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Novel natural mutations in the hepatitis B virus reverse transcriptase domain associated with hepatocellular carcinoma.

Wu Y, Gan Y, Gao F, Zhao Z, Jin Y, Zhu Y, Sun Z, Wu H, Chen T, Wang J, Sun Y, Fan C, Xiang Y, Qian G, Groopman JD, Gu J, Tu H - PLoS ONE (2014)

Bottom Line: The entire sequence of HBV RT was first compared between 29 HCC and 35 non-HCC cases, and candidate mutations were then evaluated in two independent validation sets.There were 15 candidate mutations identified from the discovery set, with A799G and T1055A being consistently associated with HCC across all studies.Our study provides evidence the first that HBV RT contains naturally occurring mutations that can be used as predictive markers for HCC.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT

Background/aim: Hepatitis B Virus (HBV) mutations play a role in the development of hepatocellular carcinoma (HCC). However, the association between HBV polymerase gene mutations and HCC has not been reported. In this study, we conducted a multi-stage study to identify HCC-related mutations in the reverse transcriptase (RT) domain of the HBV polymerase gene.

Methods: A total of 231 HCCs and 237 non-HCC controls from Qidong, China, were included in this study. The entire sequence of HBV RT was first compared between 29 HCC and 35 non-HCC cases, and candidate mutations were then evaluated in two independent validation sets.

Results: There were 15 candidate mutations identified from the discovery set, with A799G and T1055A being consistently associated with HCC across all studies. A pooled analysis of samples revealed that A799G, A987G, and T1055A were independent risk factors for HCC, with adjusted odds ratios of 5.53 [95% confidence interval (CI), 1.69-18.10], 4.20 (95%CI, 1.15-15.35), and 3.78 (95%CI, 1.45-9.86), respectively. A longitudinal study showed that these mutations were detectable 4-5 years prior to HCC diagnosis.

Conclusions: Our study provides evidence the first that HBV RT contains naturally occurring mutations that can be used as predictive markers for HCC.

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Related in: MedlinePlus

Frequency of nucleotide mutations and amino acid substitutions within HBV RT domain (n = 64).The values are presented as the percent nucleotide mutations (upper panel) or amino acid substitution (lower panel) compared to the wild-type genotype C hepatitis B virus (HBV) DNA sequence. The locations of the domains (A–F) are shown at the top of the figure. The pre-S/S-overlapping region in the polymerase gene is identified by grey areas. Abbreviation: HBV, hepatitis B virus; RT, reverse transcriptase.
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pone-0094864-g002: Frequency of nucleotide mutations and amino acid substitutions within HBV RT domain (n = 64).The values are presented as the percent nucleotide mutations (upper panel) or amino acid substitution (lower panel) compared to the wild-type genotype C hepatitis B virus (HBV) DNA sequence. The locations of the domains (A–F) are shown at the top of the figure. The pre-S/S-overlapping region in the polymerase gene is identified by grey areas. Abbreviation: HBV, hepatitis B virus; RT, reverse transcriptase.

Mentions: DNA sequences of the HBV RT domain(nt 130–1161)were determined by direct PCR sequencing using the samples from 29 HCC patients and 35 HBV carriers. According to the results of the phylogenetic tree, all of these subjects were infected with the genotype C virus. Thus, a prototype sequence of HBV genotype C from a CHB patient was used as the reference to perform the alignment. A total of 101 nucleotide mutations were found in the HBV RT domain. As depicted in Figure 2, the majority of these mutation sites were located in the 3′-terminal region of RT (nt 836–1161), which does not overlap with the HBV preS2/S gene (nt 3205–835). The average mutation rate in this RT 3′-terminal region was significantly higher than that of the RT 5′-terminal region spanning from nt 130 to nt 835 (for HCC, 16.9±8.7 per 1000 nts vs. 4.4±2.6 per 1000 nts, P<0.001; for non-HCC, 17.7±12.6 per 1000 nts vs. 3.5±2.6 per 1000 nts, P<0.001). At the protein level, the total number of aa substitution sites was 80; the distribution and frequency of the aa substitutions in the RT domain are presented in Table S2 in File S1. The conservative-function G domain showed no aa substitutions, whereas domains A, B, C, D, E and F exhibited 4, 7, 1, 3, 2, and 1 substitution, respectively. After normalization with the size of each domain, domain E displayed the highest aa substitution rate (20.41 per 1000 aa), followed by the region from domain E to the end of RT (18.86 per 1000 aa) and the space between domain D and domain E (18.75 per 1000 aa). It is worth noting that we did not observe any of the common NA-related resistance mutations including rtM204V/I, rtS202C/G/I, rtL180M, rtA181T/V, rtT184A/I/L/G/C/M, rtA194T, rtI169T, rtV173L, rtL80I, rtN236T, and rtM250I/V [12], [13], [24], suggesting that these variants were either absent or present at a very low frequency in our study subjects.


Novel natural mutations in the hepatitis B virus reverse transcriptase domain associated with hepatocellular carcinoma.

Wu Y, Gan Y, Gao F, Zhao Z, Jin Y, Zhu Y, Sun Z, Wu H, Chen T, Wang J, Sun Y, Fan C, Xiang Y, Qian G, Groopman JD, Gu J, Tu H - PLoS ONE (2014)

Frequency of nucleotide mutations and amino acid substitutions within HBV RT domain (n = 64).The values are presented as the percent nucleotide mutations (upper panel) or amino acid substitution (lower panel) compared to the wild-type genotype C hepatitis B virus (HBV) DNA sequence. The locations of the domains (A–F) are shown at the top of the figure. The pre-S/S-overlapping region in the polymerase gene is identified by grey areas. Abbreviation: HBV, hepatitis B virus; RT, reverse transcriptase.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4006920&req=5

pone-0094864-g002: Frequency of nucleotide mutations and amino acid substitutions within HBV RT domain (n = 64).The values are presented as the percent nucleotide mutations (upper panel) or amino acid substitution (lower panel) compared to the wild-type genotype C hepatitis B virus (HBV) DNA sequence. The locations of the domains (A–F) are shown at the top of the figure. The pre-S/S-overlapping region in the polymerase gene is identified by grey areas. Abbreviation: HBV, hepatitis B virus; RT, reverse transcriptase.
Mentions: DNA sequences of the HBV RT domain(nt 130–1161)were determined by direct PCR sequencing using the samples from 29 HCC patients and 35 HBV carriers. According to the results of the phylogenetic tree, all of these subjects were infected with the genotype C virus. Thus, a prototype sequence of HBV genotype C from a CHB patient was used as the reference to perform the alignment. A total of 101 nucleotide mutations were found in the HBV RT domain. As depicted in Figure 2, the majority of these mutation sites were located in the 3′-terminal region of RT (nt 836–1161), which does not overlap with the HBV preS2/S gene (nt 3205–835). The average mutation rate in this RT 3′-terminal region was significantly higher than that of the RT 5′-terminal region spanning from nt 130 to nt 835 (for HCC, 16.9±8.7 per 1000 nts vs. 4.4±2.6 per 1000 nts, P<0.001; for non-HCC, 17.7±12.6 per 1000 nts vs. 3.5±2.6 per 1000 nts, P<0.001). At the protein level, the total number of aa substitution sites was 80; the distribution and frequency of the aa substitutions in the RT domain are presented in Table S2 in File S1. The conservative-function G domain showed no aa substitutions, whereas domains A, B, C, D, E and F exhibited 4, 7, 1, 3, 2, and 1 substitution, respectively. After normalization with the size of each domain, domain E displayed the highest aa substitution rate (20.41 per 1000 aa), followed by the region from domain E to the end of RT (18.86 per 1000 aa) and the space between domain D and domain E (18.75 per 1000 aa). It is worth noting that we did not observe any of the common NA-related resistance mutations including rtM204V/I, rtS202C/G/I, rtL180M, rtA181T/V, rtT184A/I/L/G/C/M, rtA194T, rtI169T, rtV173L, rtL80I, rtN236T, and rtM250I/V [12], [13], [24], suggesting that these variants were either absent or present at a very low frequency in our study subjects.

Bottom Line: The entire sequence of HBV RT was first compared between 29 HCC and 35 non-HCC cases, and candidate mutations were then evaluated in two independent validation sets.There were 15 candidate mutations identified from the discovery set, with A799G and T1055A being consistently associated with HCC across all studies.Our study provides evidence the first that HBV RT contains naturally occurring mutations that can be used as predictive markers for HCC.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.

ABSTRACT

Background/aim: Hepatitis B Virus (HBV) mutations play a role in the development of hepatocellular carcinoma (HCC). However, the association between HBV polymerase gene mutations and HCC has not been reported. In this study, we conducted a multi-stage study to identify HCC-related mutations in the reverse transcriptase (RT) domain of the HBV polymerase gene.

Methods: A total of 231 HCCs and 237 non-HCC controls from Qidong, China, were included in this study. The entire sequence of HBV RT was first compared between 29 HCC and 35 non-HCC cases, and candidate mutations were then evaluated in two independent validation sets.

Results: There were 15 candidate mutations identified from the discovery set, with A799G and T1055A being consistently associated with HCC across all studies. A pooled analysis of samples revealed that A799G, A987G, and T1055A were independent risk factors for HCC, with adjusted odds ratios of 5.53 [95% confidence interval (CI), 1.69-18.10], 4.20 (95%CI, 1.15-15.35), and 3.78 (95%CI, 1.45-9.86), respectively. A longitudinal study showed that these mutations were detectable 4-5 years prior to HCC diagnosis.

Conclusions: Our study provides evidence the first that HBV RT contains naturally occurring mutations that can be used as predictive markers for HCC.

Show MeSH
Related in: MedlinePlus