Review on hepatitis B virus precore/core promoter mutations and their correlation with genotypes and liver disease severity

Of 350 million people worldwide are chronically infected with hepatitis B virus(HBV)and are at risk of developing cirrhosis and hepatocellular carcinoma(HCC)later in life.HBV is the most diverse DNA virus,and its genome is composed of four open reading frames:Presurface antigen/surface antigen gene(preS/S),precore/core gene(preC/C),polymerase gene(P),and theχgene(χ).HBV produces quasispecies naturally or in response to antiviral agents because of the absence of proofreading activity amid reverse transcription and a high replication rate.The virus has 10 genotypes(A to J)with different geographical distributions.There are various HBV mutations in the HBV genome,including preC/C mutations,preS/S mutations,P gene mutations,andχgene mutations.The core promoter region plays a vital part in the replication,morphogenesis and pathogenesis of the virus.The precore region also plays a crucial role in viral replication.Both core promoter and precore mutations rescue the virus from host immune surveillance and result in the formation of mutated strains that may have altered pathogenicity.preC/C mutations are associated with liver disease progression.Precore mutations stop hepatitis B e antigen(HBeAg)production and basal core promoter mutations downregulate HBeAg production.Mutations in the basal core promoter are also associated with increased HBV replication and an increased incidence of advanced liver diseases such as cirrhosis and HCC.The emergence of antiviral-resistant mutations is the main reason for treatment failure.This review focuses mainly on preC/C promoter mutations and their correlation with genotypes and liver disease severity.Thorough perception and knowledge of HBV genetic variety and mutants could be vital to discover techniques for the prognosis and control of HBV infection.

Relationship between the different replication status of HBV and mutations in the core promoter in mothers and their children infected via mother-to-infant transmission

OBJECTIVE: To study the relationship between the different replication status of hepatitis B virus (HBV) and mutations in the core promoter (CP) in mother and her child infected by mother-to-infant transmission. METHODS: The core promoter was amplified by PCR and cloned into pGEM-T vector with the T-A choning technique. The recombinant plasmid pGEM-CP was confirmed by digestion with restriction enzyme Apa I and Sac I. Two clones were selected to be sequenced in each patient. RESULTS: Every pair of mother and child had same serotype and genotype and the homology of nucleotides encoding 'a' determinant was 98%-100%. The number of mutations in the core promoter of patients with a high replication status was less than that in those with a low replication status. Mutations were mainly distributed in basia core promoter (BCP) and the inbibitor region of Kunitz-type serine protease. This difference was not associated with mother or child. CONCLUSION: The different replication status of HBV is caused by mutations in the core promoter in mother and child infected hy mother-to-infant transmission and appears to be not associated with the status of development of the infection.

Quasispecies groups in the core promoter region of hepatitis B virus

Objectives: To investigate the mutation of the basic core promoter (BCP) of hepatitis B virus (HBV) and clarify the significance of HBV quasispecies groups in patients with chronic HBV infection. Methods: A set of specific primers was synthesized according to the HBV DNA sequence of a Chinese strain. The BCP was amplified by PCR method from the serum of 40 patients with chronic HBV infection, and the PCR products of 2 patients were subcloned into pGEM Teasy vectors. Polyacrylamide gel elec- trophoresis (PAGE) was employed to display the de- letion mutations, and clones with differential length were selected to be sequenced. Sequence comparison was made to find the difference. Results: Two or three bands were displayed by PAGE in 60% patients. The results of sequence anal- ysis showed that there are some kinds of mutations in the BCP region. The substitution always occurs in TATA-like boxes, especially from T to C on 140 site. The deletion mutations were detected in TA1, TA2 and TA3. The 8bp, 20bp deletion mutations fre- quently happened. Conclusions: There is a hot deletion region in the BCP. The deletion and the substitution in the TATA- like box may influence the expression of preC/C pro- tein. The sequencing results indicate that there are HBV quasispecies groups in patients with chronic HBV infection.

Etiological roles of core promoter variation in triple-negative breast cancer

Abnormal geneexpression playskeyrole in cancerdevelopment.Acorepromoter is located around the transcriptional start site.Through interaction between core promoter sequences and transcriptional factors,core promoter controls transcriptional initiation.We hypothesized that in cancer,core promoter sequences could be mutated to interfere the interaction with transcriptional factors,resulting in altered transcriptional initiation and abnormal gene expression and cancer development.We used triple-negative breast cancer(TNBC)as a model to test our hypothesis.We collected genome-wide core promoter variants from 279 TNBC genomes.After extensive filtering of normal genomic polymorphism,we identified 19,427 recurrent somatic variants in 1,238 core promoters of 1,274 genes and 1,694 recurrent germline variants in 272 core promoters of 294 genes.Many of the affected genes were oncogenes and tumor suppressors.Analysis of RNA-seq data from the same patient cohort identified increased or decreased gene expression in 439 somatic and 85 germline variantsaffected genes,and the results were validated by luciferase reporter assay.By comparing with the core promoter variation data from 610 unclassified breast cancer,we observed that core promoter variants in TNBC were highly TNBC-specific.We further identified the drugs targeting the genes with core promoter variation.Our study demonstrates that core promoter is highly mutable in cancer,and can play etiological roles in TNBC and other types of cancer through influencing transcriptional initiation.

Focused transcription from the human CR2/CD21 core promoter is regulated by synergistic activity of TATA and Initiator elements in mature B cells

Complement receptor 2 （CR2/CD21） is predominantly expressed on the surface of mature B cells where it forms part of a coreceptor complex that functions, in part, to modulate B-cell receptor signal strength. CR2/CD21 expression is tightly regulated throughout B-cell development such that CR2/CD21 cannot be detected on pre-B or terminally differentiated plasma cells. CR2/CD21 expression is upregulated at B-cell maturation and can be induced by IL-4 and CD40 signaling pathways. We have previously characterized elements in the proximal promoter and first intron of CR2/CD21 that are involved in regulating basal and tissue-specific expression. We now extend these analyses to the CR2/CD21 core promoter. We show that in mature B cells, CR2/~D21 transcription proceeds from a focused TSS regulated by a non-consensus TATA box, an initiator element and a downstream promoter element. Furthermore, occupancy of the general transcriptional machinery in pre-B versus mature B-cell lines correlate with CR2/CD21 expression level and indicate that promoter accessibility must switch from inactive to active during the transitional B-cell window.

Conserved Curvature of RNA Polymerase Ⅰ Core Promoter Beyond rRNA Genes: The Case of the Tritryps

In trypanosomatids, the RNA polymerase I （RNAPI）-dependent promoters controlling the ribosomal RNA （rRNA） genes have been well identified. Although the RNAPI transcription machinery recognizes the DNA conformation instead of the DNA sequence of promoters, no con- formational study has been reported for these promoters. Here we present the in silico analysis of the intrinsic DNA curvature of the rRNA gene core promoters in Trypanosoma brucei, Trypanosoma cruzi, and Leiskmania major. We found that, in spite of the absence of sequence conservation, these promoters hold conformational properties similar to other eukaryotic rRNA promoters. Our results also indicated that the intrinsic DNA curvature pattern is conserved within the Leishmania genus and also among strains of T. cruzi and T. brucei. Furthermore, we analyzed the impact of point mutations on the intrinsic curvature and their impact on the promoter activity. Furthermore, we found that the core promoters of protein-coding genes transcribed by RNAPI in T. brucei show the same conserved conformational characteristics. Overall, our results indicate that DNA intrinsic curvature of the rRNA gene core promoters is conserved in these ancient eukaryotes and such con- served curvature might be a requirement of RNAPI machinery for transcription of not only rRNA genes but also protein-coding genes.

Systematic evaluation of HBV BCP/PC mutations on the risk of hepatocarcinogenesis

Objective:To evaluate of the effects of mutations in BCP-A1762T/G1764A and PC-G1896A genes on hepatocarcinogenesis.Methods:Computer searches for PubMed,SCI,CNKI,VIP and WanFang Data databases were conducted to collect literature on the role of mutations in the disease process associated with HBV infection from database creation to July 1,2021.Two researchers independently screened the articles,extracted information and evaluated the quality of the studies.Review Manager software version 5.4 was used for Meta-analysis.Results:A total of 40 articles were included,with a total of 12423 cases and 3710 cases of hepatocellular carcinoma.Meta-analysis showed that mutations in BCP-A1762T/G1764A gene were associated with the disease process of HBV infection and promoted hepatocellular carcinogenesis.mutations in BCP/PC gene were significant in the process of HBV infection in BCP-A1762T/G1764A in HCC vs non-HCC[OR=4.05,95%CI=2.64~6.22],CHBC[OR=3.90,95%CI=2.13~7.17],CHB[OR=2.77,95%CI=1.78~4.32],LC[OR=1.64,95%CI=0.95~2.84],which were statistically significant;in PC-G1896A mutation HCC vs non-HCC[OR=1.49,95%CI=1.02~2.17],CHBC[OR=1.56,95%CI=0.89~2.72],CHB[OR=1.80,95%CI=1.17~2.77]were statistically significant,while the difference was not statistically significant when comparing HCC with LC(P=0.4).The BCP-A1762T/G1764A mutation in the B genotypes/genotyped versus the C genotype[OR=0.36,95%CI=0.20~0.64],with a statistically significant difference,and no statistically significant difference in the PC-G1896A mutation.BCP-A1762T/G1764A mutation in the C gene in HCC versus non-HCC[OR=3.71,95%CI=1.82~7.61]and PC-G1896A mutation in HCC vs non-HCC[OR=2.81,95%CI=1.34~5.91],the differences were statistically significant.Conclusions:Current evidence suggests that mutations in the BCP-A1762T/G1764A and PC-G1896A genes have a significant effect on the increased risk of hepatocellular carcinoma and are genotype dependent.However,due to the limitation of the number and quality of included studies,these findings need to be validated by more high-quality studies.

Precore mutation enhances viral replication to facilitate persistent infection especially in HBeAg-negative patients

Naturally occurred precore(PC,G1896A)and/or basal core promoter(BCP,A1762T/G1764A)mutations are prevalent in chronic HBV-infected patients,especially those under HBeAg-negative status.However,the replicative capacity of HBV with PC/BCP mutations remains ambiguous.Herein,meta-analysis showed that,only under HBeAg-negative status,the serum HBV DNA load in patients with PC mutation was 7.41-fold higher than those without the mutation.Both PC mutation alone and BCPþPC mutations promoted HBV replication in cell and hydrodynamic injection mouse models.In human hepatocyte chimeric mouse model,BCPþPC mutations led to elevated replicative capacity and intrahepatic core protein accumulation.Mechanistically,preC RNA harboring PC mutation could serve as mRNA to express core and P proteins,and such pgRNA-like function favored the maintenance of cccDNA pool under HBeAg-negative status.Additionally,BCPþPC mutations induced more extensive and severe human hepatocyte damage as well as activated endoplasmic reticulum stress and TNF signaling pathway in livers of chimeric mice.This study indicates that HBeAg-negative patients should be monitored on HBV mutations regularly and are expected to receive early antiviral treatment to prevent disease progression.

Gene heterogeneity of hepatitis B virus isolates from patients with severe hepatitis B

BACKGROUND: The pathogenesis of severe hepatitis B remains unknown. Reports have indicated that hepatitis B virus (HBV) mutations are important factors in the pathogenesis of this disease. This study was to investigate the genetic heterogeneity of HBV strains from serum samples of patients with fulminant hepatitis B. METHODS: Full-length HBV genomes from 4 patients with severe hepatitis B were cloned and sequenced to observe mutations in every open reading-frame ( ORF). Serum samples of another 25 patients with severe hepatitis B, 30 patients with chronic hepatitis B, and 25 HBV carriers were collected for sequencing and comparison of mutations in preS2, preC and core promoter regions. RESULTS: Of 4 HBV full-length genome sequences, 3 had a G to A mutation at nucleotide A1896 in the preC region and 1 had double mutations of T1762-A1764 in the core promoter region. The 4 sequences showed mutations in the known B or T cell epitopes of the preS2 and C regions. For the other 3 groups, more mutations were seen in the preS2 region in the HBV isolates from the patients with severe hepatitis B than those from the patients with chronic hepatitis B and HBV carriers (P <0.01). There was a significant difference of mutations in the T cell epitope region of preS2 between the patients with severe hepatitis B and those with chronic hepatitis B or HBV carriers (P <0.01). In the preC and core promoter regions, the mutation frequencies of T1653 and C1753 were 48.0% and 24.0% respectively in the patients with severe hepatitis B, but none of these mutations were observed in the patients with chronic hepatitis B group or HBV carriers (P <0.01). The mutation frequency of T1762-A1764 was 76.0% in the patients with severe hepatitis B, 40.0% in the patients with chronic hepatitis B (P <0. 01) , and 16. 0% in the HBV carriers ( P < 0. 01). There was a significant difference in A1896 mutation between the patients with severe hepatitis B and the patients with chronic hepatitis B (P < 0. 05 ) or the HBV carriers (P<0.05). CONCLUSION: Our observations suggest that the accumulation and persistence of high frequency mutations or complex mutations may be associated with the development and deterioration of HBV infection.

Characterization of hepatitis B virus genotypes/subgenotypes in 1301 patients with chronic hepatitis B in North China

Background There is still a paucity of data on hepatitis B virus （HBV） subgenotype prevalence in North China based on sequencing of large-size samples. In addition, whether HBV genotypes impact drug-resistance-associated and HBV e antigen （HBeAg）-Ioss-associated mutations in patients with chronic hepatitis B （CHB） is still under investigation. This study aimed to disclose clinical prevalence of HBV genotypes/subgenotypes in North China and the clinical implications of HBV genotype classification in respect to HBeAg loss and drug-resistant occurrence. Methods Sera were collected from 1301 nucleos（t）ide analog-experienced CHB patients. Viral DNA was extracted and used as template for HBV genome amplification by nested PCR. DNA sequencing was performed for the analysis of HBV genotypes/subgenotypes, drug-resistance-associated mutations in polymerase gene and HBeAg-loss-associated mutations in precore/basal core promoter （BCP） regions. Results HBV/B, HBV/C, and HBV/D were detected in 190 （14.6%）, 1096 （84.2%）, and 15 （1.2%） patients, respectively. HBV/B2 （182/190）, HBV/C2 （1069/1096）, and HBV/D1 （12/15） were predominant subgenotypes within individual genotypes. By contrast, C2 prevalence is relatively lower in Beijing area （77.2%） than in other north areas （84.9%-87.4%）. HBV/C-infected patients had an older age and a lower serum albumin level but similar HBV DNA and alanine aminotransferase （ALT） levels compared to HBV/B-infected patients. HBV/C infection had a higher incidence of lamivudine-resistant mutations rtM2041N （44.9% vs. 30.2%, P 〈0.01） and BCP mutations A1762T＋G1764A （65.8% vs. 40.0%, P〈0.01） compared with HBV/B infection. Conclusions C2 is the most prevalent HBV subgenotype followed by B2 in CHB patients in North China; and HBV genotype prevalence is influenced by immigrant population. HBV/C infection is likely to have longer disease duration and severer liver functional impairment and might be more susceptible to develop lamivudine resistance compared to HBV/B infection.