dlDNA marks progression of HBV-related liver disease
Published on June 30, 2015 at 9:15 AM · No Comments
By Shreeya Nanda, Senior medwireNews Reporter
The level of serum duplex-linear DNA (dlDNA) increases markedly with liver disease progression and development of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B virus (HBV) infection, suggests research published in Gut.
Viral dlDNA has been shown to be the primary precursor of HBV DNA integration into host chromosomes, a process that can have oncogenic consequences, explain the researchers, adding that their main result “supports the notion that dlDNA may play a role in HCC oncogenesis and suggests that therapeutic reduction of dlDNA may reduce the risk of HCC development.”
Using a peptide nucleic acid-mediated quantitative real-time polymerase chain reaction clamping assay developed for the purpose of detecting dlDNA, the proportion of serum dlDNA relative to total HBV DNA was found to be a median of 7.24% in the 143 chronic HBV patients.
This rose significantly to a median of 14.14% in the 20 patients with liver cirrhosis (p=0.001) and to 20.30% in the 55 patients with liver cirrhosis-related HCC (p<0.001).
Among patients with chronic HBV, median serum dlDNA proportion was significantly higher in those with abnormal compared with normal alanine aminotransferase (ALT) levels, at 8.18% and 4.84% (p<0.001), respectively. The difference between groups remained significant when patients were matched by gender, age, and HBV DNA level and genotype (p<0.001).
When just those patients treated with pegylated interferon-α-2b were considered, serum dlDNA proportion was significantly higher in the 10 patients who responded to treatment than in the 20 who did not (11.54 vs 6.64%; p=0.028)
And experiments using HBV transfected HepG2 cells showed that treatment with inflammatory cytokines, such as interleukin (IL)-10, tumour necrosis factor-α and interferon-γ, had an effect on dlDNA levels, with IL-10 resulting in a dose-dependent increase in dlDNA proportion while the other cytokines had a “bell-shaped effect”.
“This might reveal the mechanism of association between abnormal alanine aminotransferase levels and elevated dlDNA proportion, and further provide evidence that chronic inflammation might contribute to the development of liver cancer by increasing the level of dlDNA”, speculate Xiao-Ben Pan (Peking University People’s Hospital, Beijing, China) and co-investigators.
Gut. 2015 Jun 4. pii: gutjnl-2014-308989. doi: 10.1136/gutjnl-2014-308989. [Epub ahead of print]
Serum viral duplex-linear DNA proportion increases with the progression of liver disease in patients infected with HBV.
Zhao XL1, Yang JR2, Lin SZ3, Ma H1, Guo F4, Yang RF1, Zhang HH1, Han JC1, Wei L1, Pan XB1.
1Peking University People's Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing, P.R. China.
2Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China.
3Department of Hepato-Biliary-Pancreatic Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, P.R. China.
4Peking University People's Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing, P.R. China Department of Microbiology and Immunology, Drexel Institute for Biotechnology and Virology Research, Drexel University College of Medicine, Doylestown, Pennsylvania, USA.
Abstract
OBJECTIVE:
HBV has two forms of genomic DNA, relaxed-circular DNA (rcDNA) and duplex-linear DNA (dlDNA). Compared to rcDNA, dlDNA has been demonstrated to integrate more frequently into host cellular chromosomes, which may have oncogenic consequences. However, the dlDNA proportion relative to total HBV DNA and its clinical significance in patients remain to be investigated.
DESIGN:
Based on the structural difference between rcDNA and dlDNA, we developed a peptide nucleic acid (PNA)-mediated quantitative real-time PCR (qPCR) clamping assay to measure the proportions of dlDNA in total HBV DNA in sera obtained from patients with chronic hepatitis B (CHB), liver cirrhosis (LC) or LC-developed hepatocellular carcinoma (HCC). The factors that influence the proportion of dlDNA were also investigated.
RESULTS:
The average dlDNA proportion was approximately 7% in the sera of chronic HBV-infected patients and was elevated in CHB patients with abnormal levels of alanine aminotransferase. The sera dlDNA proportions increased to approximately 14% and 20% in the patients with LC and HCC, respectively. Interferon-α treatment slightly increased the dlDNA proportion in the responders; and nucleotide analogue therapy spuriously elevated the proportion. Moreover, treatment of human hepatoma cells supporting HBV replication with inflammatory cytokines significantly altered the dlDNA proportion in vitro.
CONCLUSIONS:
Using a novel PNA-mediated qPCR clamping assay, we first showed that serum dlDNA proportions progressively increased during the development of HBV-related liver diseases. The dlDNA proportion can be regulated by inflammatory cytokines, suggesting an association among inflammation, increased production of HBV dlDNA and development of HCC.