Relationship Between Single Nucleotide Polymorphisms In IL28B Gene And Response To Interferon Treatment In Chronic Hepatitis B Patients

Introduction Hepatitis B virus (HBV) is one of the most serious and prevalent health problems, effecting more than2billion people worldwide. There are still more than350million chronic carriers,75%of whom reside in the Asia Pacifi c region. People with HBV are at increased risk of developing hepatic decompensation, cirrhosis, and hepatocellular carcinoma. The estimated worldwide mortality is0·5to1·2million a year. HBV has extremely complicated natural history, from self-limited infection to chronic infection, from acute hepatitis to chronic hepatitis, and even in a proportion of servere patients, HBV can cause liver failure, cirrhosis, and hepatocellular carcinoma.Chronic Hepatitis B (CHB) account for the majority in the patients who should be acccept antiviral therapy, Hepatitis B virus replication is the key to liver injury and disease progression,and, therefore, the main aims of treatment are to suppress the virus to achieve HBeAg seroconversion or undetectable viral-DNA levels, or both; stop or reduce hepatic necroinflammation; and prevent the development of hepatic decompensation. Long term goals are to reduce cirrhosis and hepatocellular carcinoma development, and ultimately extend survival. Viraemic patients with an ALT concentration of twice the upperlimit of normal or more, or substantial liver disease, are candidates for drug therapy.Two strategies are available for treatment of chronic hepatitis B:interferon and nucleos(t)ide analogues, which mechanism of action are totally different. Nucleos (t) analogues directly suppress the virus effectively. When compared to IFN-a. nucleos(t)ide analogues have several advantages and a few pitfalls. The first issue lies in the fact that the length of treatment for nucleos(t)ide analogues is as yet undefined, and may possibly last for life. Nucleos(t)ide analogues induce HBeAg seroconversion rates that are proportional to the length of treatment, but it has been reported that most patients (-80%) serorevert to HBeAg once the treatment is stopped. Patients also have to face the risk of emergence of drug resistance at the same time. Once it occurs, the initial clinical and histological benefits of antiviral therapy diminish. Interferon. including Interferon alfa and pegylated interferon, have immunomodulating activity, and influence the immune system of host. Not only does the HBeAg seroconversion rate following IFN-a2a therapy continue to increase during the six months after the discontinuation of treatment, but the seroconversion status remains stable over time in70-87%of patients who had achieved it at the end of therapy. Theoretically, interferons are the ideal treatment for patients with chronic hepatitis B.Interferon-based therapy is preferred in patients with compensated liver disease particularly in young patients, women of childbearing age, and those with low ALT values because of the finite duration of treatment, sustained response, and long term benefits including prevention of hepatocellular carcinoma. But the response rate after4-6months of interferon alfa is only30-40%in HBeAg-positive patients, while22-30%in HBeAg-negative patients.However, the question remains unresolved as to why only a certain percentage of patients respond to therapy. Hence, the search for predictive factors determining therapeutic responses continues. In HBeAg positive chronic HBV patients, the most important predictors of good response to IFN-a treatment include high ALT levels (>200U/L), low serum HBV DNA (<108copies/mL), female gender, and active fibrosis on liver biopsy pecimens. Patients with genotype A (vs D) or B (vs C) infection tend to have a better response.In focusing on the host genetic background, the role of single nucleotide polymorphisms (SNPs) in relation to disease and treatment response has become increasingly supported in a variety of illnesses. In particular to hepatitis B disease. MHC I and MHC II class polymorphisms, as well as TNF-a and mannose-binding protein (an opsonizing protein), the SNPs have been suggested to affect host immune and antiviral responses and, thus, are associated with variable disease progression (ie., resistance and chronic infection) and treatment response. Hence, looking into such a topic may lead to important predictions of treatment response for HBV patients, especially for IFN therapy, given many displeasing side effects associated with this medical regimen, and may avoid the unpleasant side effects that commonly accompany the treatment when it is unlikely to be beneficial, and reduce overall treatment costs.IL28B (interfere n-λ3) produces an antiviral state by triggering a cascade through the JAK-STAT pathway that up-regulates the IFN-stimulated genes (ISGs)., IL28B has been identified as a key modulator of the immune response to hepatitis C virus (HCV), because some SNPs (single-nucleotide polymorphism) of the IL28B gene, specially SNPrs12979860and SNP rs8099917, was associated with spontaneous and treatment-induced HCV clearance. Whether the SNPs affects the outcome of hepatitis B virus (HBV) wherein IFN-α and ISGs are important in the host response need to be further expounded.Objective To explore the predictions of IFN treatment response for HBV patients by studying the relationship between IL28B gene polymorphism, including SNPrs12979860and SNPrs8099917.Methods Peripheral blood samples were from82subjects with interferon treatment we recollected(38interferon response,44interferon nonresponse). Chromosomal DNA was extracted and IL28B gene was amplified. SNP rs8099917was typed using a protocol based on PCR-RLFP. PCR-RFLP is a SNP-genotyping method based on nucleic acid-specific restriction enzyme digestion reaction principle and PCR. which is simple, fast, economic, and accurate. Restriction endonuclease study of genetic variation in the molecular biology is a scalpel, which can cleave the recognition of base sequence, the use of the characteristics of gene fragments can be detected a certain point whether there is variation. IL28B gene SNP rs8099917corresponding endonuclease restriction sites BsrDl the restriction, when the point base for G, there is endonuclease cleavage sites, and when the G variant T restriction site disappear, therefore, digested with three kinds of situations:First, the digested PCR product length did not change, polyacrylamide gel electrophoresis only one band, indicating that this amplified fragment is no such enzymatic cleavage sites is the TT genotype; heterozygous chromosome of a cut point of this enzyme, the allele on another chromosome does not have this restriction sites, the electrophoresis pattern with three, that is, the CG genotype. The product was cut completely, the electrophoresis pattern only two with the tangent point of two chromosomes containing this enzyme is the GG genotype for homozygous.Some sample were randomly selected to confirm the PCR-RLFP result by PCR-direct sequence analysis. While SNP rs12979860was typed by sequencing. Identify the genotype of HBV from each patient by PCR and sequencing. Odds ratio and chi-square test were applied in statistical analysis.Results The IL28B gene region including rs8099917locus and rs12979860locus were successfully amplified respectively. The PCR-RLFP results were easy to be distinguished and can be confirmed by sequence analysis of the randomly selected-samples after cloning. All of the HBV DNA samples were amplified and the genotypes were confirmed successfully. Distribution of genotype and allele of the rs12979860locus and rs8099917locus were in accordance with Hardy-Weinberg equilibrium in different groups or with the total population. The frequencies of the rs8099917TT, TG+GG genotypes were81.8%(31/38) and18.4%(7/38), and the frequency of allele T and G accounted for90.8%and9.2%. respectively in response group. While97.7%(43/44)2.3%(1/44) and98.9%(87/88),1.1%(1/88) in nonresponse group. Differences between response group and nonresponse group was statistically significant (genotype P=0.014; allele P=0.025).The frequencies of the rs12979860CC, TT+CT genotypes were15.8%(6/38) and4.2%(32/38), and the frequency of allele C and T accounted for92.1%(70/76) and7.9%(6/76), respectively in response group. While4.5%(2/44),95.5%(42/44), and97.7%(86/88) and2.3%(2/88) in nonresponse group. There were no statistically significant differences between response group and nonresponse group.Conclusion SNP rs8099917may be associated with response to interferon treatment on hepatitis B e antigen-positive chronic hepatitis B patients. Allele G may be associated with interferon treatment success. Genotyping on this SNP may be predicting valuable for interferon therapy for chronic HBV infection.