| Background and Objective: Hepatitis B virus(HBV)infection is one of the major infectious diseases with more than 350 million chronic carriers worldwide, which causes a broad spectrum of liver diseases ranging from asymptomatic carrier, fulminant hepatitis, chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Persistent HBV infection has been considered as a multifactorial and polygenic disorder with viral, environmental and genetic components. HBV genomic variability and a number of conventional risk factors, including age, gender, and concurrent infection with other viruses, are clearly the important factors contributing to the incidence of persistent HBV infection. However, segregation analysis and twin studies strongly support the role of host genetic components in determining the chronicity of HBV infection. Genetic association analyses based on the Gambia, European and Asian cohorts have implicated the HLA allele DRB1?1302, tumor necrosis factorα(TNF-α),mannose binding protein(MBP)and vitamin D receptor were associated with the persistent HBV infection or HBV clearance. An unknown number of other unidentified genes are likely to modify the susceptibility to persistent HBV infection. For chronic HBV infection, genetic associations are likely to provide some clues to viral persistence and disease progression, and might lead to a new therapeutic approach.The influences of genomic background are confirmed in more and more diseases along with the developing of genomic medicine. Single nucleotide polymorphisms (SNPs)occupy about 90% of human DNA variation, and are regarded as most important genomic tag. The role of cytokines in the viral clearance and the host immune response to the virus has been investigated. Several proinflammatory cytokines such as Th1 cytokines(including IL-2 and IFN-γ) and TNF-αare believed to participate in elimination of HBV. In contrast, IL-10, a Th2 cytokine, acts as a potent inhibitor of Th1 effector mechanisms. There is some evidence that the capacity for cytokine production in individuals has a major genetic component. These have been ascribed to polymorphisms within the regulatory regions or signal sequences of cytokine genes. The aim of the present study was to clarify whether the inheritance of TNF-α, IFN-γ, IL-10 and IL-18 gene promoter or coding regions SNPs in Han nationality from Hubei Province of Chinese could serve as a candidate for determining clinical outcomes of the disease caused by chronic HBV infection. In addition, IL-18 is involved in infectious and immuno-inflammatory diseases. Different individuals may have different capacities for IL-18 production. This might determine a predisposition to development some complications or phenotypes of these diseases. The aims of our study were to explore if single-nucleotide polymorphisms(SNPs)at position–137,-607 in the promoter and at position 105 in the coding region of IL-18 gene can influence its gene expression.Methods: A case-control panel was undertaken to study the polymorphisms of TNF-αgene,IFN-γgene,IL-10 gene and IL-18 gene. The single nucleotide polymorphisms(SNPs)in TNF-αgene promoter marked as–238G/A, -308G/A, -857C/T and–863C/A, in promoter of IL-10 gene at position -1082G/A,-819T/C,-592A/C and in coding region 105A/C of IL-18 gene were detected with polymerase chain reaction with restriction fragment length polymorphism(PCR-RFLP). The SNPs in the first intron +874T/A of IFN-γgene and in the promoter region of IL-18 gene at position–607C/A and–137G/C were detected with polymerase chain reaction with sequence specific primers(PCR-SSP)and sequencing. The genotypes and alleles frequencies were calculated and analyzed. Peripheral blood mononuclear cells(PBMCs)of healthy individuals were separated and incubated for 24hours after LPS stimulation, finally, the production of IL-18 was determined by ELISA assay and IL-18 mRNA on PBMCs measured by reverse transcription polymerase chain reaction(RT-PCR).Results:Part I. Polymorphism of tumor necrosis factor-αgene promoter and its association with susceptibility to chronic hepatitis B in Chinese Han people1. Twelve different promoter genotypes were detected from all of 126 control subjects and 131 patients with chronic hepatitis B, and GG·GG·CC·CC, GG·GG·CC·CA, GG·GG·CT·CC, GG·GA·CC·CC genotypes accounted for about 85% of genotypes in these subjects.2. By analyzing the four promoter genotypes of TNF-α, the results showed that–238G/A and–857C/T SNP sites polymorphisms were not associated with susceptibility to chronic hepatitis B, whereas the genotypes of–308G/A and–863C/A SNP sites were associated with susceptibility to chronic hepatitis B in Chinese Han people.3. In respect to the alleles frequencies of the four SNP sites respectively, the Chinese Hans were similar to Japanese and HongKong people, but different from whites.Part II. Association of the +874T/A polymorphisms in the first intron of interferon-γgene with susceptibility of hepatitis B virus infection1. A highly significant difference in the distribution of IFN-γgene polymorphism at position +874 was observed between patients with chronic HBV infection(TT, TA and AA frequencies were 9.1%, 12.1% and 78.8%, respectively) and controls(TT, TA and AA frequencies were 12.6%, 23.0% and 64.4%, respectively)(χ2=9.60, P=0.008), but there was no significant difference between individuals who spontaneously recovered from HBV infection(TT, TA and AA frequencies were 13.9%, 23.7% and 62.4%, respectively) and controls(χ2=0.16, P=0.92).2. No statistically significant difference in frequencies of IFN-γgenotypes and alleles at position +874 were found between patients with chronic HBV infection with low- and high-level HBV replication. 3. Compared with other ethnic individuals, A highly statistically significant difference in the distribution of the IFN-γgene polymorphism(intron 1 at position +874 )was observed between Chinese Hans and Italian(χ2=33.8, P=0.001), and Chinese Hans and Israel(χ2=13.1, P=0.001).Part III. Association of interleukin-10 gene promoter polymorphisms with hepatitis B virus infection in Chinese Han population1. No significant difference was found in frequencies of genotypes and alleles of IL-10 gene promoter region at position -1082G/A,-819T/C,-592A/C among normal controls, individuals spontaneously recovered from HBV infection and patients chronically infected with HBV(P>0.05).2. No significant difference was also found in frequencies of genotypes and alleles of IL-10 gene promoter region at position -1082G/A,-819T/C,-592A/C between patients with HBV infection with HBV-DNA <1×103 copies/mL and those with HBV-DNA≥1×103 copies/mL(P>0.05).3. However, frequencies of TT genotype at position–819T/C and AA genotype at position–592A/C in chronic hepatitis B were significantly higher than that in a symptomatic HBV carriers(P<0.05).Part IV. Association of interleukin-18 gene polymorphisms with disease progression in patients chronically infected with hepatitis B virus1. The frequency of CC genotype in IL-18 gene promoter region at position–607 was 22%(66/300)and 27%(62/231)in 300 normal controls and 231patients respectively. The frequency of CA genotype was 53%(160/300)and 50%(116/231)in normal controls and patients respectively. The frequency of AA genotype was 25%(74/300)in normal controls and 23%(53/231)in patients. The frequencies of GG,GC and CC genotype in IL-18 gene promoter region at position–137 were 67%, 30% and 3% in normal controls respectively, while in patients chronically infected with HBV were 79%, 19% and 2%. While the frequencies of AA,AC and CC genotype in the coding region 105 site of IL-18 gene were 69%, 29% and 2% in normal controls respectively, in patients chronically infected with HBV were 78%, 21% and 1%. Theχ2 test showed that the genotype frequencies of–137GG in the promoter(χ2=8.55,P=0.003)and 105AA in coding regions(χ2=6.734,P=0.034) in chronic hepatitis B virus infection groups was significantly higher than that in normal controls, but the frequencies of–607C/-137C and–607A/-137C haplotypes in chronic hepatitis B virus infection groups were significantly lower than that in normal controls.2. The relation of IL-18 gene promoter and coding regions polymorphisms with HBV copies and disease progression showed that the frequencies of–607AA genotype in low HBV-DNA copies group and a symptomatic carriers were higher than that in high HBV-DNA copies group(χ2=6.03,P=0.014)and chronic hepatitis B group(χ2=7.77,P=0.021).3. Compared with Sweden, Poland and Australia population, there was significant different in the distributions of the SNP(P< 0.05). But compared with Japanese, there was similar in the distributions of the SNP at position -137 in the promoter of IL-18 gene(P> 0.05).4. The concentrations of IL-18 in individuals PBMCs with CC,CA and AA genotypes of IL-18 gene promoter at position–607 were(11.54±6.48)ng/mL,(10.92±5.16)ng/mL and (11.79±3.18)ng/mL, respectively, and the levels of IL-18 mRNA were 0.878±0.633,0.877±0.521and 0.881±0.400. There were no significant differences among IL-18 productions of individuals PBMCs with different genotypes at position -607 in the promoter of IL-18 gene. The concentrations of IL-18 in individuals PBMCs with GG,GC or CC genotypes of IL-18 gene promoter at position–137 were(11.27±5.42)ng/mL,(11.31±4.62)ng/mL, respectively, and the levels of IL-18 mRNA were 0.835±0.485 and 0.984±0.613. There were no significant differences among IL-18 productions of individuals PBMCs with different genotypes at position–137 of IL-18 gene. The concentrations of IL-18 in individuals PBMCs with AA and AC genotypes in coding region 105A/C of IL-18 gene were(11.26±5.38)ng/mL and(11.31±4.59)ng/mL, respectively, and the levels of IL-18 mRNA were 0.846±0.493 and 0.966±0.613.There were no significant difference among IL-18 productions of individuals PBMCs with different genotypes at position 105 in the coding region of IL-18 gene.Conclusion:1. There is an association between polymorphisms of the promoter region–308G/A and–863C/A of TNF-αand chronic hepatitis B. Subjects with TNF-α-308GA, -863CA genotypes may be resistant to HBV infection, which gives some new clues to the study of pathogenesis of chronic hepatitis B.2. There is an association between the +874T/A SNP in the first intron of IFN-γgene and chronic HBV infection. This study suggested the possibility that IFN-γgene polymorphism at position +874 might be important in determining an individual's susceptibility to development of chronic HBV infection.3. It is probable that genetic polymorphisms of interleukin-10 promoter region are not associated with both susceptibility of HBV infection and HBV-DNA replication after infected HBV in Chinese Han population, but polymorphisms of the promoter region of IL-10 gene at position -819T/C and -592A/C are related to inflammatory reaction in the liver of the patients chronically infected with HBV.4. The polymorphisms in the promoter region at position–607C/A,–137G/C and in coding region 105A/C of IL-18 gene are associated with the development of chronic hepatitis B. The people with -137C allele in the promoter and /or 105C allele in the coding region of IL-18 gene may be protected against HBV infection, and the IL-18–607AA genotype may be linked to HBV-DNA copy. However, SNPs at position–137,-607 in the promoter and at position 105 in the coding region of IL-18 gene could not influence its gene expression. |
PDF Full Text Request