Even decades since the start of international coverage of hepatitis B virus (HBV) vaccine, HBV infection has still remained persistent in about 250 million individuals worldwide [1]. The chronic HBV infection remains asymptomatic or actively progresses toward cirrhosis and hepatocellular carcinoma [2]. The host factors including age, gender, race, and genetic background in addition to viral sequence diversity have been proposed to have ian mpact on the disease progression [3].

The DNA genome of HBV encompasses 4 open reading frames (ORF), which encodes 7 different proteins [4]. The virus polymerase, reverse transcriptase (RT) is an error-prone enzyme that leaves significant mutations alongside the viral genome during productive replication [5]. Interestingly, RT gene is overlapped by the small surface protein, Hepatitis B virus surface antigen (HBsAg) that constitutes as the major part of virus genome. As a result, mutations in the small HBsAg region interchangeably affect RT protein and vice versa [6].

HBsAg variants have been involved in some medical issues including failure of passive immunization by immunoglobulin (IG), disability of diagnostic method for detection, and immune evasion [7,8]. Variations that contribute to this issue are mainly located at the dominant epitope of HBsAg inside the “a” determinant (aa 124–147), within the major hydrophilic region (MHR, aa 99–169) [7].

The importance of HBsAg variations in immune evasion and disease progression has already been taken into consideration [7,9–13]. The detailed analysis of immunologic regions of HBsAg showed an association between some point mutations including substitution and deletion with immune escape phenotypes [7,14]. On the other hand, amino acid change in HBsAg structure could ultimately alter the virus secretion and replication [15]. While the point mutations that attributed to immune evasion have been defined clearly, those that are responsible for disease progression need to be investigated further. In fact, some substitutions such as T53C, L94S, S98T, P120T/S, A181T, L176, P203Q, S210R, and L213I are suggested to be involved in disease outcome [7,10,12]. Furthermore, the significance of some HBsAg stop mutations including L95*, Q104*, W172*, W182*, W196*, and L216* that are implicated in disease progression [12,16].

More precisely, some HBsAg mutations have been suggested to contribute to hepatocellular carcinoma (HCC)/cirrhosis development, although controversial issues remain to be elucidated. Regarding this issue, a genotype-dependent pattern of variation has been suggested inside the HBV genome [17,18]. For example, in some studies with the aim to find occult HBV-related mutations, the “a” determinant variations were significantly correlated with virus genotype [7,18]. Therefore, in order to justify this investigation, HBsAg mutations in disease progression should be conducted separately for each genotype.

On the basis of genome sequence, eight (A–H) different HBV genotypes were determined by Norder et al. [19]. HBV genotype D has been detected as the dominant genotype in Iran [20].

There is limited data regarding the comparison of HBsAg variations between HCC/cirrhotic cases and asymptomatic individuals amongst a pure population of patients with genotype D. Therefore, in this study the HBsAg mutation patterns between these groups were investigated and some new critical substitutions and stop codon mutations were suggested to be responsible for disease progression.

HBsAg variants have been suggested to be involved in some issues, such as vaccine escape, failure of IG protection, a shortcoming in the detection of carriers, and even in disease progression (10). While the correlation of some HBsAg variations with disease progression has been suspected (10), it still remains to be clarified further.

As the main aim of this study, the comparison of the variation pattern between groups showed that some substitutions such as of Y134N/F/L and P120T/S were significantly more prevalent in the HCC/Cirrhotic cases than the asymptomatic group. In a similar finding, P120 position was shown to be a critical point for HCC/cirrhosis prediction [4]. However, Y134N/F/L has not been reported as an important position for HCC/cirrhosis prediction. Instead, a correlation between this mutation and HBV reactivation following the immune suppression was reported [4,21,22]. In spite of immune evasion mutations, the controversial issue still remain unclear for the role of HBsAg variations in disease progression. The studies that were in agreement with the role of HBsAg variation in disease progression suggested a genotype-dependent manner [17,18]. It was shown that occult HBV related substitutions strongly follow a genotype-dependent pattern [17,18]. Mutations in T53, L94, S98, P120, A181, L176, P203, S210, and L213 positions have been suggested to be involved in the disease outcome [7,10,12]. However, none of them was detected as a remarkable variation in our experiment.

As an interesting finding, a noticeable number of stop codons were detected among the HCC/cirrhotic group. In total, 12 stop codon mutations including C69*, C107*, G159*, W182*, L94* and L95* were detectable, but only C69* was significantly important. Regarding the length of HBsAg, it is predictable that truncation from gene end such as L216 leaves an ineffective alteration in the structure when compared to longer deletions of protein (e.g. C69 and C107) in the HCC/Cirrhotic group. This is not the first report claiming the importance of HBs truncation in disease progression. These long truncations lead to deletion of carboxyl terminal and creation of new HBsAg versions with different activities. Interestingly, stop codons including L95*, Q104*, W172*, W182*, W196* and L216* have been indicated to be responsible for disease progression toward tumor and cirrhosis [12,16,23]. The stop codon mutations like L95*, W182*, and L216* were shown to activate cell proliferation and induce transformation through transactivation of different oncogenes [16,23]. Similarly, the presence of stop codons in our HCC/cirrhotic group might also emphasize on their molecular roles in enhancing the disease progression toward cirrhosis and HCC by unknown mechanisms. The correlation of C69* stop mutation with cirrhosis has also been described by Veazjalali et al. on a pure genotype D infected population [24]. However, since there is no similar report, a genotype dependency for these kinds of mutations is predictable.

In addition, truncating stop codons are suspected to correlate with failure in HBsAg detection and with virus diagnosis [11,25]. On the contrary, all of our cases showed a detectable amount of HBsAg when assayed by an ELISA method. Hence, it could be concluded that at least in our population with genotype D, the role of variations in detection failure is ignorable. By modeling and in vitro assessment, Sadeghi et al. showed that variations in MHR might have an impact on diagnosis, but not as much to entirely destroy the detection process [15].

In the MHR region, substitutions of Y134N/F/L and P120T/S were detected more in the HCC/Cirrhotic cases compared to the asymptomatic carriers. Several mutations in the MHR region and HBsAg have been described, which leads to the emergence of immune escapes mutant [8,12,27]. The more frequent immune evasion mutations reported so far include P120T, R122K/T, Q129H, M133I, F/Y134N, S143L, D144E/A, G145R, E164D, A168V, W172*, W182*, and I195M [6,26–28]. Amongst these mutations, Y134 and P120 positions showed a higher prevalence of variations in our study. All these mutations cause the virus to escape from vaccine, IG, and commercial detection assays [26]. In our study, the well-known G145R substitution, as a remarkable vaccine escape mutation, was not detected. In line with our study, G145R has been reported in a very low frequency (<0.5%) in other studies [6,9,26,27]. It is suggested that mutations including G119R, C124Y, I126S, Q129R, S136P, C139R, T140I, K141E, D144A, and G145R significantly impaired the virus and S protein secretion in vitro[7,11,15]. Therefore, mutations in the MHR or in other immunological regions might also impact the virus replication, and consequently the disease outcome.

In the case of cellular immune epitopes, our analysis revealed that S193 variation at T-helper epitope as well as some substitutions including Y206F/L/S/H/C, S207R, I208T, P214L/Q in T-cytotoxic epitopes were more prevalent. These findings also showed that substitution of Y206S/F/L/H in the CTL epitopes was detected more frequently in our study population. Some mutations in HBsAg immunologic region, like G159 and L213, were also detected with a lower frequency. In a similar report on genotype D samples, substitution at S207, I208 and L213 was determined as the most prevalent immune escape mutations [29]. The limit in HBsAg detection, cellular immune evasion and consequent viral persistence might be associated with both Th or CTL epitopes variations [31].

The substitution at position Y206 was detected more frequently, but was not significant in asymptomatic patients in comparison with the HCC/cirrhosis group. Its correlation with disease outcome was poorly described; however, the association with lower levels of virus titer has already been suggested. Mirabelli et al. suggested that specific HBsAg-mutations (M197T, S204N, and Y206C/H) were significantly associated with lower (<2000IUml−1) viral titer in the sera [30].

The analysis of RT drug resistance showed a low frequency of resistance-related substitutions. In all cases, just one primary resistance mutation (A194T) was confirmed, while other mutations such as Q215S/H were defined as secondary (potential resistance) [8,31]. It has been reported that the genotype D of HBV is associated with an increased risk of adefovir resistance. The position of Q215 from RT has been detected as more frequent spontaneous mutation arising among genotype D, and its mutants are potentially resistant to adefovir[32]. The low frequency of all RT mutations can be explained by the drug avoidance of the selected groups, which was reported in several studies [27,32].

In agreement with our results, several studies from the southern region of Iran have implied the dominant prevalence of genotype D throughout the country [20,29]. However, our study showed that serotype ayw1 was dominant amongst the investigated population. Another report indicated that serotypes ayw2 (94.4%) followed by ayw1 and ayw3 were more frequent than other types in our region [33].

Generally, this study highlighted the significant prevalence of some mutations inside the HBsAg in terms of their possible effects on disease progression; however, our findings should be evaluated in a larger population to meet the predictive marker criteria. Hence, participants must be age-matched to prevent bias with respect to the effect of disease duration. Moreover, traditional sequencing does not have enough sensitivity to detect mutations that are the minority. Therefore, in order to precisely define medically important mutations, performing clonal sequencing or next-generation ones could be the preferred choice.

In conclusion, the higher frequency of substitutions in MHR and immune epitopes at positions such as Y134 and P120 as well as stop codons such as C69* might have the predictive value to determine the HBV infection prognosis due to higher numbers in the HCC/cirrhotic group.AbbreviationsHBV

hepatitis B virus