Impact Of Mutation In HCV ISDR On The Efficacy Of Pegylated Interferon/ribavirin Therapy In Patient With Chronic Hepatities C Virus Genotype 1b Infection

BACKGROUDHepatitis C virus (HCV) infection is one of the leading causes of chronic liver diseases. It was reported that 5% to 25% HCV infection would develop to cirrhosis during a period of 20 to 30 years. Patients with HCV-related cirrhosis are at risk for developing to the end-stage liver disease (a risk of approximately 30% over 10 years) as well as hepatocellular carcinoma (HCC) (a risk of approximately 1% to 3% per year). There is still no effective vaccine for prevention. Effective antiviral therapy can improve long-term survival and quality-of-life of the patients. Therefore, the goal of treatment is to prevent complications of HCV infection; this is principally achieved by eradication of the infection. Because of the slow evolution of chronic HCV infection over several decades, it has been difficult to demonstrate that therapy prevents complications of liver disease. Accordingly, treatment responses are defined by a surrogate virological parameter rather than a clinical endpoint. Short-term outcomes can be evaluated biochemically, virologically, and histologically. The most important is the sustained virological response (SVR), defined as the absence of HCV RNA from serum by a sensitive PCR assay 24 weeks of following up after discontinuation of therapy. This is generally regarded as a "virological cure".The currently recommended therapy of chronic HCV infection is the combination of a pegylated interferon alfa and ribavirin (RBV). Interferon plus RBV is the chronic hepatitis C (CHC) standard regimen of antiviral therapy. Several large clinical studies showed that pegylated interferon combined with RBV can approaches the SVRs of 65%, significantly better than non-prgylated.HCV genotyping is useful in epidemiological studies and clinical management. The optimal duration of treatment should be based on HCV genotypes. AASLD guidelines recommend that patients with genotype 1 should be treated for 48 weeks with peg-interferon alfa-2a plus standard weight-based RBV, whereas those with genotype 2 and 3 could be treated with peg-interferon alfa-2a plus low dose RBV for 24 weeks. It is highly recommended that HCV genotyping should be performed in all HCV-infected persons prior to interferon-based treatment. HCV genotypes have a geographic and population distribution. Different genotypes have different clinical manifestations and correlations with the severity of liver disease. However, there is still no commercially available HCV genotyping kit in China. Therefore, It is greatly significant to develop a simple, sensitive, reliable and practical genotyping method for the clinical and molecularly epidemiological studies.There are many parameters for estimating the response to interferon-based therapies. Of which, genotype and pretreatment viral load are two major predictors. Other indicators include gender, age, body weight, with or without insulin resistance and fatty liver disease and fibrosis, alcohol abuse and intravenous drugs or not. These predictors not only can help clinicians and patients evaluate the possible outcomes, but also can be considered as the basis of individualized treatment.HCV-1b, the prevalent genotype in China, Japan, Southern and Eastern Europe, commonly indicates the poor response to antiviral therapy. SVR is achieved in 10 to 40 percent of cases after conventional IFN monotherapy and in approximately 40 to 50 percent of cases after combination therapy using Peg-IFN plus RBV in patients with HCV-lb infection.Since Peg-IFN-based therapy is expensive and may lead to serious adverse effects, it would be useful for clinicians to choose the optimal case by the detection of virus-related factors such as genotypes and predictors for HCV-lb infection with the aim of improving the effect of IFN-based therapies.Enomoto et al found a significant correlation between the response to interferon in patients with HCV-1b infection and the number of amino acid (aa) substitutions that were present before therapy in a small region of NS5A2209-2248. The NS5A2209-2248 region was therefore named the interferon sensitivity determining region (ISDR). Patients infected with genotype-1b HCV harbouring more than three mutated sites in this region are more likely to become sustained responders (SR) than those whose HCV quasispecies harbour few ISDR mutations. There were 3 groups according to their ISDR pattern into:no mutation (wild-type; WT); one to three mutations (intermediate-type; IT); more than three mutations (mutant-type; MT). These initial findings have been further confirmed by other Japanese studies. M Pascu et al found, by a meta-analysis of all available studies, the number and patterns of mutations within the ISDR can be considered as an independent factor to assess the response to IFN in patients with HCV genotype lb infection.However, controversial data were reported from mainland China. Tang Zhenya et al preformed a study including 12 patients in Wuhan who received IFN treatment and found that the ISDRs of isolates highly conserved, and the wild strains is sensitive to IFN. Hu Yunwen et al found none of them was mutant type in Shanghai.3 of 6 sustained responders were infected with wild-type isolates, the rest with intermediate type isolates. ISDR sequence alone could not predict outcome of IFN treatment. However, their study sample size was too small and not for statistical analysis. And Shen et al analyzed retrospectively 20 cases and supported the position that the mutations within the ISDR of NS5A gene affected the effect of interferon therapy. Yen et al found the mutations of the ISDR significantly correlated with SVR to Peg-IFNa-2b plus RBV in patients with HCV-lb in Taiwan. However, the correlation between ISDR mutations and the response to Peg-IFN-based therapies in HCV-1 patients is rarely reported in China.We studied the HCV virological factors that estimating the response to interferon-based therapies, and this thesis consisted of three studies. The first study aimed to develop a rapid and specific HCV genotyping method with the reverse dot hybridization technique and investigate the distribution of HCV genotypes and subtypes in Guangdong area, China. The second study was to establish a reverse transcription polymerase chain reaction (RT-PCR) method to amplify the HCV lb ISDR in Guangdong area, then the amino acid sequence of NS5A 2209-2248 was determined by direct sequencing, and to investigate the mutation number of amino acid in ISDR compared with HCV J strain. The last study was to perform a retrospective analysis on 44 patients who had been tested by ISDR and treated with Peg-IFN and RBV in order to discuss the primarily predictive factor that affected the therapeutic effect and the clinical significance of ISDR by analyzing clinical characters before treatment. Details are as follows:1. A novel hepatitis C virus genotyping method using RT-PCR reverse dot hybridization technique and its applicationObjective:To develop a rapid and specific HCV genotyping method with the reverse dot hybridization technique and investigate the distribution of HCV genotypes and subtypes in Guangdong area, China. Methods:The primers and the probes targeting the 5'untranslated region (5'UTR) and Core region of HCV genotypes 1b,2a,3a,3b and 6a were specially designed. And then the RT-PCR Reverse Dot Hybridization (PCR-RDH) method for HCV genotyping was established. A total of 115 patients with hepatitis C from Nanfang Hospital were genotyped by the method, and 38 of them were also genotyped by sequencing and phylogenetic analysis to evaluate the accuracy and specificity of the method. Results:Of 115 patients,111 (96.5%) were successfully genotyped to be 1b,2a, 3a,3b,6a and mix-infection of lb/2a at frequencies of 56.8%,8.1%,3.6%,5.4%, 25.2% and 0.9% respectively.15 healthy control samples were all negative. The accuracy and reliability of genotyping method of PCR-RDH was confirmed by amplification of HCV Core and NS5B regions from 38 cases followed by DNA sequencing and phylogenetic analysis. The newly developed HCV genotyping method was proved to be reliable and specific, and may be suitable for clinical and epidemiological investigations. The prevalence of HCV genotypes 1b and 2a declined although lb was still the dominant genotype in Guangdong area, and the prevalence of 6a significantly increased when compared with 10 years ago.2. Establishment of test method on ISDR of HCV genotype lbObjective:To establish a establish a reverse transcription polymerase chain reaction (RT-PCR) method based on the special primers of the interferon sensitivity determining region (ISDR) of hepatitis C virus (HCV), and to detect the sequence of the HCV ISDR and to investigate the mutation number of amino acid in ISDR compared with HCV J strain in Guangdong area, China. Methods: According to the total length sequences of HCV genotype lb D90208 in GenBank and the ISDR primer sequences of HCV lb NS5A in references, we selected 55 blood serum samples from patients with HCV-lb infection from out-patients and in-patients in the department of infectious diseases, Nanfang Hospital. We adopted nested PT-PCR to enlarge ISDR of HCV lb NS5A and compared sequences with HCV J strain in order to define the mutation numbers of amino acid in ISDR and to analyze the distribution of ISDR patterns among patients with HCV genotype lb in Guangdong area. Result:The size of ISDR-amplified products was 249bp, which was determined clearly in the electrophoretic pattern and consistent with the expected molecular size. There were no any non-specific bands. The sequencing results were in accord with the target fragments confirmed by BLAST. We could find out that,25.5%(14/55) of HCV genotype lb were wild-type (no mutation), while 72.7% (40/55) were intermediate-type (1 to 3 mutation numbers) and 1.8%(1/55) was mutant-type isolates (more than 3 mutation numbers) in Guangdong area.3. Impact of Mutation in HCV ISDR on the Efficacy of pegylated interferon/ribavirin Therapy in Patients with Chronic Hepatitis C Virus Genotype lb InfectionObjective:To assess whether ISDR mutations correlate with the response to Peg interferon plus RBV therapy among patients with HCV genotype lb in Guangdong area. Methods:We retrospectively analyzed 44 patients with HCV genotype lb infection who had received Peg-IFN/RBV for 24 weeks to 48 weeks. Pretreatment serum samples were collected. The amino acid sequence of ISDR was determined by direct sequencing of the HCV genome amplified by the RT-PCR and was compared with the established sequence for HCV-J. Result: The sustained virological response (SVR) rate was 43.2%(19/44). There was no significant difference in SVR between Pegasys Group and Peg-intron Group (P =0.797). The statistical analysis indicated significant correlations between the mutation number (over 2) in ISDR of HCV-lb and SVR/EVR (P=0.005/P=0.018). However, no significant difference could be found between mutate number and EVTR (P=0.056). In young group (age≤40), we could find out that the younger, the more mutate numbers in ISDR (P=0.012). But ISDR was not significantly related with blood transfusion and non-blood transmission (P=0.117). The mutation of ISDR amino acid 2218 was prevalent in mutate group, and there was no statistical difference between SVR group (14/18,77.8%) and Nonresponse group (11/15,73.3%) (P=0.767). Multivariate regression analysis indicated that the independent variables related to SVR were the mutation number in ISDR (odds ratio,17.038; 95% confidence interval,1.923～150.944; P=0.011), age group (≤40 years) (odds ratio,12.259; 95% confidence interval,0.012～0.559; P=0.011) and AST (odds ratio,1.021; 95% confidence interval,1.003～1.039; P=0.020).Conclusion:1. The newly developed HCV genotyping method was proved to be reliable and specific, and it might be suitable for clinical and epidemiological investigations.2. The prevalence of HCV genotypes 1b and 2a declined although lb was still the dominant genotype in Guangdong area, and the prevalence of 6a significantly increased in comparison with 10 years ago.3. Amplification of ISDR of HCV was carried out successfully without any non-specific bands. The sequencing results were in accord with the target fragments. It shown that, the distribution of ISDR of HCV genotype lb in Guangdong area was mainly wild strains and intermediate strains, while mutant strains accounted for a small proportion (1.8%).4. The SVR rate of Guangdong patients with HCV genotype lb who was treated by Peg-IFN/RBV was 43.2%(19/44). There was no significant difference in SVR between Pegasys Group and Peg-intron Group.5. The mutation number of ISDR (over 2) was significantly associated with EVR and SVR. ISDR was not significantly related with blood transfusion and non-blood transmission.6. The mutation of ISDR amino acid 2218 was the prevalent in Guangdong area; but there was no statistical difference for SVR. Further studies with an expanded sample size are needed.7. The mutations number in ISDR, age less 40 years and AST are independent predictors of the response to antiviral therapy in chronic hepatitis C lb patients treated with peg-interferon plus RBV.8. The impct of ISDR amino acids mutation on SVR might be correlated with evolution of HCV.