution of cytokine gene polymorphisms . therefore , variation in the study population could account for the different frequencies in our population and these contradictions might be due to different study populations . increased distribution of the -857 ( cc + ct ) genotypes in the hbv patients might be due to lower transcriptional activities in subjects with the c allele . it was revealed that -857 tt genotype is associated with higher expression of tnf- and inhibition of hbv infection in subjects spontaneously recovered from hbv ( 45 ) . it was also noted that , tnf- -857 c / t polymorphism was located in the binding sequence of the octamerbinding protein ( oct1 ) . -857 c / t snp might affect the interaction between the oct1 and binding sequence , leading to a change in the transcriptional function of the gene ( 46 ) . in the line with our findings , hohjoh et al . ( 48 ) reported that -857 c allele is associated with the reduced production of the tnf- and increased the risk of disease because of the afore mentioned mechanism . also , it is probable that the combination of transcriptional regulators of hbv to the gene promoter changes with the presence of snps , which causes different trans activities . our study indicated the -863 a / c polymorphism as a risk factor for chronic hbv infection . we found that the existence of at least one -863 a allele in subjects increased the severity of the infection , which was associated with lower serum tnf- levels ( 49 ) . also , we observed that tnf- production reduced in subjects with a allele compared to the subjects with the c allele among the hbv patients . in agreement with our findings , in vivo studies with the hepatoblastoma cell line indicated that -863 a allele is associated with the reduction of tnf- receptor expression ( 49 , 50 ) . it should be noted that -863 a allele can bind to the p50/p65 complex of the downstream nuclear factor - kb ( nf-b ) transcription factor , and c allele can bind to both p50/p50 and p50/p65 . this different affinity leads to the change in the downstream nf-b activation ( 51 ) . therefore , -863a / c various genotypes might be responsible for the different tnf- serum levels and susceptibility to the hbv infection . ( 52 ) did not find statistically signicant difference in tnf- -863 polymorphism between hbv patients and healthy controls . the haplotype ( -238 a / g , -308 a / g , -857 c / t , and -863 a / c ) analysis revealed that the ggca haplotype with low production of tnf- was a potential risk factor for hbv infection . interestingly , haplotype analysis showed a strong association between tnf- and hbv , and emphasized that the susceptibility to disease may be due to the influence of tnf- polymorphisms on the promoter region of the gene . consequently , we can obtain a reasonable evidence of the relationship between chronic hepatitis b and tnf- polymorphisms . our results demonstrate an increased risk for chronic hbv infection with tnf- -308 gg , -857 cc , and -863 aa genotypes . also , the carriers of -238 g , -308 g , -857 c , and -863 a alleles with lower amount of tnf- have a serious risk of infection compared to the subjects who do not have these alleles . the identication of appropriate diagnostic biomarkers would develop the best diagnostic approach and clinical management for hbv . some disagreements with the current study indicate the possible impact of genetic background of different populations . on the other hand , several factors , such as sample size , patients selection , different cultural backgrounds , epidemiological and geographical factors , study conditions ( such as number and characteristics of the subjects and hbv genotype variations ) and different gene - gene interactions also can cause importing results . additional studies with regard to the abovementioned limitations should provide additional understanding of tnf- gene variant in chronic hbv infection and provide good predictors of disease and therapeutic tools for subjects who are at risk .
backgroundthe host genetic background regulates the natural history of chronic hepatitis b virus ( hbv ) infection.objectivesthe aim of this study was to investigate the association between tnf- gene polymorphism in the promoter region and susceptibility to chronic hepatitis b virus infection.methodsfour polymorphisms of tnf- gene including -238 a / g , -308 a / g , -857 c / t , and -863 a / c were analyzed by pcr - rflp in 100 chronic hbv infected patients ( hbv group ) , 40 spontaneously recovered hbv subjects ( sr group ) , and 100 healthy controls ( c group ) . also , serum levels of protein were monitored.resultsthe study showed that the existence of -308 g , -857 c , and -863 a alleles signicantly increased susceptibility to chronic hbv infection . 
 in addition , ggca haplotype had a higher frequency in hbv patients than c and sr groups that might be related to the natural history of the infection . 
 chronic hbv patients with -308 gg , -857 cc , and -863 aa genotypes had lower serum levels of tnf- compared to those with other genotypes.conclusionsthe results indicated that there was a positive association between susceptibility to chronic hbv infection and tnf- polymorphism . 
 in addition , hbv patients carrying -308 gg , -857 cc , and -863 aa genotypes with lower serum levels of tnf- had an increased risk of infection .