Analysis of the reverse transcriptase domain of HBV polymerase in 310 patients with chronic hepatitis B under Lamivudine therapy
REN Wei-hong,SUN Lei,ZHAO Su-ling,et al. (Department of Clinical Laboratory,the First Affiliated Hospital of Henan College of Traditional Chinese Medicine Zhengzhou Henan 450000,China)
Objective To study the nucleotide substitution sites and patterns of the reverse transcriptase CRT) domain of HBV polymerase of the patients with chronic hepatitis B(CHB) by means of LAM therapy in order to find the HBV resistance early. Methods the serum were selected from the patients with chronic hepatitis B,who have received the treatment of Lamivudine therapy more than 6 months,and P genes of HBV isolated from serum were amplified by means of one-step PCR and then sequenced. The difference of nucleotide,aminoacid and mutation patterns of RT region was classified by using Chromas223 software. Results 135 mutations in the HBV DNA polymerase gene of 310 patients was determined by using PCR and direct sequence.Mutation of 24 nucleotide substitution sites were found by sequencing,and the primary mutations and frequence are rtM204 v/I(n=111, 82.20%),rtS213T(n = 8,5.92%),rtC256S(n = 4,2.96%),and there is a significant difference between 3 primary mutations,mutation pattern and frequence:rtM204 I(n= 39,28.89%),rtM204V+rtL180M(n = 25,18.51%),rtM204V+rtM204I+rtL180M(n =11,8.15%),rtM204I+rtL180M(n=6,4.44%),rtM204V+rtL180M+rtV173L/M(n=5,3.70%),rtS213T(n=5,3.70%), rtM204V+rtL180M+rtV207M/L/I(n = 4,2.96%),rtM204V(n = 4,2.96%)etc.rtM204V mutation can also be accompanied by rtT184 I/S/M、rtA181T、rtC256S、rtS213T、rtL229V et al compensatory mutation.Conclusion The major mutations with LAM are rtM204V/I and usually companied by tL180M、rtV173L、rtS213T、rtC256S、rtV207M/L et al compensatory mutations after CHB patients received long term LAM treatment.Not only rtM204I but also rtS213T、rtM204V is of one nucleotide substitution site pattern. Most of the multi-nucleotide substitution site patterns contain rtM204,at the same time rtS213T+rtL187I or rtC256S+ rtF221Y can also be detected by means of sequencing.