Hepatitis B surface gene 145 mutant as a minor population in hepatitis B virus carriers

Abstract

Background: Hepatitis B virus (HBV) can have mutations that include the a determinant, which causes breakthrough infection. In particular, a single mutation at amino acid 145 of the surface protein (G145) is frequently reported in the failure of prophylactic treatment. The aim of this study was to evaluate the frequency of the a determinant mutants, especially the G145 variant, in Japan, where universal vaccination has not been adopted.

Methods: The present study was a retrospective study. The study cohorts were defined as follows: group 1, children with failure to prevent mother-to-child transmission despite immunoprophylaxis (n = 18, male/female = 8/10, age 1-14 years; median 6 years); group 2, HBV carriers who had not received vaccination or hepatitis B immunoglobulin (n = 107, male/female = 107, age 1-52 years; median 16 years). To detect the G145R and G145A mutants in patients, we designed 3 probes for real-time PCR. We also performed direct sequencing and cloning of PCR products.

Results: By mutant-specific real-time PCR, one subject (5.6%) was positive for the G145R mutant in group 1, while the G145 mutant was undetectable in group 2. The a determinant mutants were detected in one (5.6%) of the group 1 subjects and 10 (9.3%) of the group 2 subjects using direct sequencing, but direct sequencing did not reveal the G145 mutant as a predominant strain in the two groups. However, the subject who was positive according to the mutant-specific real-time PCR in group 1 had overlapped peaks at nt 587 in the electropherogram. In group 2, 11 patients had overlapped peaks at nt 587 in the electropherogram. Cloning of PCR products allowed detection of the G145R mutant as a minor strain in 7 (group 1: 1 subject, group 2: 6 subjects) of 12 subjects who had overlapped peaks at nt 587 in the electropherogram.

Conclusions: The frequency of the a determinant mutants was not high in Japan. However, the G145R mutant was often present as a minor population in children and adults. HBV carriers might have the a determinant mutants as a minor form.

Figure 1

Mutant-specific real-time PCR was performed in duplicate for each probe. (A) HBV DNA from serum with an escape mutant (single mutation at nt 587, A to G) was amplified using the wild-type and mutant 1, 2, and 3 probes. (B) Constructed plasmid mutant-type (single mutation at nt 587 G to A) DNA and wild-type DNA were mixed. The amount of the mutant-type plasmid DNA was equal to that of the wild-type DNA. The curves of the amplification plots are indicated by arrows. (C) Constructed plasmid mutant-type (single mutation at nt 587 G to A) DNA and wild-type DNA were mixed. The mutant:wild ratio in plasmid DNA was 1:10. (D) Constructed plasmid mutant-type (single mutation at nt 587 G to A) DNA and wild-type DNA were mixed. The mutant:wild ratio in plasmid DNA was 1:100.

Figure 2

Serum HBV DNA from a girl who became positive for HBsAg and negative for anti-HBs despite immunoprophylaxis was amplified by mutant-specific real-time PCR in duplicate. (A) Before antiviral therapy (May 2007), the Ct values of the amplification plots in the mutant 1 probe were the same as those for the wild-type probe. In addition, the amplification plots of both probes were similar in the upward slopes. Although the signals of the mutant 2 and 3 probes were detectable, the curves of the amplification plots were not steep. There was an overlapped peak at nt 587 in the electropherogram. The larger peak was G, and the smaller peak was A. Of the 20 clones, 5 had a mutation at nt 587 from G to A. (B) At the cessation of antiviral therapy (May 2009), the girl was negative for serum HBV DNA by real-time PCR. 4 months after the completion of antiviral therapy, however, she became positive again for serum HBV DNA. The signals of the mutant 1 probe as well as the wild-type probe were detected in the amplification plots (the mutant 2 and 3 probes were not used). The curves of the amplification plots were steep. There was an overlapped peak at nt 587 in the electropherogram. Of the 18 clones, six had a mutation at nt 587 from G to A.