Sequence analysis of Pre-S gene in asymptomatic chronic HBV carriers with low-level HBsAg

Objective To analyze the sequence of Pre-S gene in asymptomatic chronic HBV carriers (ASCs) with low-level HBsAg. Methods The serum samples were collected from 654 ASCs in the First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou Sixth People’s Hospital and the 903th Hospital of PLA. According to the level of HBsAg, ASCs were divided into low-level HBsAg group (≤10 IU/mL) and high-level HBsAg group (>10 IU/mL). The pre-S/S gene amplification and sequencing were performed in 138 ASCs with low-level HBsAg and 100 age-matched ASCs with high-level HBsAg. A phylogenetic tree was constructed to determine the genotype, based on the successful sequencing results of Pre-S gene in the high level HBsAg group, the Pre-S gene reference sequences of the main ASCs genotypes in Eastern China were established. The sequence of Pre-S gene in low-level HBsAg group was analyzed and compared with the reference sequences. SPSS 12.01 statistical software was used to analyze the data. Results Sixty-three cases of Pre-S/S were successfully sequenced in 138 ASCs of low-level HBsAg group, including 52 cases of B genotype and 11 cases of C genotype. Among the 100 cases of high-level HBsAg group, 94 cases of Pre-S/S were successfully sequenced, including 48 cases of B genotype and 46 cases of C genotype. The sequence analysis indicated that in the B genotype, 81 amino acid mutation sites were found in the Pre-S protein of the low-level HBsAg group, including 4 significant mutations: F56I/V, T76A/N/P in the Pre-S1 region, P15L/S/T and Y21T/F/H/N in the Pre-S2 region; while 47 amino acid mutation sites were found in Pre-S protein of high-level HBsAg group, including 3 significant mutations: L34F, V49A and P59S/L in Pre-S1 region. The total number of amino acid mutation sites in the low-level HBsAg group of B genotype was higher than that of the high-level HBsAg group (χ2=14.008, P<0.05). In the C genotype, 19 amino acid mutation sites were found in the Pre-S protein of the low-level HBsAg group, including 3 significant mutations: W66V/G and A79V in the Pre-S1 region, V32A in the Pre-S2 region; while 39 amino acid mutation sites were found in Pre-S protein of the high-level HBsAg group, including 2 significant mutations: A79V in Pre-S1 region and T49I in Pre-S2 region. The total number of amino acid mutation sites of Pre-S protein in the C genotype was significantly different between the two groups (χ2=7.571, P<0.05). Conclusion Significant mutations in Pre-S gene may be associated with the persistent expression of low-level HBsAg in ASCs. Key words: Hepatitis B virus; Asymptomatic chronic HBV carriers; Low-level HBsAg; Pre-S gene reference sequences