BACKGROUND: To provide a scientific basis for clinical genetic diagnosis before lamivudine treatment in hepatitis B virus (HBV) patients, the rate of natural YMDD mutations in Western China and the correlations between YMDD mutations and several factors were investigated in this study. METHODS: A pyrosequencing approach for detecting YMDD mutations was first developed. The sensitivity of the pyrosequencing approach was determined by assaying polymerase chain reaction (PCR) products generated from 10-fold serial dilutions of HBV DNA and PCR products from mixed control plasmids with different ratios. Natural YMDD mutations in Western China were evaluated by analyzing the clinical samples from HBV patients who had no experience of using lamivudine, and the existence of YMDD mutants was further confirmed by real-time PCR. RESULTS: With the developed pyrosequencing approach, YMDD mutations could be determined within 2 h after PCR amplification. About 10 copies of HBV DNA per reaction were required to obtain sufficient PCR products to produce clear and accurate pyrosequencing patterns. The pyrosequencing approach developed had the capacity to detect minor mutants; most HBV mutants in samples were minor ones. The rate of natural YMDD mutations in Western China was 15.56%. The hepatitis B e antigen (HBeAg) level in serum was correlated with YMDD mutations. CONCLUSIONS: In Western China, natural YMDD mutations occur at a rate of 15.56%, and it is suggested that pyrosequencing be performed to detect YMDD mutations before lamivudine treatment in patients with a chronic HBV infection.
BACKGROUND: To provide a scientific basis for clinical genetic diagnosis before lamivudine treatment in hepatitis B virus (HBV) patients, the rate of natural YMDD mutations in Western China and the correlations between YMDD mutations and several factors were investigated in this study. METHODS: A pyrosequencing approach for detecting YMDD mutations was first developed. The sensitivity of the pyrosequencing approach was determined by assaying polymerase chain reaction (PCR) products generated from 10-fold serial dilutions of HBV DNA and PCR products from mixed control plasmids with different ratios. Natural YMDD mutations in Western China were evaluated by analyzing the clinical samples from HBVpatients who had no experience of using lamivudine, and the existence of YMDD mutants was further confirmed by real-time PCR. RESULTS: With the developed pyrosequencing approach, YMDD mutations could be determined within 2 h after PCR amplification. About 10 copies of HBV DNA per reaction were required to obtain sufficient PCR products to produce clear and accurate pyrosequencing patterns. The pyrosequencing approach developed had the capacity to detect minor mutants; most HBV mutants in samples were minor ones. The rate of natural YMDD mutations in Western China was 15.56%. The hepatitis B e antigen (HBeAg) level in serum was correlated with YMDD mutations. CONCLUSIONS: In Western China, natural YMDD mutations occur at a rate of 15.56%, and it is suggested that pyrosequencing be performed to detect YMDD mutations before lamivudine treatment in patients with a chronic HBV infection.