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. 2022 Feb 12;4(4):100449.
doi: 10.1016/j.jhepr.2022.100449. eCollection 2022 Apr.

Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B

Nicholas van Buuren  1 , Ricardo Ramirez  1 , Cameron Soulette  1 , Vithika Suri  1 , Dong Han  1 , Lindsey May  1 , Scott Turner  1 , P C Parvangada  1 , Ross Martin  1 , Henry L Y Chan  2 , Patrick Marcellin  3 , Maria Buti  4 , Nam Bui  1 , Neeru Bhardwaj  1 , Anuj Gaggar  1 , Li Li  1 , Hongmei Mo  1 , Becket Feierbach  1
Affiliations

Targeted long-read sequencing reveals clonally expanded HBV-associated chromosomal translocations in patients with chronic hepatitis B

Nicholas van Buuren et al. JHEP Rep. .

Abstract

Background & aims: HBV infects over 257 million people worldwide and is associated with the development of hepatocellular carcinoma (HCC). Integration of HBV DNA into the host genome is likely a key driver of HCC oncogenesis. Here, we utilise targeted long-read sequencing to determine the structure of HBV DNA integrations as well as full isoform information of HBV mRNA with more accurate quantification than traditional next generation sequencing platforms.

Methods: DNA and RNA were isolated from fresh frozen liver biopsies collected within the GS-US-174-0149 clinical trial. A pan-genotypic panel of biotinylated oligos was developed to enrich for HBV sequences from sheared genomic DNA (∼7 kb) and full-length cDNA libraries from poly-adenylated RNA. Samples were sequenced on the PacBio long-read platform and analysed using a custom bioinformatic pipeline.

Results: HBV-targeted long-read DNA sequencing generated high coverage data spanning entire integrations. Strikingly, in 13 of 42 samples (31%) we were able to detect HBV sequences flanked by 2 different chromosomes, indicating a chromosomal translocation associated with HBV integration. Chromosomal translocations were unique to each biopsy sample, suggesting that each originated randomly, and in some cases had evidence of clonal expansion. Using targeted long-read RNA sequencing, we determined that upwards of 95% of all HBV transcripts in patients who are HBeAg-positive originate from cccDNA. In contrast, patients who are HBeAg-negative expressed mostly HBsAg from integrations.

Conclusions: Targeted lso-Seq allowed for accurate quantitation of the HBV transcriptome and assignment of transcripts to either cccDNA or integration origins. The existence of multiple unique HBV-associated inter-chromosomal translocations in non-HCC CHB patient liver biopsies suggests a novel mechanism with mutagenic potential that may contribute to progression to HCC.

Lay summary: Fresh frozen liver biopsies from patients infected with HBV were subjected to targeted long-read RNA and DNA sequencing. Long-read RNA sequencing captures entire HBV transcripts in a single read, allowing for resolution of overlapping transcripts from the HBV genome. This resolution allowed us to quantify the burden of transcription from integrations vs. cccDNA origin in individual patients. Patients who were HBeAg-positive had a significantly larger fraction of the HBV transcriptome originating from cccDNA compared with those who were HBeAg-negative. Long-read DNA sequencing captured entire integrated HBV sequences including multiple kilobases of flanking host sequence within single reads. This resolution allowed us to describe integration events flanked by 2 different host chromosomes, indicating that integrated HBV DNA are associated with inter-chromosomal translocations. This may lead to significant transcriptional dysregulation and drive progression to HCC.

Keywords: CCS, circular consensus sequence; CHB, chronic hepatitis B; Chromosomal translocations; Chronic HBV; Clonal expansion; DNA-Seq, DNA sequencing; DR1, direct repeat 1; FFPE, formalin-fixed paraffin-embedded; HCC, hepatocellular carcinoma; Integrated HBV DNA; Long-read sequencing; N/A, nucleos(t)ide analogue; NHEJ, non-homologous end-joining; PEG-IFNα, pegylated interferon α; RIN, RNA integrity number; RNA-Seq, RNA sequencing; TDF, tenofovir disoproxil fumarate; TERT, telomerase reverse transcriptase; Targeted sequencing; WGS, whole genome sequencing; cccDNA, covalently closed circular DNA; contig, contiguous sequence; dslDNA, double-stranded linear DNA; gDNA, genomic DNA; pgRNA, pre-genomic RNA; rcDNA, relaxed circular DNA; targeted Iso-Seq, targeted long-read RNA-sequencing.

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Conflict of interest statement

All authors are employed by Gilead Sciences Inc. Please refer to the accompanying ICMJE disclosure forms for further details.

Figures

None
Graphical abstract
Fig. 1
Fig. 1
A combination of genomics approaches were developed and utilised to characterise HBV integrations in CHB patient liver biopsies. (A) Liver biopsies obtained through the Gilead 174-0149 clinical trial were processed to extract both DNA and RNA. Bulk short-read RNA-Seq was performed and multiple reads that were chimeric for host and HBV sequences were identified and mapped across the human genome (x-axis) and HBV genome (y-axis). Triangle orientation reflects the directionality of each chimeric junction. (B) WGS was performed on 19 patients with corresponding RNA-Seq and chimeric reads were mapped using the same algorithm as shown in part A. (C) We developed a custom target enrichment panel that was coupled with PacBio sequencing to generate long reads containing HBV integrations. (D) Chimeric reads were obtained from all 42 samples. Chimeric reads were mapped across the HBV genome (y-axis) and human genome (x-axis). Triangle orientation reflects the directionality of each chimeric junction. (E) Targeted PacBio recovered HBV reads that were chimeric with host sequences and HBV reads that were not. These 2 read types were quantified and correlated to HBeAg status as well as peripheral HBV DNA levels. Statistical analyses were performed using unpaired t tests and linear regression analysis. CHB, chronic hepatitis B; RNA-Seq, RNA sequencing; WGS, whole genome sequencing.
Fig. 2
Fig. 2
Heterogeneity observed amongst HBV integration patterns within and between patients. (A) Schematics for a collection of contigs generated from individual integration events assembled using targeted PacBio. The contigs are those with the highest level of read support. (B) HBV-associated chromosomal translocations where HBV sequences are flanked by sequences from 2 different human chromosomes. The contigs highlighted were those with the highest level of read support. (C) Length distributions of the HBV sequences from integrated HBV DNA vs. those that were associated with inter-chromosomal translocations. Statistical comparisons were performed using an unpaired t test. (D) Assessment of the ORFs associated with integrated HBV DNA. Data were only generated from contigs where both chimeric ends were validated by sequencing. Plotted are the number of contigs with full, partial, or no coverage for a given HBV ORF. contig, contiguous sequence; ORF, open reading frame.
Fig. 3
Fig. 3
Differentiation and quantification of the transcriptional burden from cccDNA vs. integrated HBV DNA. (A) Differentiation of transcripts from cccDNA vs. integrated HBV DNA by characterising unique 3′ tails. (B) Quantification of each transcript type as cccDNA, chimeric integrated, or non-chimeric integrated in each of the 31 baseline samples. Transcriptional signatures are correlated to serum HBV DNA as well as the ratio of chimeric to non-chimeric HBV sequences obtained from targeted DNA-Seq. (C) Correlation of cccDNA transcription to corresponding peripheral HBV DNA and HBsAg levels. Additionally, we summed all HBsAg transcripts from cccDNA and integrated origins and demonstrate a strong correlation to the corresponding peripheral HBsAg levels. Patients who are HBeAg-positive are represented by purple circles and those who are HBeAg-negative by white triangles. Correlations were calculated using a linear regression. (D) HBsAg and HBV DNA decline in the periphery at Week 48 in all patients from 174-0149 treated with TDF alone for 120 weeks (Arm C). Statistical comparison was performed using an unpaired t test. cccDNA, covalently closed circular DNA; DNA-Seq, DNA sequencing.
Fig. 4
Fig. 4
Differentiation and quantification of all HBV RNA isoforms in CHB liver biopsies. (A) All HBV isoforms including PreCore, Core, PreS1, and PreS2/S RNAs are expressed at the highest levels in biopsies from patients who were HBeAg-positive. Statistical comparison was performed using an unpaired t test. (B) HBV transcript isoform analysis from an individual patient sampled longitudinally at baseline and Week 96. This patient experienced HBeAg loss before Week 96. (C) Serum HBsAg and HBV DNA levels from the same patient from baseline through Week 96. (D) Identification of all known HBV splice isoforms in CHB liver biopsies. The ratio of spliced to non-spliced RNAs as well as the types of splice isoforms expressed were variable between patients. CHB, chronic hepatitis B.
Fig. 5
Fig. 5
Identification of a clonally expanded and transcriptionally active HBV-associated chromosomal translocation. (A) Matching FFPE and fresh frozen baseline biopsies were analysed from a 36-year-old White male who was HBeAg-negative. This patient had HBV DNA load of 7.22 log10 IU/ml and HBsAg load of 3.90 log10 IU/ml. We obtained dozens of unique reads confirming an HBV-associated chromosomal translocation between chromosome 1 and chromosome 18. We also obtained targeted Iso-Seq and bulk RNA-Seq reads indicating transcriptional activity from the same integration. Two FFPE slides were stained. As part of a multiplex panel, HBcAg, HBsAg, and NaKATPase were overlaid. In addition, a chromogenic HBsAg stain was performed. (B) Matching FFPE and fresh frozen baseline biopsies were analysed from a 24-year-old Asian male who was HBeAg-positive. This patient had a HBV DNA load of 7.54 log10 IU/ml and a HBsAg load of 3.74 log10 IU/ml. We obtained a number of reads confirming an integration consisting of 4,119 bp of continuous HBV sequence. We also obtained targeted Iso-Seq reads indicating transcriptional activity from the same integration. Two FFPE slides were stained. As part of a multiplex panel, HBcAg, HBsAg, and NaKATPase were overlaid. In addition, a chromogenic HBsAg stain was performed. FFPE, formalin-fixed paraffin-embedded; RNA-Seq, RNA sequencing; targeted Iso-Seq, targeted long-read RNA-sequencing.
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