Bajunaid, Huda Ahmad
(2013)
Genetic variability of Hepatitis B virus.
MPhil thesis, University of Nottingham.
Abstract
We detected HBV-DNA in two out of 15 (13.3%) archival liver biopsies from Saudi HCC patients.
Our real-time quantitative assay of HBV by SYBR green was more accurate and has detection range from 102 to 1010 copies/ml. The melting temperature varied 80.5 - 81.7 oC in different genotypes. Mean HBV DNA level was significantly higher in UK than Saudi hepatitis B patients at 5.09 (95% CI 1.43-8.75) and 4.52 (95% CI 0.14-8.90) log10 copies/ml, respectively (p: 0.007). Mean HBV DNA in patients with HBeAg was higher at 7.18 and 6.44 log10 copies/ml in UK and Saudi patients, respectively. HBV genotypes A, B, C and D were mostly detected in UK patients, A and D in Saudi patients. In UK patients, genotypes C and D showed higher mean HBV DNA than genotypes A or B. Saudi patients had higher mean HBV DNA in genotypes D than genotypes A or B. Within genotype D, UK patients had even higher HBV DNA levels than Saudi patients at 6.10 and 5.10 log10 IU/ml, respectively. All UK HBV genotype A had subgenotype Ae, genotype B had subgenotype Ba, and genotype C subgenotype C1 (Cs). HBV detected in Saudi Samples had HBV subgenotype D1, while UK had subgenotype D2 mostly in Asian patients.
Core promoter region/X gene sequences showed clear correlation to HBV genotypes and subgenotypes unlike the conserved precore region. Sequencing of the S gene, Pre S1 region, Pre S2 and beginning as well as the end of S gene overlapping the YMDD region of the reverse transcriptase part of pol gene showed accurate discrimination between genotypes, and genotype-related sequence variations. In contrast, the more conserved regions at the `a` determinant and the rt part of the Pol gene. That can contribute to differences in fibronectin binding site to liver sinusoids, and the expression efficiency of HBsAg between different genotypes. Furthermore, the genotype-specific contribution of preS1 epitopes favours vaccination using the large HBsAg of the prevalent HBV genotypes in specific geographical areas. We found Single nucleotide polymorphism II (SNPs) at S/pol gene that are genotype-specific, and can be of benefit in new HBV molecular assays and DNA arrays.
BCP mutants 1762-1764 were seen in 19/26 (73%) Saudi patients compared to 9/23 (39%) UK patients (p: 0.003). In Saudi patients, BCP mutants were highest in genotype A and genotype D at 7/9 (78%) and 17/20 (85%), respectively. All possibilities were seen a change of AGG to TGG in 5 patients, 1762/1764 A/A, and the double mutant T/A were seen in 2 and 7 patients, respectively. In addition, two patients had G1762/T1764, one had C1764, one T1764 and one case showed 12 bp-deletion in BCP region. In UK patients, one BCP variants 1762-1764 had AGT. A/A, and the double mutant T/A were also seen in 3 and 5 patients, respectively. C1766T, T1768A and C1773T C1799G and C1858 were present. All patients had chronic hepatitis and one case presented with HCC
Significant proportion of Saudi patients 37/56 (66.1%) had precore (PC) mutants (p: 0.004), especially in genotype D 35/48 (73%),. Of those, codon 28 mutant (G1896A) was present in 30/37 (81.1%), resulting in a change of tryptophan to a stop codon. Double mutants in both codons 28 and 29 were seen in 15/37 (40.5%) of total mutants. G1899A mutation, which alters codon 29 from glycine to aspargine, was detected mostly in Saudi genotype D patients 22/37 (61%). In contrast, UK patients, precore mutations found in only 5/23 (21.7%), of HBV genotypes A, B and D. Their implications need to be verified in detailed studies. The mean HBV DNA of wild type in both BCP and PC was higher than mutants. Coexisted BCP and PC mutants occurred mainly in Saudi patients 21/25 (84%) versus only one UK patient. In this study, HBV X gene mutations lysine to methionine (K130M) was seen in a total of 6/22 UK and 11/25 Saudi patients. Valine has changed to isoleucine (V131I) present in 8/22 UK patients and 10/25 Saudi patients. Other X-gene variants; I127N and F132Y were seen. F132Y was evident in two genotype A out of 10 (20%) patients, and 6/20 (30%) genotype D patients.
Clinically, BCP mutants were present in liver disease patients were much higher in Saudi patients 18/21 (90%) than in UK patients 8/21 (38%). Saudi patients with inactive hepatitis had comparable rates of PC mutants 9/13 (69%) to liver disease 28/43 (65%) (P: 0.019); as acute hepatitis 4/5 and chronic hepatitis 17/29 (59%).
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