| BackgroundInfection of hepatitis B virus is one of the most serious and prevalent global health problems. More than350million people worldwide are chronically infected with HBV. HBV related acute-on-chronic liver failure (ACLF) is a main cause of death for patients with chronic hepatitis. The mortality of ACLF is60-80%, resulting in22,600deaths a year. In addition to host factors, viral factors per se could also play an important role in determining clinical outcomes of chronic hepatitis B. HBV is a small enveloped DNA virus of the Hepadnaviridae family with approximately3200nucleotides. The virus has a partially double-stranded DNA genome made of four overlapping open reading frames that encode hepatitis B surface (S) antigen, a core protein, a polymerase, and a multifunctional nonstructural protein named X. Since HBV replicates through the reverse transcription of pregenome RNA, mutations occur more frequently in HBV than in other DNA viruses. Therefore, mutations in the HBV genome may accumulate with time, and some of them might serve as viral markers for predicting the development of HBV-associated liver failure. Recently, several studies have indicated that patients with basal core promoter (BCP) mutations (A1762T/G1764A) had higher risks of ACLF. However, BCP mutations are also frequently found among patients with mild chronic hepatitis B and asymptomatic carriers. In addition to these two mutations, mutations at nt.1846, nt.1896, and nt.1913have also been found to be associated with the development of ACLF. All of these studies focused mainly on the particular portions of the HBV genome (e.g. BCP and precore (PC) regions), and rarely analyzed the other locus that might play important roles. Hence, it remains unclear whether other predictive markers might be found by comparative analysis of the complete HBV genomes between different stages of hepatitis B. The aims of present investigation were to screen new variants of HBV related to the risk of ACLF development and to find some specific biomarkers in the HBV genome for chronic hepatitis B progression.Methods1Illumina high-throughput sequencing1.1The samples from two groups (12ACLF patients and12mild chronic hepatitis B (CHB-M)) were analyzed with Illumina high-throughput sequencing. There were no significant differences of age, sex and genotype distribution between two groups. The plasma HBV DNA levels of these patients were>100,000copies/mL. HBV DNA was extracted from200μl serum and three primers pairs were used to amplify3overlapping HBV full-length fragments. PCR products were utilized for the high-throughput sequencing, which was carried out on a Genome Analyzer II platform. Then the high-throughput sequencing data were analyzed.1.2The hepatic function (ALT, AST, TBIL, PTA), serum HBV markers and the titer of HBV DNA were measured.1.3Analysis of mutations.258full genome sequences of HBV subimitted by Chinese were found from the Genbank database and analyzed.2HBV mutations were validated by direct sequencing in large samples2.1The samples were from438subjects (80asymptomatic carriers (ASC),152CHB-M patients,102severe chronic hepatitis B (CHB-S) patients and104ACLF patients).2.2HBV DNA was extracted from200μl serum, and semi-nest PCR was used to amplify the target regions of HBV genome. The nucleotide sequences of PCR products were carried out by Sanger sequencing.2.3The hepatic function (ALT, AST, TBIL, PTA), serum HBV markers, the titer of HBV DNA in438subjects were measured. Results1Result of Illumina high-throughput sequencing:1.1An examination of the qualities revealed that lower values were more common near the end of the reads (positions20and higher in reads of length36), so we trimmed the last16nuclotieds before the alignment. The overall plasmid sequence error rate was0.49%, and the mean number of nucleotides with>4%mismatch error rate per one hundred nucleotides was2.4. According to this result,4%was empirically served as the limit to distinguish the authentic variants from technological artifacts.1.2In ACLF group, the mutations at nine sites (T216C, G285A, A1846T, G1896A, C1913A/G, G2095A, A2159G, A2189C and G2345A) were frequently found.1.3Mutation frequencies at nt.216, nt.285, nt.1846, nt.1896, nt.1913, nt.2095, nt.2159, nt.2189and nt.2345of twelve ACLF patients were significantly higher than those of twelve CHB-M (P<0.05or P<0.01)1.4The cases with mutation at nt.1762/nt.1764were six in both ACLF group and CHB-M group. Mutation frequencies of BCP mutations (A1762T, G1764A) were not significantly higher in12ACLF patients than in12CHB-M (P>0.05).1.5Based on the analysis of258HBV genomic sequences downloaded from Genbank database, we found that in genotype B virus, the prevalence of T216C, G285A, A1846T, G1896A and C1913A/G were significantly higher in ACLF as compared with CHB-M; in genotype C virus, the prevalence of A1846T, C1913A/G, A2159G, A2189C and G1764A were significantly higher in ACLF. The prevalence of A1762T/G1764A double mutations was not much higher in ACLF as compared with CHB-M.2Result of the validation2.1A significantly higher ratio of genotype B to C was found in patients with ACLF than in patients with non-ACLF.2.2The prevalence of seven mutations (T216C, G285A, A1846T/G, G1896A, C1913A/G, A2159G/C, and A2189T/C)in ACLF group enhanced significantly, as compared with ASC, CHB-M, and CHB-S groups (P<0.05).2.3The prevalence of five mutations (T216C, G285A, A1846T/G, G1896A and C1913A/G) was significantly higher in genotype B virus than in genotype C virus. 2.4As compared with ASCs, mutations at nt.216, nt.285, nt.1896, nt.1913in genotype B were associated significantly with an increased risk of CHB-S and ACLF. respectively. In subjects with genotype C virus, prevalence of mutations at nt.1846, nt.1896, nt.1913, and nt.2159were significantly higher in ACLF and CHB-S than those in ASC. As compared with patients with CHB-M, mutations at nt.216, nt.285, nt.1896, nt.1913and nt.2159were significantly associated with an increased risk of ACLF in genotype B, whereas mutations at nt.285, nt.1846, nt.1896, and nt.1913were significantly associated with an increased risk of ACLF in genotype C. As compared with the patients with CHB-S, mutations at nt.2159and nt.2189in genotype B were significantly associated with an increased risk of ACLF.2.5T216C, G285A, A1846T/G, G1896A, C1913A/G, A2159G/C, and A2189T/C in genotype B were each associated with ACLF compared with non-ACLF, whereas G285A, A1846T/G, G1896A, C1913A/G, and A2159G/C in genotype C were each associated with ACLF compared with non-ACLF.2.6Multivariate analysis showed that T216C, G1896A, C1913A/G and A2159G/C were independent risk factors for ACLF.2.7Combinations with any2or more of T216C, G1896A, C1913A/G and A2159G/C were significantly associated with the development of ACLF.2.8C216in any combination, A/G1913in any combination, and G/C2159in any combination had high specificity for ACLF.Conclusions1The patients with CHB infected with genotype B virus were more prone to development of ACLF than those with genotype C virus.2T216C, G1896A, C1913A/G and A2159G/C were independent factors associated with ACLF. T216C and A2159G/C mutations were novel independent risk factors related to ACLF development, which highlighted the influence of genetic variants in the HBV S gene and core gene on the progression of hepatitis B.3A combined examination of different viral mutations could predict more precisely the progression of liver disease. C216in any combination, A/G1913in any combination, and G/C2159in any combination were specific for ACLF.4These virus mutations, as molecular biomarkers, may be useful for preventing ACLF development and predicting prognosis of patients with hepatitis B. |
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