Clinical significance of pre-S mutations in patients with genotype C hepatitis B virus infection

We investigated the overall and site-specific prevalence of pre-S mutations and its clinical significance in patients with genotype C hepatitis B virus (HBV) infection. Three hundred subjects were included: 50 asymptomatic carriers (AC), 87 chronic hepatitis (CH), 91 liver cirrhosis (LC) and 72 hepatocellular carcinoma (HCC). Pre-S mutations were determined by nucleotide sequence analysis. Possible correlations between pre-S mutations and clinical/virological parameters were examined. Pre-S mutations were detected in 82 cases (27.3%); it was more frequently found in HCC (43.1%) and LC (35.2%) group than in the CH (20.7%) and AC (2.0%) group. Pre-S2 deletion was the most commonly found mutation (10.7%), followed by pre-S2 start codon mutation (9.7%), pre-S1-S2 deletion (3.0%) and both pre-S2 deletion and start codon mutation (2.7%). Pre-S2 deletion and pre-S2 start codon mutation were more frequently detected in advanced diseases (LC and HCC). Pre-S mutations were associated with older age and higher rates of positive HBV DNA (>/=0.5 pg/mL). Advanced disease and positive HBV DNA were shown to be independent predictors of pre-S mutations by logistic regression analysis. These findings suggest that pre-S mutations, especially pre-S2 deletions and pre-S2 start codon mutations, are common in patients with genotype C HBV infection and are associated with advanced liver disease and active viral replication.     

中国贵州地区乙型肝炎病毒前S基因变异的研究

目的研究HBV前S基因变异与基因型及疾病类型的关系。方法建立聚合酶链反应-限制性片段长度多态性检测HBV前S2起始码变异的方法;用聚丙烯酰胺凝胶电泳分析HBV前S区缺失变异;用直接测序验证方法的准确性。用S基因聚合酶链反应-限制性片段长度多态性确定HBV基因型。检测160份HBV感染者血清,并结合基因型、疾病类型分析。结果160份标本中B基因型81份,C基因型79份。C基因型前S2起始码变异的检出率为43.04%,高于B型的1.23%(P<0.05)。前S区缺失变异在C基因型中的检出率也高于B型(36.71%与19.75%,P<0.05)。前S2起始码变异在HCC、LC组的检出率分别为50.00%、39.47%,明显高于慢性肝炎组的8.00%、慢性无症状乙型肝炎表面抗原携带者组的0(P值均<0.05)。前S区缺失变异检出率在HCC、LC组分别为53.13%和42.11%,也高于慢性肝炎组的18.00%及慢性无症状乙型肝炎表面抗原携带者组的7.50%(P值均<0.05)。经多因素Logistic回归分析,C基因型和严重肝病是影响前S基因变异的重要因素(OR分别为6.26、11.99,P值均<0.01)。测序结果与酶切,聚丙烯酰胺凝胶电泳结果一致。结论与B基因型相比,C型HBV感染者更易发生前S2起始码、前S区缺失变异;前S基因变异与肝病进展及预后相关。     

表面抗原和抗体双阳性慢性乙型肝炎病毒感染者病毒S基因的变异分析

目的 了解HBsAg和抗-HBs双阳性慢性HBV感染者的S基因变异情况.方法 分别对8例HBsAg和抗-HBs双阳性(实验组)及9例HBsAg阳性、抗-HBs阴性慢性HBV感染者(对照组)的S基因进行PCR扩增并测序,将测序结果进行对比分析.基因型和血清型分布比较、主要亲水区变异位点数比较采用Fisher'S精确检验,核苷酸和氨基酸序列的同源性比较采用t检验.结果 感染HBV的基因型分布:实验组为B型2例、C型6例,对照组为B型6例、C型3例,两组间差异无统计学意义(P>0.05);血清型分布:实验组为adw 2例、adr 5例、ayr 1例,对照组为adw 6例、adr 3例,两组间差异无统计学意义(P>0.05).实验组和对照组HBV前S1区的核苷酸替换率(2.29%比1.8%)和氨基酸替换率(2.66%比1.59%)差异无统计学意义(t值分别为1.56和1.39,P值均>0.05),前S2区的核苷酸替换率(1.74%比0.91%)差异有统计学意义(t=4.68,P<0.01),氨基酸替换率(3.18%比2.05%)差异无统计学意义(t=1.85,P>0.05);S区的核苷酸替换率(2.13%比0.81%)和氨基酸替换率(4.37%比1.52%)差异有统计学意义(t值分别为6.00和5.32,P值均<0.01).主要亲水区内外均存在氨基酸的替换,"a"决定簇变异相对较高(P<0.05).结论 HBsAg和抗-HBs双阳性慢性HBV感染者的S基因变异率相对较高.     

Hepatitis B virus pre-S2 start codon mutations in Indonesian liver disease patients

AIM: To identify the prevalence of pre-S2 start codon mutations and to assess their association with liver disease progression. METHODS: The mutations were identified by direct sequencing from 73 asymptomatic carriers, 66 chronic hepatitis (CH), 66 liver cirrhosis (LC) and 63 hepatocellular carcinoma (HCC) patients. Statistical significances were determined using Fisher's exact test, χ 2 test, and t -test analyses whenever appropriate. Pre-S mutation as a risk factor for advanced liver disease was estimated by unconditional logistic regression model adjusted with age, sex, and hepatitis B e antigen (HBeAg). P 0.05 was considered significant. RESULTS: Mutation of the hepatitis B virus (HBV) pre-S2 start codon was found in 59 samples from 268 subjects (22.0%), with higher prevalence in patients with cirrhosis 27/66 (40.9%) followed by HCC 18/63 (28.6%), chronic hepatitis 12/66 (18.2%) and asymptomatic carriers 2/73 (2.7%) (P 0.001). Logistic regression analysis showed that pre-S2 start codon mutation was an independent factor for progressive liver disease. Other mutations, at T130, Q132, and A138, were also associated with LC and HCC, although this was not statistically significant when adjusted for age, sex, and HBeAg. The prevalence of pre-S2 start codon mutation was higher in HBV/B than in HBV/C (23.0% vs 19.1%), whilst the prevalence of T130, Q132, and A138 mutation was higher in HBV/C than in HBV/B. The prevalence of pre-S2 start codon mutation was higher in LC (38.9%) and HCC (40.0%) than CH (5.6%) in HBeAg(+) group, but it was similar between CH, LC and HCC in HBeAg(-) group. CONCLUSION: Pre-S2 start codon mutation was higher in Indonesian patients compared to other Asian countries, and its prevalence was associated with advanced liver disease, particularly in HBeAg(+) patients.     

Clustering of Different Subtypes of Hepatitis B Surface Antigen in Families of Patients with Chronic Liver Diseases

Clustering of hepatitis B surface antigen (HBsAg) with both subtypes adr and adw in three families of patients with chronic liver diseases or hepatocellular carcinoma was demonstrated in Taiwan where adw is the main subtype. The subtype in the children was similar to that in their mothers, suggesting maternal transmission. In all the family units clustered with different subtypes, the same pattern occurred, invariably with fathers carrying HBsAg/adr and the children carrying HBsAg/adw. The subtype difference clearly rules out the transmission of hepatitis B virus (HBV) from father. Horizontal infection with the locally dominant adw-subtyped HBV in the children of fathers carrying HBsAg/adr explains the discrepancy of the subtypes in these families. Clustering of two HBsAg-positive brothers with hepatocellular carcinoma in one of the families was found. That both adr-subtyped and adw-subtyped HBV are capable of inducing chronic active hepatitis in another family suggests that host factors are probably more important in determining the clinical course of HBV infection.     

Genotypes and subtypes of hepatitis B virus in Thailand

Hepatitis B virus exhibits considerable variability evident in its various antigenic subtypes, which complicates the characterization of epidemiological factors, particularly in areas endemic for hepatitis B. Our group investigated the genotypes and subtypes prevalent in Thailand employing nested PCR and sequencing of the a determinant, as well as the sub-determinants located on the S gene. The sera examined originated from a mixed range of HBV-infected individuals. The results were mostly consistent with those reported for Southeast Asia in that genotype C (54.4%) dominates over genotypes A (22.1%) and B1 (23.5%). Regarding the subtypes, we have exclusively found adw2 (45.6%) and adr (54.4%) as expected for this area, with one case of subtype adw representing the exception. While genotype and/or subtype of HBV do not predispose to clinical disease, they nevertheless may account for those few cases reported in which a mutation, particularly within the a determinant of the S gene, causes evasion of routine detection by commercial kits, particularly as long as the respective individuals remain asymptomatic carriers solely expressing anti-HBc.     

Prevalence and significance of hepatitis B virus (HBV) pre-S mutants in serum and liver at different replicative stages of chronic HBV infection

Several types of naturally occurring pre-S mutants in sera or liver tissues in patients with chronic hepatitis B virus (HBV) infection have been identified. To clarify the prevalence and significance of emergence of pre-S mutants, 140 sera and 18 resected livers from patients with HBV were studied. Replicative status was designated as high, intermediate, and low based on the HBV-DNA levels in serum or the expression of HBV antigens in liver. In vitro transfection and Western blot analysis were performed to characterize expression and secretion of HBsAg by the mutant constructs. Five major types (I to V) of pre-S deletion mutants in serum and liver and 2 types (VI and VII) in liver were identified. Pre-S mutant was 6.4% at high replicative phase, 13% at intermediate, and 37.5% at low or nonreplicative phases in serum. In livers, the same tendency existed: pre-S2 deletion mutants emerged and prevailed at a low replicative phase in hepatocytes that expressed a novel marginal pattern of HBsAg and usually clustered in groups. The deletion sequence of pre-S2 region coincides with human leukocyte antigen-restricted T- and B-cell epitopes. In vitro HBsAg was retained in the hepatocytes and synthesis and secretion of major surface antigen decreased for most of the pre-S mutants. Pre-S mutants prevailed with evolution of chronic HBV, probably under immune pressure. Emergence of pre-S mutants may account for the life-long persistence and discrepancy of HBsAg in serum and liver in HBV and may confer growth advantage in view of the clustering proliferation of hepatocytes harboring pre-S2 mutant.     

HBsAg和HBsAb双阳性检测结果的初步分析

目的对临床检测中少见的HBsAg和HBsAb双阳性结果进行分析,寻找可能的产生原因。方法对3家医院初筛HBsAg和HBsAb双阳性的81份血标本进行同种和不同种试剂盒复检,并检测HBsAg145位氨基酸变异（G145R变异）、HBVDNA基因型和血清型分析。结果81份双阳性标本经复检有30例（37％）仍然为双阳性;对30例复检双阳性标本应用不同试剂盒再次检测,HBsAg／HBsAb仍为双阳性者18例（22．2％）;18例双阳性标本G145R检测全部为阴性;其中8例HBV DNA阳性血清的基因型分布为B型2例和C型6例,血清型分布为adw2例、adr5例和ayr1例,与9份对照血清的检测结果比无显著性差异。结论HBsAg和HBsAb双阳性检测结果大多数与操作或试剂的质量有关,少数可能为S基因变异或不同血清型的再次感染等有关。     

Significance of the minor i and t determinants of hepatitis B virus in hepatocellular carcinoma

Stored sera from asymptomatic hepatitis B virus (HBV) carriers and hepatitis B virus surface antigen-positive hepatocellular carcinoma (HCC) patients were tested for HBV subtypes, such as subtype determinants d, y, w, r and also antigenic determinants isoleucine (i) and threonine (t) by direct S gene nucleotide sequencing. Significant changes in minor i and t determinants in hepatocellular carcinoma patients with adr hepatitis B carriers were seen. The adr subtype with t determinant was present in 14/25 (56%) of HCC patients compared with only two of 28 (7%) in asymptomatic hepatitis B carriers ( P < 0.001). However, the adr subtype with i determinant was present in nine of 25 (36%) of the HCC patients and also present in 24/28 (86%) of asymptomatic carriers ( P < 0.001). No significant changes were seen with the adw subtypes. These results show that i and t minor determinant changes are more common with adr subtypes associated with HCC than with the adw subtype. Whether these subtle changes are pathologically relevant or only a polymorphism of hepatitis B genotypes will depend on subsequent follow-up studies.     

A molecular analysis of viral persistence in surface antigen-negative chronic hepatitis B

To identify the mechanisms of viral persistence in patients with chronic hepatitis B after the acquisition of anti-hepatitis B surface antigen antibodies (antiHBs), we serially analyzed the nucleotide sequence of the envelope region in a cohort of infected patients. Four patients with histological diagnoses of chronic hepatitis B who had at least 5 years of observance by our hospital staff were studied. All but one showed normalization of serum alanine aminotransferase (ALT) concentration after clearance of the hepatitis B surface of antigen (HBsAg) and the appearance of anti-HBs. Hepatitis B virus (HBV) DNA was still detectable by polymerase chain reaction (PCR) amplification assay in serum specimens from two patients, even in the presence of circulating anti-HBs. The envelope gene was amplified by PCR in serum samples obtained both before and after seroconversion, and direct cycle sequencing of the PCR products was performed. A mutation resulting in a premature stop codon was found in the pre-S1 region of one patient just prior to clearance of HBsAg. Two years later, the stop codon was converted to a leucine codon and three mutations developed in the "a" loop. In the other patient, 16 amino acids had been deleted between amino acids 8 and 23 in the pre-S2 region before clearance of HBsAg. After the appearance of circulating anti-HBs, the pre-S2 gene reverted to the wild type but three additional mutations appeared inside the "a" loop. These results suggest that HBV mutates when HBsAg is cleared, which may contribute to viral persistence due to an evasion of the host immune surveillance. (Hepatology 1996 Mar;23(3):389-95)     

Clinical significance of hepatitis B surface antigen mutants

Hepatitis B virus (HBV) infection is a major public health problem in many countries, with nearly 300 million people worldwide carrying HBV chronic infection and over 1 million deaths per year due to cirrhosis and liver cancer. Several hepatitis B surface antigen (HBsAg) mutations have been described, most frequently due to a single amino acid substitution and seldom to a nucleotide deletion. The majority of mutations are located in the S region, but they have also been found in the pre-S1 and pre-S2 regions. Single amino acid substitutions in the major hydrophilic region of HBsAg, called the “a” determinant, have been associated with immune escape and the consequent failure of HBV vaccination and HBsAg detection, whereas deletions in the pre-S1 or pre-S2 regions have been associated with the development of hepatocellular carcinoma. This review article will focus on the HBsAg mutants and their biological and clinical implications.     

Expression of pre-S gene-encoded proteins in liver and serum during chronic hepatitis B virus infection in comparison to other markers of active virus replication

Pre-S gene-encoded proteins of the hepatitis B virus (HBV) were studied in the liver by immunofluorescence and in serum by radioimmunoassay in 30 patients with chronic HBV infection. The results were compared with molecular hybridization analysis of HBV-DNA in liver and serum, with serum hepatitis B e antigen/antibody (HBeAg/anti-HBe) status and with underlying liver histology. Pre-S peptides were detected in the serum of 11 patients, 10 of whom were positive for serum HBV-DNA and/or liver hepatitis B core antigen. Only 4 of these patients were HBeAg positive. The prevalence of serum pre-S among HBV replicating carriers was 59% (10/17) compared to only 8% (1/13) among those with non-replicating virus (P less than 0.01). All patients with circulating pre-S peptides had active liver disease. Anti-pre-S was detected in the serum of only 4 patients, 3 with integrated HBV-DNA. In contrast to serum findings, pre-S peptides were detected in the liver of all patients with histochemically demonstrable hepatitis B surface antigen (HBsAg), regardless of HBV replicative status. HBsAg carriers with integrated HBV-DNA had abundant cytoplasmic pre-S1 and pre-S2 localized in numerous ground-glass hepatocytes. It is concluded that pre-S peptides are usually displayed in the liver simultaneously with histochemically detectable HBsAg; they are secreted in the serum in association with high HBV replication and release of HBV particles, but in the absence of episomal HBV replication, pre-S peptides seem to be largely retained within hepatocytic membranes.     

A mutation specific polymerase chain reaction for detecting hepatitis B virus genome mutations at nt551

AIM: The hepatitis B surface antigen (HBsAg) is considered to be one of the best markers for the diagnosis of acute and chronic HBV infection. But in some patients, this antigen cannot be detected by routine serological assays despite the presence of virus. One of the most important explanations for the lack of detectable HBsAg is that mutations which occur within the “a” determinant of HBV S gene can alter expression of HBsAg and lead to changes of antigenicity and immunogenicity of HBsAg accordingly. As a result, these mutants cannot be detected by diagnosis assays. Thus, it is essential to find out specific and sensitive methods to test the new mutants and further investigate their distribution.This study is to establish a method to investigate the distribution of the HBsAg mutant at nt551.METHODS: A mutation specific polymerase chain reaction (msPCR) was established for amplifying HBV DNA with a mutation at nt551. Four sets of primer pairs, P551A-PPS,P551G-PPS, P551C-PPS and P551T-PPS, with the same sequences except for one base at 3‘ terminus were designed and synthesized according to the known HBV genome sequences and the popular HBV subtypes, adr and adw, in China. At the basis of regular PCR method, we explored the specific conditions for amplifying HBV DNAs with a mutation at nt551 by regulating annealing temperature and the concentration of these primers. 126 serum samples from patients of hepatitis B were collected, among which 16 were positive for HBV S DNA in the nested PCR amplification.These 16 HEN S DNAs were detected by using the msPCR method.RESULTS: When the annealing temperature was raised to 71℃, nt551A and nt551G were amplified specifically by P551A-PPS and P551G-PPS, At 72 ℃ and 5 pmole of theprimers (each) in reaction of 25μl volume, nt551C and nt551T were amplified specifically by P551C-PPS and P551T-PPS. 16 of HBV S gene fragments were characterized by using this method. 14 of them were positive for nt551A,one was positive for nt551G, and the other one was positive for nt551T. The results were confirmed by nucleotide sequencing.CONCLUSION: The mutation specific polymerase chain reaction is a specific and sensitive method for detecting the mutations of HBV genome at nt551.     

Association of pre-S deletion mutant of hepatitis B virus with risk of hepatocellular carcinoma

BACKGROUND: Pre-S deletion mutant of hepatitis B virus (HBV) affects the expression of middle and small surface proteins, resulting in intracellular accumulation of large surface protein. The correlation between pre-S deletion mutant and risk of hepatocellular carcinoma (HCC) in hepatitis B virus carriers remains unclear. METHODS: Using molecular assays, pre-S deletion mutant of HBV were determined in 266 patients with chronic HBV genotype B or C infection. They included 202 asymptomatic carriers and 64 HCC patients. RESULTS: The overall prevalence of pre-S deletion mutant was 16.5%. Hepatocellular carcinoma (odds ratio [OR], 3.23; 95% confidence interval [CI], 1.23-8.48, P = 0.02) and genotype C (OR, 3.19; 95%CI, 1.54-6.62, P = 0.002) were independently associated with the presence of pre-S deletion mutant. The prevalence of pre-S deletion mutant was comparable between HCC patients with genotype B and C infection. Nevertheless, in asymptomatic carriers, patients with genotype C infection were significantly associated with the presence of pre-S deletion mutant compared to those with genotype B infection (20.8% vs 7.2%, P = 0.007). Compared with age- and genotype B-matched asymptomatic carriers, young HCC patients (<50 years of age) had a significantly higher frequency of pre-S deletion (3.4% vs 20%, P = 0.04). CONCLUSIONS: Pre-S deletion mutant is more frequent in HBV carriers with genotype C infection, and those with pre-S deletion mutant may be associated with the development of HCC, irrespective of HBV genotype.     

Hepatitis B surface antigen disappearance and hepatitis B surface antigen subtype: a prospective, long-term, follow-up study of Japanese residents of Okinawa, Japan with chronic hepatitis B virus infection.

To determine the natural course of hepatitis B surface antigen (HBsAg) disappearance in chronic hepatitis B virus (HBV) infection and the factors related to its disappearance, 946 HBsAg carriers in Okinawa, Japan were prospectively followed for up to 19 years (mean = 9.2 years). The disappearance of HBsAg, as determined by radioimmunoassay (RIA), was observed in 62 (6.6%) and the overall annual disappearance rate was 0.79%/year. Its disappearance was more frequent in 60 (7.4%) of 815 serum samples negative for hepatitis B e antigen (HBeAg) by RIA at entry compared with only two (1.5%) of 131 serum samples that were HBeAg positive by RIA at entry (P < 0.05). Stepwise logistic regression analysis showed that age and HBsAg subtype were significantly associated with HBsAg disappearance (both P < 0.05), and that carriers with subtype adr (odds ratio = 2.87) had an increased probability of clearing HBsAg compared with carriers with subtype adw. Conversely, HBeAg disappearance was earlier in those with the adw subtype than in those with adr. Hepatitis B virus DNA was not detected by the polymerase chain reaction after HBsAg disappearance in any of the 62 from whom it had disappeared. The HBsAg titer, as measured by reverse passive hemagglutination, was related to the time to its disappearance; the higher the titer, the longer the time to disappearance. These findings suggest that HBeAg negativity, a more advanced age, and low titers of HBsAg are favorable factors for HBsAg disappearance in the natural course of chronic HBV infection. Moreover, HBsAg subtype adr was a predictive factor for HBsAg disappearance, whereas subtype adw was predictive of early HBeAg disappearance.     

Relationship of HBsAg Subtypes with HBeAg/anti-HBe Status and Chronic Liver Disease. Part I: Analysis of 1744 HBsAg Carriers

A total of 1744 HBsAg carriers were investigated to determine whether there are clinical differences among HBsAg subtypes or not. Although adr was more predominant than adw in 1078 asymptomatic carriers as well as in 666 carriers with liver dysfunction, the adr carriers had liver dysfunction more frequently than the adw carriers (p = 0.005). In addition, the adr carriers were more often positive for HBeAg and less often positive for anti-HBe than the adw carriers (p less than 0.001). Multivariate analyses indicated that the HBsAg subtypes were associated with liver dysfunction not directly but through the relationship between the HBsAg subtypes and HBeAg/anti-HBe status. HBeAg/anti-HBe status of each age bracket in the adr carriers and in the adw carriers suggested that adr carriers are seroconverted later than adw carriers. In conclusion, HBsAg subtypes may affect the development of chronic liver disease, through their association with HBeAg/anti-HBe status.     

Impact of hepatitis B virus (HBV) preS/S genomic variability on HBV surface antigen and HBV DNA serum levels

To evaluate whether hepatitis B virus (HBV) preS/S gene variability has any impact on serum hepatitis B surface antigen (HBsAg) levels and to analyze the replication capacity of naturally occurring preS/S variants, sera from 40 untreated patients with HBV-related chronic liver disease (hepatitis B e antigen [HBeAg]-positive, n = 11; HBeAg-negative, n = 29) were virologically characterized. Additionally, phenotypic analysis of three different preS/S variant isolates (carrying a 183-nucleotide deletion within the preS1 region, the deletion of preS2 start codon, and a stop signal at codon 182 within the S gene, respectively) was performed. HBV infecting 14 (35%) patients had single or multiple preS/S genomic mutations (i.e., preS1 and/or preS2 deletions, preS2 start codon mutations, C-terminally truncated and/or "a" determinant mutated S protein). Presence of preS/S variants negatively correlated with HBsAg titers (r = -0.431; P = 0.005) and its prevalence did not significantly differ between HBeAg-positive and HBeAg-negative patients. No correlation was found between HBsAg and HBV DNA levels in patients infected with preS/S mutants, whereas a significant correlation was found between HBsAg and viremia levels (r = 0.607; P = 0.001) in patients infected with wild-type HBV strains. HepG2 cells replicating the above-mentioned three preS/S variants showed significant reduction of HBsAg secretion, retention of envelope proteins in the endoplasmic reticulum, less efficient virion secretion and nuclear accumulation of significantly higher amounts of covalently closed circular DNA compared with wild-type HBV replicating cells. CONCLUSION: In patients infected with preS/S variants, HBV DNA replication and HBsAg synthesis/secretion appear to be dissociated. Therefore, the use of HBsAg titer as diagnostic/prognostic tool has to take into account the frequent emergence of preS/S variants in chronic HBV infection.