The YMDD motif mutant of the polymerase gene is frequently found in patients who used lamivudine on a long term basis, and it is usually reported to emerge after 8 months of therapy (2
). The selection of a mutation in the YMDD motif is known to occur several months earlier than the phenotypic resistance, however the information about how early the genotypic resistance occurs and what causes the resistance is limited due to the small number of subjects and differences in detection methods (3
). Hence the rate of hepatitis B resistance to lamivudine might increase further when more sensitive tests are used to detect the small amount of YMDD mutants among the wild type HBV, which would impact upon detection and monitoring the resistance.
This study demonstrated by oligonucleotide chip technology that the YMDD mutants exist naturally in the patients with chronic hepatitis B infection who had not received lamivudine before.
YMDD mutants have a significantly low replication ability. It is thought that naturally occurring YMDD mutants occupy very small portion of total HBV, and very sensitive investigative tools are required to prove it. Oligonucleotide chip technology is a very useful tool for the rapid and accurate identification of pathogenic microbes and the detection of drug-resistant and point mutations. This method has such sensitivity that it can find rifampin-resistant tuberculosis resultant from point mutation in amounts of less than 1% (9
). Oligonucleotide chip technology has been expected to be clinically applicable to detect minor variants in viral quasispecies and to increase our knowledge of spontaneous viral genome variability and selection of mutants by antiviral therapy (10
). We developed the oligonucleotide chip for the detection of mutants resistant to lamivudine. In the experiment using reference sequences generated by site-directed mutagenesis, we confirmed the sensitivity and specificity of probes. The oligonucleotide chips could specifically detect mutations in the YMDD motif (codon 552) of the HBV DNA polymerase. The chips included three types of probe (five species): wild-probe (YMDD), valine-probe (YVDD) and isoleucineprobes (YIDD1, YIDD2 and YIDD3). We could accurately detect YMDD motif mutants of YMDD, YVDD, YIDD2 and YIDD3 types in sera from chronic HBV-infected patients. These chip results were in accordance with results from RFLP, sequence analysis and allele-specific PCR. We could detect easily as little as 10% of coexisting mutant viruses in wild type viruses.
The oligonucleotide chip method detected 3 YIDD mutants in the 40 patients included in this study. Only ATT (YIDD3) was detected in all three samples. This chip result was consistent with that of the RFLP analysis. The mutants were not detected by direct-sequencing and this is reasonable when taking into consideration that naturally occurring YMDD mutants are a minor portion of the total HBV and that the standard base sequence determinant method requires more than 25% of mutants among HBV.
Our study supports that oligonucleotide chip technology may have an important role as a reliable and useful diagnostic tool for the early detection of mutants resistant to lamivudine.