| The importance of lamivudine resistance has been well recognized by doctors and patients during the course of lamivudine treatment. Two hundred and forty seven chronic hepatitis B patients who received lamivudine in our department were followed-up. The liver function and HBV virology including HBV DNA level, HBV marker, YMDD mutant (after nine months treatment) were tested every three months. The study showed that after six months lamivudine treatment, HBV DNA level decreased to below 1x105 copies/ml in 94% patients, liver function normalized in 76% patients. The HBeAg seroconversion rate increased with the duration of treatment from 16.08% at 1 year, 33.75% at 2 years to 47.00% at 3 years. Patients with pretreatment ALT over three times ULN had a significantly higher rate of HBeAg seroconversion compared with patients with pretreatment ALT lower than three times ULN (p=0.0316). With the extended treatment of lamivudine, the accumulated incidence of YMDD mutant rised from 8.33% at 1 year, 25.79% at 2 years, 41.30% at 3 years to 51.97% at 4 years. The incidence rate was significantly higher in patients with pretreatment HBV DNA over 25 pg/ml than patients with pretreatment HBV DNA lower than that (p=0.0224). Among the 42 patients with the apperance of YMDD mutant, 32 patients experienced ALT flare with ALT over 2 times ULN, 35 patients showed DNA breakthrough with DNA over 1x105 copies/ml. However, the mean level of HBV DNA after the appearance of YMDD mutant was significantly lower that that before lamivudine treatment (p=0.005). The peak level of ALT flare after the appearane of YMDD mutant was higher in HBeAg positive patients than HBeAg negative patients (p=0.049) which may be related to the more active liver diseases in HBeAg positive patients.Nineteen patients were sequenced to confirm the result of RFLP and to find mutations outside YMDD motif of polymerase gene. It showed that RFLP was an accurate and easy method for the detection of YMDD mutant. In our patients, YVDD mutant was rarely accompanied by rtL180M change which may be related to genotype distribution in our country. We need to screen more patients to confirm the result. Ten out of nineteen patients had mutations outside YMDD motif. Five kinds of mutations (rtL80I, rtV84M, rtG 172E, rtG 174C and G172EMG174C) occurred in over two patients.These mutations were not detected in consensus sequence of HBV. In vitro replication and drug susceptibility assay are needed to investigate the biological significance of these mutants. As polymerase gene overlapped with surface gene, mutations in polymerase may affect these two proteins. Mutations in YMDD motif lead to change of I195M or W196S/L in surface protein. Two kinds of polymerase gene mutation (rtD83N and rtM204I) lead to stop codon in surface protein.Five kinds of plasmid with different polymerase gene mutations were constructed with the method of site-directed mutagenesis. The plasmids were transfected into HepG2 cells by calcium phosphate precipitation method. Co-transfection of report plasmid pSEAP was applied to monitor the transfection efficiency. Supernatant and core particle associated HBV DNA was detected with Abbott AXSYM and Real-time PCR respectively. The result showed that every plasmid can express and secrete HBsAg and HBeAg with similar level. The replication efficiency of YIDD or YVDD was only ten percent of that of wild type. The replication efficiency of rtG172E, rtG174C and rtG172E/rtG174C mutant was a little lower than that of wild type. Our result needs to be confirmed by southern blot.
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