Applications of next-generation sequencing analysis for the detection of hepatocellular carcinoma-associated hepatitis B virus mutations

Abstract

BackgroundNext-generation sequencing (NGS) is a powerful and high-throughput method for the detection of viral mutations. This article provides a brief overview about optimization of NGS analysis for hepatocellular carcinoma (HCC)-associated hepatitis B virus (HBV) mutations, and hepatocarcinogenesis of relevant mutations.Main bodyFor the application of NGS analysis in the genome of HBV, four noteworthy steps were discovered in testing. First, a sample-specific reference sequence was the most effective mapping reference for NGS. Second, elongating the end of reference sequence improved mapping performance at the end of the genome. Third, resetting the origin of mapping reference sequence could probed deletion mutations and variants at a certain location with common mutations. Fourth, using a platform-specific cut-off value to distinguish authentic minority variants from technical artifacts was found to be highly effective. One hundred and sixty-seven HBV single nucleotide variants (SNVs) were found to be studied previously through a systematic literature review, and 12 SNVs were determined to be associated with HCC by meta-analysis. From comprehensive research using a HBV genome-wide NGS analysis, 60 NGS-defined HCC-associated SNVs with their pathogenic frequencies were identified, with 19 reported previously. All the 12 HCC-associated SNVs proved by meta-analysis were confirmed by NGS analysis, except for C1766T and T1768A which were mainly expressed in genotypes A and D, but including the subgroup analysis of A1762T. In the 41 novel NGS-defined HCC-associated SNVs, 31.7% (13/41) had cut-off values of SNV frequency lower than 20%. This showed that NGS could be used to detect HCC-associated SNVs with low SNV frequency. Most SNV II (the minor strains in the majority of non-HCC patients) had either low (< 20%) or high (> 80%) SNV frequencies in HCC patients, a characteristic U-shaped distribution pattern. The cut-off values of SNV frequency for HCC-associated SNVs represent their pathogenic frequencies. The pathogenic frequencies of HCC-associated SNV II also showed a U-shaped distribution. Hepatocarcinogenesis induced by HBV mutated proteins through cellular pathways was reviewed.ConclusionNGS analysis is useful to discover novel HCC-associated HBV SNVs, especially those with low SNV frequency. The hepatocarcinogenetic mechanisms of novel HCC-associated HBV SNVs defined by NGS analysis deserve further investigation.

Advanced Therapeutics, Vaccinations, and Precision Medicine in the Treatment and Management of Chronic Hepatitis B Viral Infections; Where Are We and Where Are We Going?

“…NGS is also applied for the characterization of viruses in situ, in animals [123] and in humans [124]. Recently, NGS was used to detect pre-S mutants in the plasma of HBV-related HCC patients, suggesting that NGS may provide better accuracy for the detection of pre-S deletions, potentially improving prediction outcomes for patients with HBV-related HCC [125]. NGS analysis was used to assess HBV mutations and the relevance of mutations to HCC development.…”

“…NGS analysis was used to assess HBV mutations and the relevance of mutations to HCC development. In addition, ultradeep sequencing was used to examine the diversity between intrahepatic HBV strains and those circulating in the serum [125]. This same technique was also used for the analysis of HBV reverse transcriptase quasi-species heterogeneities, to identify not only those host genes that are frequent sites for HBV integration but also to study the effects of HBV integration on the genomes of HCC patients [126].…”

Hepatitis B Virus: From Diagnosis to Treatment

“…The mutations in the gene C that encode for precore and core proteins are significantly correlated with liver disease progression in CHB patients (Al-Qahtani et al 2018). The changes in the amino acid sequences: W28*, G29D, G1896A, G1899A, G1862T in the precore proteins that affect HBeAg, and F24Y, E64D, E77Q, A80I/T/V, L116I, E180A in the core proteins mutations are commonly identified and related to clinical severity (Kim et al 2016;Wu et al 2018).…”

Differences in HBV Replication, APOBEC3 Family Expression, and Inflammatory Cytokine Levels Between Wild-Type HBV and Pre-core (G1896A) or Basal Core Promoter (A1762T/G1764A) Mutants

“…HBV-related oncogenesis is complex and is influenced by viral characteristics such as genetic variants particularly within the X/basal core promoter (BCP)/pre-core (PC) region and integration into the host chromosomes contributing to genomic instability and hepatocarcinogenesis. Through next-generation sequencing (NGS), our group and others have demonstrated the variability of HBV within CHB carriers either with or without end-stage liver disease (cirrhosis and cancer) ( Yan et al, 2015 ; Lau et al, 2018 ; Wu et al, 2018 ). Moreover, we have shown HBV genome integration in both liver and lymphoid cells in individuals with hepatic and extrahepatic malignancy ( Lau et al, 2019 , 2020 ).…”