Research ArticleHepatitis B virus splicing is enhanced prior to development of hepatocellular carcinoma
Introduction
Despite the availability of a preventative vaccine, chronic hepatitis B virus (HBV) infection remains a global health issue, affecting more than 400 million people worldwide and is associated with more than two million deaths annually [8], [14]. Chronic HBV infection is a leading risk factor for the development of hepatocellular carcinoma (HCC) and 40% of all HCCs are directly attributable to HBV [8]. HCC itself remains the 5th most common cancer and 3rd highest cause of cancer-related mortality worldwide [15].
HBV has a 3.2 kB partially double-stranded, relaxed circular genome organised into four overlapping open-reading frames from which all viral genes are transcribed, including the polymerase protein (Pol) [20]. Production of progeny virus requires the completion of the double-stranded genome, a function performed by Pol through reverse transcription [10], [20]. The HBV genome also encodes specific sequence elements which promote splicing of viral DNA [1], [10]. Spliced (sp) HBV variants are incapable of autonomous replication as they do not encode a functional Pol, however, they may replicate in the presence of wild type (wt) HBV [1], [3], [24]. To date, fourteen different spHBV variants have been identified in cells or serum obtained from patients with chronic HBV [23] (Fig. 1A).
The most frequently detected spHBV variant is a 2 kB molecule termed Sp1, generated through the removal of a 1.3 kB intron from the HBV pregenomic RNA (pgRNA) [1]. Excision of this large intron removes the promoters and coding sequences for the PreS1 and PreS2 proteins as well as the terminal protein, spacer and much of the reverse transcription domains of Pol [22]. However, the promoters and coding sequences for core, precore, and the HBV X proteins remain intact, and expression of the resultant HBV proteins and HBV replication itself are suggested to increase during co-infection with wild type (wt) and spHBV compared to wt infection alone [1].
Cell culture experiments have further shown that up to 30% of all HBV RNA transcripts are spliced and support the notion that expression of spliced variants independently increases wtHBV replication [1], [16]. A doubly-spliced HBV variant; Sp10, was isolated from the serum of a patient with HCC and reported to increase wtHBV replication in vitro [16]. This is important as increased HBV replication is a major risk factor for development of HCC [4], [5], [15].
In spite of this, the true nature of the relationship between spHBV and development of HCC remains unknown. Previous studies have suggested a relationship between splicing and liver disease, although the results remain contentious [1], [9], [13], [16], [21], [25]. One possible explanation for this is the inclusion of patients with advanced cirrhosis, which is well recognised as a precancerous state [4], [21]. Similarly, co-infection of patients with HIV and HCV accelerates the progression of liver disease [4], [31] and thus represents a potential confounder. The lack of available samples and commensurate clinical data from patients prior to diagnosis of HCC, which would allow for more inclusive multivariate analysis of the factors contributing to changes in spHBV, has further compounded the difficulties associated with tracking changes in spHBV over time.
In this study, we have analysed changes in spHBV over time; providing the first longitudinal analysis of spHBV in relation to the development of HCC. This information has been correlated with clinical data to provide a holistic examination of the factors affecting spHBV both before and after clinical diagnosis of HCC.
Section snippets
HCC positive patients
One hundred and thirty serum samples with positive HBV viral load were obtained from 58 individuals (47 male; 11 female) with chronic HBV monoinfection who were clinically diagnosed with HCC between 1997 and 2012 (Table 1). This included 47 samples from time points prior to clinical diagnosis of HCC, 38 samples from time points which correlated with clinical diagnosis of HCC (within three months) and 45 samples from time points following clinical diagnosis of HCC. For each patient, between two
Case control study for the evaluation of the utility of real-time PCR testing of serum spHBV
spHBV was detectable via real-time PCR in 95% (36/38) of serum samples taken from HCC patients at the time of HCC diagnosis and 85% (91/107) of clinically matched control samples (p = 0.16). Median serum spHBV levels were significantly higher in the HCC patients than HCC-free control patients (6.693% (0–60.89%) vs. 0.424% (0–26.68%) for HCC and HCC-free respectively; p <0.001, where spHBV is expressed as a percentage of wtHBV). When spHBV levels were compared across HCC and HCC-free controls with
Discussion
This study is the first to identify quantitative changes in spHBV prior to the development of HCC. Among a cohort of 58 HBV monoinfected patients who later developed HCC, we demonstrated the presence of spHBV in over 80% of serum samples, echoing earlier findings of constitutive spHBV expression during chronic HBV infection [9], [21], [25]. We further detected an increase in spHBV of approximately 0.1% each year prior to the development of HCC.
Our finding that spHBV is increased prior to HCC
Financial support
Funding for this study was provided by the Watt Geyer Grant in Aid for Liver Cancer Research through Melbourne Health.
Conflict of interest
The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.
Acknowledgements
The authors acknowledge the assistance of Dr. Uma Devi and Ms. Margaret Nash in collecting serum samples used throughout this study.
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