The variations of pre-core/core would possibly determine the fate of HBV infection as they modify encapsidation, HBeAg production, immune response, inflammation, ER stress and DNA damage(15). Some studies reported that HBV infected patients with the presence of precore/core mutations were significantly developed to severe liver disease and HCC(15, 23).
The result of our study showed that a point mutation of pre-core, G1896A that convert the tryptophan to a stop codon (W/*28) was the most common variation in the patients, as detected in chronic HBV in Asia and the Mediterranean region (15). This mutation destroyed the HBeAg production and was suggested to be effective on virus replication and somehow disease progression toward HCC(24). The number of this mutation was higher in the C/HCC group than the IC and CA groups but not significantly. In the same line that, in another study in Shiraz, Iran, Taghavi et al. has reported 31.8% (14/44) of patients had mutations in the precore region (G1896A)(25). Moreover, Kim et al. reported no significant correlation between W/*28 and higher susceptibility to HCC(22). In addition, recently it has been reported that chimeric mice expressing recombinant virus containing the PC G1896A or BCP/PC/2051 mutations demonstrated higher levels of virus replication and viral protein expression than the mice expressing wild-type strain(21). Also, microarray analysis of the liver transcriptome of these mice showed increased expression of the genes involved in cell proliferation and hepatocarcinogenesis in comparison with the control group(21).
Our result also showed G29D precore mutation at the highest frequency in the C/HCC group among the groups, but the differenc ewas not statistically significant. In this regard, a meta-analysis study showed a significant correlation between G29D mutation and higher risk of HCC(26).
On the core protein, types of substitutions including inside and outside of epitopes were suggested to be impressive. This investigation indicated that numerous substitutions in the immune epitopes were coincidently associated with more severe liver disease. The substitution such as E180A which was not mapped in an epitope region was suggested to be significantly associated with disease progression by another strategies (27). There were some variations such as E77Q, E113Q, S181P/H and Q182K/*Stop outside of the epitope regions which have previously been reported to accumulate by the disease progression (28–30). However, in spite of their prevalence, our data showed that E80Q/D, E113D/Q, S181P/R and Q182K/*Stop variations were not statistically significant among the groups. Instead, as a new finding, the prevalence of E40D/Q was significantly higher in the IC subjects than CA and C/HCC groups. Further mechanisms accounting for these mutations in infection progression require additional molecular and virologic investigations.
The core substitutions of epitopes which are associated with severe liver disease have been reported by others. Jia Ja et al. investigated the association between HBc mutations and the post-operative prognosis of HBV-related HCC, which demonstrated that the HBc E77 mutation was more associated with shorter overall survival than other mutations(2). Al-Qahtani et al. reported 6 core mutations (F24Y, E64D, E77Q, A80I/T/V, L116I, and E180A) related with the progression of the disease to cirrhosis and HCC(27). They also showed that F24Y, E64D, and V91S/T mutations were located in the T-cell epitope regions and E77Q, A80I/T/V, and L116I were located within the B-cell epitope regions(27). The comparison of core sequence among our groups indicated that substitutions at residues S21, E40 and I105 were possibly accelerating the development of IC state. This was also supported by amino acid changes at the CTL epitopes including S21, P25 and V149 that were significantly more frequent in the IC group. In line with this, Soad Ghabeshi et al. reported that some mutation of HBc gene sequence in the T helper, CTL and B cell epitopes in asymptomatic HBV infected blood donors can cause a decrease in HBc and HBe antigenicity and increase in escape mutants(31). The same mutations were found in our study in the amino acids at positions 21 ( T/A to S), 25 (A/Q/T to P) in the CTL epitopes ,40 (D/P to E), 64 (D to E) in the T helper epitopes and 79 (N/R to P), 80 (T to I),109 (A/ I /M to T),113 (P/Q to E), 114 (I/N to T) in the B cell epitopes although those substitutions were different from ours in some positions. In addition, the rate of mutation at the position 21 (S/T, S/A and S/H) in the CTL epitope in IC subjects was significantly more prevalent than the CA and C/HCC groups. In theory, mutations in the core immune epitopes would subvert the immune responses to permit persistent HBV infection. It was also reported that the non-accidental distribution of core mutations detected in the immune and non-immune regions was associated with progress of the disease(15).
The present study also demonstrated that the rates of F24Y, E64D, E77Q, L116I, and E180A mutations were higher in the C/HCC patients than the other groups; however these mutations were not statically significant. Recently, Yu Zhang et al. found that HBc L60V variation was associated with higher viral loads, necroinflammation of the liver and it may be related with a poor prognosis(32). They proposed that this variation could influence both virus replication and T cell responses(32). Mohamadkhani et al. suggested that the frequency of HBc mutations in the CTL epitope regions and C-terminal domain is associated with higher stage of fibrosis(33). Likewise, similar studies indicated that mutations of CTL epitopes at C-terminal of the core might enhance the development of fibrosis (33). In sum, there are several mutations considered as effective variations to enhance the progress of the liver disease. Accumulation of these mutations during viral persistence could be the result of escape recognition of the infected cell by the immune system and progression of liver impairment. Therefore, more efforts should be made to understand these mutations’ effect on the liver disease progression during HBV infection.
Some studies have shown that the locations of effective mutations are definitely mapped more on specific regions such as 80–120 sequence (22, 34–36). In our study, there was not a similar pattern over different parts of the core. Instead, it was found that the rate of substitutions in the IC group was determined to be higher than the other groups.
Our study had some limitations such as small sample size, no available data regarding the viral replication parameters and the moiety of sampling that was cross-sectional. However, these results help us to know the frequency of precore/core mutations in HBV infected patient in Fars province and greatly improves our understanding of precore/core mutations, mostly within the immune epitopes.