A Study Of The Relations Between HBV Pre-S/S Gene Mutations And Anomalous Diagnostic Pattern Of Serological Tests

HBV infection is a global-scale issue of public health and China is one hot spot of HBV infection. HBV belongs to the family Hepadnaviridae. It is partly double-stranded circular DNA with a size of genome around 3.2kb. It can be divided into eight genotypes (A～H),each of which can be further split into different subgenotypes. Different genotype has strong geographical dependence, genotypes B and C prevail in Aisa. HBV pre-S/S region codes for the large (L), middle (M), and small (S) surface proteins. Several unique features of HBV determine its much higher mutation rate than other usually observed DNA viruses. HBV mutants often mix with wild-type viruses in HBV carrier and forming a mixed population. Genetic variability of HBV greatly challenges the sensitivity of immunologic and molecular based assays. Pre-S/S gene mutations add many practical complexity to the diagnosis, treatment, and post-assessment of hepatitis B. The immunoassay used to diagnose HBV infection is to detect a collection of HBV serological markers. The changes of phenotype as a result of have cast doubt on the immunoassay. In our daily routine laboratory diagnosis, it is not uncommon to encounter samples with anomalous diagnostic pattern of serological tests, including the coexistence of HBsAg and HBsAb, HBcAb alone, HBeAb alone, HBsAg negative but HBeAg positive, and inconsistent results with different diagnostic reagent. These anomalous results might be related to the HBV pre-S/S gene mutations. Ordinary serological detection could fail when there are gene mutations that affect conformational of epitope, expression and secretion of antigen. Pre-S/S gene mutations can lead to immune escape and occult HBV infection, which show coexistence of HBsAg and HBsAb or HBsAg negative under the serological tests. Given the fact that using HBsAg as the sign for HBV infection has been routine for many years, the influences of HBV pre-S/S gene mutants on HBsAg and other HBV serological marker and their interrelations need further study.Different conclusions might be reached regarding how HBV pre-S mutations and point mutations within"a"determinant affect immune escape and occult infection could vary, depending on the demographic and geographic distributions of the samples used in the studies. The immune escape was usually accompanied by point mutations in"a"determinant (especially G145R mutations). So far most relevant studies in East Asia were based on samples from Japan, Korean, and Taiwan region of China. Few studies included samples from mainland China. This indicates that further detailed studies are needed for the common relations between pre-S/S gene mutations and anomalous diagnostic patterns of serological tests from samples from mainland China. This study strives to study HBV genotyping and pre-S/S mutations of samples with anomalous diagnostic results of serological tests. The objectives are to provide data about the HBV genotypes and prevalence mutant strains in this region (i.e. mainland China), to explore the relations between pre-S/S gene mutations and anomalous diagnostic patterns of serological tests, to analyze the impact of pre-S/S gene mutations on diagnostic results of serological test as well as factors contributing to immune escape and occult HBV infection.In this study, samples of various anomalous diagnostic patterns of serological tests were collected and real-time PCR was used to select samples with HBV DNA positive. For comparison purpose, we randomly chose control samples with regular diagnostic patterns of serological tests. Highly efficient method for extracting virus DNA was applied to collect HBV DNA from serum. Primers were designed, then HBV pre-S/S amplification products were obtained by PCR,and amplification products were purified. Direct sequencing of PCR products and clonal sequencing were then used in order to study genotyping and the mutations of pre-S/S region. A non-parametric hypothesis test method, the Wilcox rank sum test, was use to assess the statistical significance of identified relations between pre-S/S gene mutations and anomalous diagnostic patterns of serological tests.Based on outcomes from the serological test and the nucleic acid testing, all samples are categorized into three groups for further analysis.GroupⅠcontained samples with coexistence of HBsAg and HBsAb. Sequencing disclosed pre-S deletion mutations, pre-S2 initiation codon mutations and point mutations within"a"determinant, two samples having G145R/A mutations. Contrast with the control group (group with HBsAg positive and HBsAb negative) showed statistically significant correlation between pre-S deletion mutations and the coexistence of HBsAg and HBsAb (p-value 0.024). No such statistically significant correlation were found between point mutations within"a"determinant and the coexistence of HBsAg and HBsAb(p-value 0.408), nor between pre-S2 initiation codon mutations and this coexistence (p-value 0.571). Pre-S deletion mutations mainly happened at 3' terminus of pre-S1 and 5' terminus of pre-S2,with coexisting large deletion mutant strain in 3' terminus of pre-S1 and wild type in three samples. Pre-S deletion mutations affects multitude functional sites to form immune escape in vivo, leading to the coexistence of HBsAg and HBsAb.GroupⅡcontained samples with HBsAg seronegative but HBV DNA positive,including HBsAg seronegative but HBeAg positive, HBcAb alone, and HBeAb alone. Occult HBV infection existed in samples with HBcAb alone or HBeAb alone,with a fraction of 1.6% and 3.3%, respectively. Sequencing analysis showed statistically significant correlation between pre-S deletion mutations and HBsAg serological tests failure (p-value 0.033) as well as between point mutations within"a"determinant and such failure (p-value 0.033). Such statistically significant correlation was found between pre-S2 initiation codon mutations and the tests failure (p-value 0.033) too. Two samples with point mutations within"a"determinant showed G145R/A mutations. Mutant strains had multiple mutants and mixed infection. However, analysis showed that pre-S/S mutations is not the sole reason for the failure of HBsAg detection. The other equally important reason was the levels of circulating antigen being lower than the the assay limit of detection.GroupⅢcontained samples with inconsistent HBsAg results under different diagnostic reagent. The results of NAT for these samples were all negative with no pre-S/S gene mutations. This result is likely related to the limitations in the selection of samples.Results from this thesis research indicates that, due to the long-term existence of HBV in the body and corresponding immuno-pressure, chronic HBV carriers can have immune escape by pre-S/S gene mutations, leading to a test result of coexisting HBsAg and HBsAb. There was statistical correlations between such coexistence and pre-S deletion mutations. Other published studies showed correlation between point mutations within"a"determinant and this pattern, which have not been seen in this study. The failures of detecting HBsAg in clinical immunoassay can be attributed to conformational change of epitope or decrease of secretion and expression of antigen, both caused by pre-S/S gene mutations. There was statistical correlations between such failures and pre-S deletion mutations,pre-S2 initiation codon mutations and point mutations within"a"determinant. Another equally possible explanation was the levels of circulating antigen below the assay limit of detection. Following point mutations were identified within"a"determinant in the samples of this study:I/T126S/N/L, Q129N/R/P/H, D144E/G, G145R/A, G130N, T131N and M133T/I. Under anomalous diagnostic pattern of serological tests, pre-S/S mutation is usually detected with multiple mutants and mixed infection between different mutant strains as well as between mutant strains and wild type. Different from relevant previous studies, G145R point mutation as the main mutational site for immune escape should not be common in this study. Varying results concerning the general relationship between the mutation of preS/S region and immune escape found in different studies might be explained by the variability of epidemic strains of HBV in different regions. In other words, different types of genotypes or different types of subgroups might lead to different types of mutations under immune pressure. HBV pre-S deletions and pre-S2 initiation codon mutations were found all to be genotype C.In summary, anomalous diagnostic pattern of serological tests tend to be related to HBV pre-S/S gene mutations, which highlights the importance of having DNA and mutant detection for such samples with anomalous patterns. Pre-S/S gene mutations can lead to the failure of ordinary serological tests and detection. If the levels of antigens are below detection threshold, it could also lead to false result. Occult HBV infection not only can lead to a false clinical diagnosis, but also can result in hematological pollution due to such occult infection of blood donors and failures of detection using normal serological test. It is very important to improve the detectablity of HBsAg mutants and low-level antigen.