An investigation of genome-wide promoter region cytosine-phosphate-guanine (CpG) Island methylation profiles in patients with chronic hepatitis B virus infection

Includes bibliographical references.Hepatitis B virus (HBV) is oncogenic and a major cause of hepatocellular carcinoma (HCC) in the developing world. It integrates parts of its genome such as the HBx gene, core and surface antigens into the human genome. The integrated viral DNA disrupts gene function resulting in physiological changes that cause liver disease. The viral inserts are inactivated through methylation. This is a protective innate response driven by human DNA methyltransferases triggered by the presence of viral DNA inserts. This thesis investigates the hypothesis that during the innate response to methylate integrated HBV DNA, there is unintended methylation of genomic DNA around the intercalated viral DNA that could be adjacent host promoter Cytosine-phosphateGuanine (CpG) islands. This would activate or silence genes including tumour suppressors and result in the clinical disease phenotypes of hepatic inflammation, fibrosis and HCC that characterise chronic HBV infection. Genome-wide microarray analysis was used to investigate for the presence of promoter CpG island methylation in a cohort of patients with liver disease due to HBV infection, HCC, autoimmune hepatitis which is a non-viral liver disease and normal cases with no liver disease. The study identified hypermethylation in promoter regions, transcription start sites, gene exons and introns. Only sites in the promoter region and within 100bp upstream of a transcription start site were analysed for this thesis presentation. Using an extended cohort of patients with chronic HBV infection and normal controls, bisulfite DNA sequencing was used to validate and confirm the presence of DNA methylation in a selection of some of genes identified. HBV infected patients were shown to have hypermethylation in the promoter CpG island regions of several genes that regulate hepatic metabolism, tumour suppression, ribonucleic acid splicing, vitamin D receptor binding, protein ubiquitination and the cell cycle. Many of these genes have transcriptional binding factors that are known to be affected by the transcriptional transactivator HBx protein, suggesting that HBx protein is important in the pathogenesis of liver disease. Amongst the most hypermethylated core promoter regions identified were those for cyclin kinases genes such as Cyclin D3 (CCND3). CCND3 gene is important in liver regeneration and wound healing and its abnormal function has been linked to the development of liver fibrosis and HCC. Increased methylation of CCND3 gene was associated with HBV e antigen positive status and genotype D, supporting the hypothesis that increased methylation is associated with host and viral factors. Methylation induced alteration in the function of the identified gene promoters would affect cellular signalling with effects on cell growth, differentiation, proliferation and apoptosis. These changes would explain the development of hepatic inflammation, apoptosis, fibrosis and malignant transformation seen in chronic HBV infection. Further investigation of these genes will provide new insights on mechanisms of HBV induced liver disease and the development of new molecular diagnostic tools or therapeutic interventions