Sequence analysis of persistently low level expression of hepatitis B surface antigen S gene in patients with hepatitis B virus infection

Objective To reveal the characteristics of S gene sequence of hepatitis B surface antigen (HBsAg) in hepatitis B virus (HBV)-infected patients with low HBsAg level. Methods From February 2016 to December 2017, 1 308 serum samples of inactive HBsAg carriers were collected from the 903rd Hospital of PLA and Hangzhou Jianggan District People′s Hospital.The cases were divided into high-level group and low-level group according to the level of serum HBsAg (10 IU/mL) expression. The HBV S gene was sequenced in patients with low-level HBsAg expression. In addition, in patients with high-level HBsAg, 100 patients were randomly selected (stratified sampling) for HBV S gene sequencing based on the matching of age and serological pattern (hepatitis B e antigen [HBeAg] negative) of low-level HBsAg group. A comparative analysis was conducted between HBV S gene sequences from inactive HBsAg carrier in low HBsAg expression group and the HBV reference S gene sequences from inactive HBsAg carrier in high HBsAg expression group.The results of normal distribution data were expressed as Mean±SD, and analyzed using t-test. The results of non-normal distribution data were expressed by M(QR), and analyzed using Mann-Whitney U test.Chi-square test or Fisher exact test was used to compare continuous variables and classification variables between the two groups. Results There were 276 serum samples from the low level group and 1 032 serum samples from the high level group, including 257 HBsAg/HBeAg/anti-HBc-positive cases, 753 HBsAg/anti-HBe/anti-HBc-positive cases, and 22 HBsAg/anti-HBc-positive cases. Successful HBV S gene sequencing was performed on 126 out of 276 patients in the low-level HBsAg group. According to the age inthe low-level HBsAg group, 100 samples with negative HBeAg in the high-level HBsAg group were randomly selected, among which 94 patients were genotyped and hemotyped. The results showed that there were statistically significant differences in HBV serological markers, HBV DNA level and HBV genotype distribution between the high level group (94 cases) and the low level group (126 cases) (all P 0.05). For genotype B, 12 single point mutations and 4 dual co-mutations were found in low level group. Among them, one single point mutation (S210R) and 3 dual co-mutations (G44E/V+ T45P/I, G44E/V+ L49P/R and N40S+ I208T) were not hot spot mutations, while 2 dual co-mutations and 2 single point mutations were found in high level group. The difference between two groups was statistical significant (χ2=7.533, P=0.006). For genotype C, 5 single point mutations (T5A, A45T, T47A/K, Q101R and I126S/T) were found in low level group and 1 single point mutation (N3S) in high level group. The difference in mutation frequency between two groups were statistical significant (χ2=47.914, P=0.000). Conclusions Significant mutations in multiple regions and at multiple sites (including co-mutations) on both sides of the MHR may be one of the causes of low HBsAg expression level in this population. Key words: Hepatitis B surface antigens; Genotype; HBV markers; HBV DNA; S gene; Mutation site