Journal
WORLD JOURNAL OF GASTROENTEROLOGY
Volume 24, Issue 19, Pages 2095-2107Publisher
BAISHIDENG PUBLISHING GROUP INC
DOI: 10.3748/wjg.v24.i19.2095
Keywords
Hepatitis B virus; Hepatitis B X gene; Hepatitis B X protein; Gene therapy; Next-generation sequencing; HBV conserved regions; Small interference RNA
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Funding
- Instituto de Salud Carlos III [PI15/00856]
- European Regional Development Fund (ERDF) [PI15/00856]
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AIM To detect hyper-conserved regions in the hepatitis B virus (HBV) X gene (HBX) 5' region that could be candidates for gene therapy. METHODS The study included 27 chronic hepatitis B treatment-naive patients in various clinical stages (from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeAg-negative and HBeAg-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia > 3.5 log IU/mL, the HBX 5' end region [nucleotide (nt) 1255-1611] was PCR-amplified and submitted to next-generation sequencing (NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences (haplotypes) obtained by NGS. Conservation at the HBx protein amino acid (aa) level was also analyzed. RESULTS NGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample (2487-9279, IQR 2817). In 14/27 patients (51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region (nt 1255-1286) and the other in the 5' end coding region (nt 1519-1603). This last region coded for a conserved amino acid region (aa 63-76) that partially overlaps a Kunitz-like domain. CONCLUSION Two hyper-conserved regions detected in the HBX 5' end may be of value for targeted gene therapy, regardless of the patients' clinical stage or HBV genotype.
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