Ph.D. thesis, Faculty of Health Sciences,University of the Witwatersrand, 2009 / Hepatitis B virus (HBV) infection is endemic in Africa. As many as 98% of black
Africans are infected during their lives and about 10% (65 million) have chronic
HBV infection, which is the cause of 70-80% of all hepatocellular carcinoma (HCC)
cases. Despite this high prevalence of HBV and the high incidence of HCC in
Africa, relatively few complete HBV genomes from African HCC cases have been
deposited in international data bases. In order to gain a clearer understanding of
the role of genetic variants and mutants in the development of HCC, the complete
genomes of HBV isolated from southern African HCC patients were amplified and
molecularly characterized. HBV DNA was extracted from the serum forty HBsAgpositive
HCC patients. Twenty six complete genomes were successfully amplified,
cloned and sequenced from nine HCC patients.
Phylogenetic analyses of the complete genomes and the individual open reading
frames of HBV isolates from the HCC patients, led to the classification of all the
isolates within subgenotype A1. No isolates belonging to subgenotype A2 and
genotype D were identified, even though these genotypes/subgenotypes have
been shown to circulate in South Africa. Three patients contained the uncommon
combination of serological subtype ayw1 in the subgenotype A1 strain. This
combination has been found previously in South Africa and the Phillipines.
Seventy-eight percent of the patients carried HBV strains with the double basic
core promoter (BCP) mutation (1762T/1764A), previously shown to reduce HBeAg
expression. Furthermore, complete genome sequence analysis has revealed a
complex combination of mutations, which include at least three or five of these
residues 1753C1762T1764A1766T1768A1809T1812T occurring as the dominant
HBV strains isolated from 5/9 HCC patients. These mutations have previously
been shown to regulate gene expression at various levels, to enhance viral
replication and simultaneously decrease HBeAg expression.
All five HBV genomes isolated from one patient contained novel complex BCP
rearrangements, which introduced 2 HNF1 and 1 putative HNF3 transcription
factor binding sites. These mutations can enhance viral replication and
simultaneously abolish HBeAg expression at a transcriptional level. Furthermore,
truncated core proteins would be expressed from 4/5 isolates and none would
express wild-type HBx. Several mutations were identified in the pre-S/S genes of
2/5 isolates, which would result in the expression of novel 3’ truncated medium
surface proteins (MHBst) and large surface proteins (LHBst). The majority of the
mutations would contribute to hepatocyte pathogenesis and transformation by
activating cell proliferating pathways.
Two patients also contained rare HBV variants not previously identified in HBV
strains from southern Africa. These included an HBV splice variant and a poly (dA)
variant from patient 10 and patient 6, respectively. These variants occurred in
combination with other isolates within the respective patients.
The envelope genes were characterised in a total of 18 HCC patients, the pre-S
gene of HBV contained deletions in 72% of the patients. Deletions across pre-
S1/pre-S2, pre-S2 initiation codon mutations with internal deletions, and S gene
nonsense mutations were prevalent. Mutated envelope proteins have been shown
to accumulate within the hepatocyte endoplasmic reticulum (ER) and are a
characteristic histopathological hallmark of HCC known as ground glass
hepatocytes. HBV induced ER stress has been shown to dysregulate several cell
cycle regulatory pathways, which contribute to HCC.
In addition several novel LHBst and MHBst have been described. These potential
transactivators require further investigation. The HBV mutations described in this
study have been associated with increased risk for HCC.
Despite the obvious heterogeneity HBV displays within and between patients,
there are common characteristics shared between the HBV variants which emerge
during the development of HCC. These include the BCP and pre-C
(1753C1762T1764A1766T1768A1809T1812T) mutations and the pre-S/S
mutations. These mutations are able to affect HBV replication and gene
expression, and may work synergistically to promote liver dysfunction and HCC.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:wits/oai:wiredspace.wits.ac.za:10539/7511 |
| Date | 15 February 2010 |
| Creators | Skelton, Michelle |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf, application/pdf, application/pdf |
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