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Hepatitis B virus preS1 deletion is related to viral replication increase and disease progression.

AIM
To investigate the clinical implications of hepatitis B virus (HBV) preS1 deletion.


METHODS
We developed a fluorescence resonance energy transfer-based real-time polymerase chain reaction (RT-PCR) that can detect four genotypes (wild type, 15-bp, 18-bp and 21-bp deletion). The PCR method was used in two cohorts of Korean chronic HBV subjects with genotype C infections. Cohort I included 292 chronic HBV subjects randomly selected from Cheju National University Hospital (Jeju, South Korea) or Seoul National University Hospital (Seoul, South Korea), and cohort II included 90 consecutive chronic HBV carriers recruited from Konkuk University Hospital (Seoul, South Korea); the cohort II patients did not have hepatocellular carcinoma or liver cirrhosis.


RESULTS
The method proposed in this study identified 341 of 382 samples (89.3%). Deletion variants were identified in 100 (29.3%) of the 341 detected samples. In both cohorts, the subjects with deletions had a significantly higher Hepatitis B virus e antigen (HBeAg)-positive seroprevalence [cohort I, wild (51.0%) vs deletion (75.0%), P < 0.001; cohort II, wild (69.2%) vs deletion (92.9%), P = 0.002] and higher HBV DNA levels [cohort I, wild (797.7 pg/mL) vs deletion (1678.9 pg/mL), P = 0.013; cohort II, wild (8.3 × 10(8) copies/mL) vs deletion (2.2 × 10(9) copies/mL), P = 0.049], compared to subjects with wild type HBV.


CONCLUSION
HBV genotype C preS1 deletion may affect disease progression in chronic HBV subjects through an extended duration of HBeAg seropositive status and increased HBV replications.


INTRODUCTION
Hepatitis B virus (HBV) infection is a global health problem, and more than 350 million people are chronic carriers of the virus [1] . The infection is associated with a wide spectrum of clinical manifestations ranging from acute or fulminant hepatitis to various forms of chronic infection, including asymptomatic carrier, chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC) [2] . Despite the recent significant decline in HBV chronic subjects thanks to a successful HBsAg vaccination program, South Korea is still recognized as an endemic area of HBV infection [3] . It has also been reported that genotype C2 is exclusively prevalent in South Korea [4] , which is known to be more prone to mutations and is related to more severe liver diseases, as well as having a lower antiviral response compared with genotype B [5] . Furthermore, the high prevalence of basal core promoter (BCP) double mutations and the presence of distinct immune responses against HBV proteins in the Korean population could lead to the generation of distinct HBV variants that are not (or rarely) encountered in other areas [6][7][8][9][10][11][12][13][14][15] and that result in distinct clinical manifestations in Korean chronic HBV subjects [16] . The preS proteins might have a pivotal function in virus assembly and attachment to hepatocytes [17,18] . Infections with preS/S HBV variants, particularly deletion variants, correlated with the most progressive forms of liver disease and HCC. Both preS1 and preS2 mutations reportedly contribute to hepatocarcinogenesis by inducing an endoplasmic reticulum (ER) stress pathway that is caused by the accumulation of large hepatitis B surface (LHBs) proteins in the ER or by altering the transactivating capacity [19][20][21] .
Recently, we introduced two types of preS1 mutations that are related to the progression of liver diseases: W4P/R [11] and preS1 start codon deletion [14] . The former proved to be exclusively detected in males and also proved to be related to liver disease progression in chronic HBV subjects with genotype C infections, according to our fluorescence resonance energy transfer (FRET)-based real-time polymerase chain reaction (RT-PCR) method [11] . The latter mutation proved to be prevalent in chronic HBV subjects with genotype C, according to our previous molecular epidemiologic study based on a direct sequencing protocol [14] . However, the clinical implications of HBV preS1 mutations have rarely been investigated. Furthermore, the direct sequencing protocol has a limitation that could underestimate the presence of quasispecies variants in a subject, which could interfere with the genuine interpretation of specific mutations from clinical and virological perspectives. To overcome this drawback, FRET-based RT-PCR could be used for molecular epidemiologic purposes. The FRET-based RT-PCR allows not only the simultaneous identification of coexisting quasispecies but also the direct identification of target mutations from primary specimens, such as serum through melting curve analyses of the amplification product [22,23] . Therefore, the present study aims to achieve the following goals: (1) to develop a FRET-based RT-PCR method to detect the HBV preS1 start codon deletion; and (2) to determine the clinical implications of this mutation using the developed methods on DNA from the sera of genotype C-infected chronic HBV subjects.

Subjects
In an effort to further support the clinical implications of preS1 deletion, our developed RT-PCR method was applied to two patient cohorts. Cohort Ⅰ included 292 subjects randomly selected from the chronic HBV subjects who visited Cheju National University Hospital (Jeju, Korea) in 2003 or Seoul National University Hospital (Seoul, Korea) in 2005. The cohort Ⅰ study protocol was approved by the Institutional Review Board of Seoul National University Hospital (C-1007-021-322). In cohort Ⅱ, 90 consecutive chronic HBsAg carriers without liver cirrhosis or HCC were recruited from January 2012 to March 2012 at Konkuk University Hospital (Seoul, Korea). The cohort Ⅱ study protocol was approved by the Institutional Review Board of Konkuk University Hospital (KUH-1010544). The clinical details of cohorts Ⅰ and Ⅱ are presented in Table 1.
The HBV DNA sera levels for cohort Ⅰ and cohort Ⅱ were determined using different methods. In cohort Ⅰ, the serum HBV DNA levels were determined using the Digene Hybrid Capture Ⅱ assay (Digene Diagnostic Inc., Gaithersburg, MD, United States), which has a lower limit of detection of 0.5 pg/mL. In cohort Ⅱ, the serum HBV DNA levels were assessed using the COBAS Amplicor PCR assay, which has a lower limit of detection of 300 copies/mL (Roche Molecular Systems, Branchburg, NJ, United States). Figure 1 and Table 2 present the primers designed to amplify the HBV preS1 gene fragment and the probes designed to identify the deletion sequence variants at the start codon, respectively. A total of 99 cloned HBV genes were aligned using SeqMan TM Ⅱ software (DNASTAR). A primer pair for amplification of the HBV preS1 gene fragment was designed using Oligo V 6.5 (Molecular Biology Insights), which produced a 203-224 bp amplicon. We used four pairs of hybridization probes (an anchor probe and a sensor probe; HybProbe) to detect wild type at channel 640 and three types of deletion polymorphisms (15-bp deletion, 18-bp deletion, and 21-bp deletion) at three separate channels (670, 610 and 640, respectively) using the LightCycler 2.0 RT-PCR system. The same preS1 primer set and wild type probe previously developed for the W4P/R mutation detection [11] were

Statistical analysis
All detection items in this study were repeated at least three times, and the results were expressed as percentages, the mean ± SD, or medians (range). Differences between categorical variables were analyzed using Fisher's exact test or the χ 2 test. For continuous variables, Student's t-test was used when the data showed a normal distribution, while the Mann-Whitney U test was used when the data were not normally distributed. SPSS version 21.0 software (Professional Statistic, Chicago, IL, United States) was used for all statistical analyses and a P value of < 0.05 (two-tailed) was considered statistically significant.

Ethics statement
This retrospective study was reviewed and approved by the Institutional Review Boards (IRBs) at Seoul National University Hospital (IRB Grant No. C-1007-021-322) for cohort Ⅰ and at Konkuk University Hospital (IRB Grant No. KUH-1010544) for cohort Ⅱ. The patients' medical records were anonymized and de-identified prior to the analyses. Experiments were mainly based on viral DNA extracted from isolates; therefore, the research study was conducted without informed consent. Informed consent waivers were granted by the IRBs.

Determination of the melting temperature for FRETbased RT-PCR to detect the deletion variants
Application of the developed FRET-based RT-PCR to the wild type and three deletion types of the control plasmid DNAs cloned from the subjects demonstrated that separation between the wild type and variants used. The remaining probes, which were used to detect three types of deletion variants, were designed in this study.

RT-PCR
A LightCycler 2.0 system was used, and its four detection channels were calibrated for color compensation and activated for the experiment. The LC Faststart DNA Master HP kit (Roche Diagnostic, Basel, Switzerland) was used to prepare the master mix, according to the kit protocol. A 10 µL reaction mixture was prepared for each sample and each target variant, as follows: 1 µL Taq buffer (containing dNTP mix and 10 mmol/L MgCl2), an additional 2 mmol/L MgCl2, 0.75 µmol/L forward primer (HBV_sF), 0.35 µmol/L reverse primer (HBV_sR), and a pair of 0.18 µmol/L HybProbes for the target variant (Table 2). Two separate reactions were performed for each sample: one to detect the wild type virus at channel 640, and the other to detect the deletion variants at channels 610, 640, and 670. For the relative quantification of the deletion variants vs the wild type in a subject, the melting temperature height values of the deletion variants vs wild type of 72 subjects with deletions in cohort Ⅰ were calculated and compared with subjects with severe types of disease (HCC and liver cirrhosis) and mild types of disease (carriers and chronic hepatitis). The values of each deletion probe were normalized to those of the wild type probe.

Direct sequencing
To verify the fidelity of the developed FRET based RT-PCR, 23 samples from cohort Ⅱ, 10 samples identified as mixed infections of both wild type and deletion variants and 13 samples identified as wild type-only infections, according to the RT-PCR assay, were subject to direct sequencing analyses. PCR amplification was performed using the same primer pair used for RT- was possible, in addition to separation between the three different types of variants (15-bp deletion, 18-bp deletion, and 21-bp deletion). The deletion variants exhibited distinct melting temperatures according to the respective probe. As demonstrated in the previous report [11] , the wild type formed two distinct melting with a cross and clearly distinguishable melting peaks from the other variants ( Figure 2).

Application of FRET-based RT-PCR to serum DNA from chronic Korean subjects in the two patient cohorts
In this study, to correct the bias that results from the time and location of sample collection, we applied the developed FRET-based RT-PCR to samples from two different cohorts: cohort Ⅰ (292 samples) and cohort Ⅱ (90 samples

Comparison of clinical data between the subjects with and without deletions
In the 264 subjects detected from cohort Ⅰ, no significant differences were found in age, gender, and alanine transaminase (ALT) level between the subjects with (192 subjects) and without (72 subjects) deletions. However, significant differences in Hepatitis B virus e antigen (HBeAg) serostatus and HBV DNA levels were found. The HBeAg-positive ratio was significantly higher in the subjects with wild type HBV than in those with deletions (51.0%, 98/192 subjects vs 75.0%, 54/72 subjects, P < 0.001). The median HBV DNA level was also significantly higher in the subjects with wild type HBV than in those with deletions (797.7 pg/mL vs 1678.9 pg/mL, P = 0.013) ( Table 5). Similar to cohort Ⅰ, a significant difference was also found among the 77 cohort Ⅱ subjects, in terms of two clinical indicators of HBeAg serostatus (69.2%, 29/49 subjects vs 92.9%, 26/28 subjects, P = 0.002) and HBV DNA levels (8.3 × 10 8 copies/mL vs 2.2 × 10 9 copies/ mL, P = 0.049) between the subjects with (49 subjects) and without (28 subjects) deletions (Table 5). This trend was also found in the combined analyses of the clinical data of the subjects (n = 341) detected in cohorts Ⅰ and Ⅱ. A significant difference in the HBeAg serostatus (52.7%, 127/241 subjects vs 80.0%, 80/100 subjects, P < 0.001) and HBV DNA levels (4.6 × 10 8 copies/mL vs 1.4 × 10 9 copies/mL, P = 0.014) was observed between the subjects with (241 subjects) and without (100 subjects) deletions (Table 6).

Quantification of variants vs wild type via melting temperature height value in 72 subjects with deletions in cohort I, according to the different clinical stages
Significant differences in the prevalence of preS1del were not found between the subjects with different clinical stages in cohort Ⅰ [CH (27.1%, 26/96 subjects), liver cirrhosis (20.3%, 12/59 subjects) and HCC (31.2%, 34/109 subjects)]. It is possible to estimate the relative amounts of deletion variants vs wild type in a subject by calculating the melting temperature height values [24] . We compared the mean melting temperature height values of the variants vs the wild type of the 72 subjects with deletions in cohort Ⅰ, according to the different clinical stages. Among the subjects with the three types of deletion variants, those with severe types of diseases (i.e., liver cirrhosis and HCC) exhibited significantly higher levels of melting temperature height values in deletion variants vs wild type compared with the subjects with mild types of chronic hepatitis. The latter finding suggests that increases in deletion variants in a subject may contribute to liver disease progression (Figure 3).

Detecting deletion variants: FRET-based RT-PCR compared with the direct sequencing protocol
To validate the effectiveness of FRET-based RT-PCR in detecting deletion variants, 23 samples from cohort Ⅱ (i.e., 10 samples identified as mixed infection of deletion and wild type and 13 samples of wild typeonly infection identified by FRET-based RT-PCR) were subjected to direct sequencing analyses. In direct sequencing data, samples that exhibited the presence of mixed peaks in the electropherogram were regarded as mixed infections with wild type and deletion variants. No difference was found between the two protocols in the detection of wild type-only infections. The 13 samples identified as wild type-only infection using RT-PCR were also identified as wild type using the direct sequencing protocol. However, a discrepancy between the two protocols was found in detecting the deletion variants. While only 7 (70%) of 10 samples were identified as mixed infection using RT-PCR, mixed infections with both wild type and deletion variants were proven using direct sequencing protocols, and mixed peaks were not found in the other three samples (Table 7). This discrepancy might have resulted from the sensitivity difference between the two protocols used to detect the deletion variants in the mixed samples. To address this issue, we investigated the limits of our RT-PCR method (15-bp-, 18-bp and 21-bp deletion) for detection of deletion variants mixed with various concentrations of wild type DNA. Our analysis showed that the RT-PCR could detect deletion variants even in cases of a 100-fold higher level of wild type DNA compared with deletion DNA, irrespective of the deletion types ( Figure 4).

DISCUSSION
There are clinical and virological differences between the HBV genotypes. Genotype C represents most HBV infections in Asian areas, including China, South Korea, and Japan, where HBV infections are the most prevalent worldwide. Recent studies have demonstrated that patients with genotype C are more likely to develop HCC than patients with other HBV genotypes [25][26][27] . Furthermore, higher levels of sustained HBeAg seropositivity and serum HBV-DNA, which are known to be HCC risk factors, were found in the genotype C subjects compared with the genotype B subjects [25,28] . In a previous study, we found that the preS1 start codon deletion is related to HCC in Korean HBV subjects [14] . Interestingly, comparisons of clinical information between subjects with the preS1 deletion and preS2 deletion indicated that the preS2 deletion (as with most HBV mutations) but not the preS1 deletion is positively related to HBeAg seronegative status [14] . This finding indicates that the preS1 deletion might be generated in the HBeAg-positive phase rather than in the HBeAg-negative phase. It also indicates that the preS1 deletion might be produced by factors other than the host immune response.
Despite the presence of overt, distinct traits in genotype C infection, the underlying mechanisms capable of explaining these distinct traits remain unknown to date. Therefore, in the present study, we used the FRET-based RT-PCR method to investigate the clinical implications of preS1 deletion in Korean chronic HBV subjects with genotype C infections. Thus, we demonstrated the significant roles of preS1 start codon deletions in liver disease progression among Korean  chronic HBV subjects infected with genotype C via enhanced duration of HBeAg-positivity and HBV DNA replication. Our data might provide, at least in part, an explanation for the distinct traits of genotype C infections (higher levels of sustained HBeAg-positivity and HBV DNA replication) compared with other genotypes, particularly genotype B infections. Most HBV mutations found to date, including BCP double mutations and preC mutations, have been positively related to HBV persistent infections and HBeAg seronegative status [29,30] . However, this relationship is not true for the preS1 start codon deletions. To the best of our knowledge, the preS1 deletion is the first HBV mutation related to enhanced HBV replication and HBeAg-positive serostatus.
We applied the proposed FRET-based RT-PCR method to two different cohorts. The results from both cohorts were similar, which further strengthens our data. Our FRET-based RT-PCR method demonstrated that there were no significant differences in the prevalence of the preS1 deletion between subjects with different clinical stages (Tables 5 and 7), which does not align with the previous result indicating that the preS1 deletion is significantly more prevalent in HCC subjects than in subjects with mild types of liver disease, CH, and carriers [14] . The difference between the two studies could be related to differences in the applied protocols: FRET-based RT-PCR and nesteddirect sequencing. Unlike the latter protocol, the former can detect deletion variants even if they exist in a small percentage compared to the wild type. Because three types of probes (for detecting the variants) existed in their respective independent channels, even the majority type cannot interfere with the detection of other probes. Using control plasmid DNAs, we discovered that our FRET-based RT-PCR could accurately detect the respective deletion type even in cases of mixed DNA showing a 100-fold higher level of wild type DNA than deletion type DNA ( Figure  4). These differences can also explain the disparity in sensitivity found between our FRET-based RT-PCR and the direct sequencing protocol for the detection of deletion variants, demonstrating the superiority of the former protocol over the latter as a method for early prediction of liver disease progression. Although there were no differences in the prevalence of the preS1 deletion between the different clinical stages, a comparison of the melting temperature heights across the different clinical stages demonstrated increases in the deletion variants vs the wild type in subjects with

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severe liver disease (Figure 3), which indicates the positive effect of the variants on disease progression. Interestingly, the most deletion variants (90%, 90/100 variants) coexisted with the wild type in the subjects with chronic hepatitis (Table 5). This finding indicates that the preS1 deletion event might occur sequentially after a wild type infection in HBeAgpositive stages instead of a de novo horizontal infection of deletion variants. Our phylogenetic study based on the quasispecies sequences demonstrated the same coherent clustering of wild type and deletion variants in a subject (data not shown), thus supporting this notion.
In particular, irrespective of the deletion types, all preS1 start codon deletions led to 11-N terminal loss from the preS1 of the otherwise 119 amino acids, as shown in genotype D, with 108 amino acids of preS1. Although the function of N-terminal region remains unknown, its absence in the genotype D strain indicates that it is dispensable in the HBV life cycle. However, together with a previous report demonstrating that deletions leading to 11 amino acids in the preS1 5' region were observed more frequently in HCC subjects [14] , our data strongly support that the N terminal loss of 11 amino acids plays a primary role in the progression of liver disease through enhanced HBeAg production and DNA replication. Alternatively, the N terminal deletion region could lead to simultaneous deletion of the overlapped polymerase region. Therefore, the molecular mechanism for enhanced HBeAg production and DNA replication induced by deletion variants should be elucidated in a future study that focuses on the modified function of the deleted LHBs or deleted polymerase.
In conclusion, the HBV genotype C preS1 deletion has the potential to lead to disease progression in chronic HBV subjects through the extended duration of HBeAg seropositive status and increases in HBV replications. This study provides novel insight into the higher infectivity and virulence of genotype C compared with other genotypes. In addition, the FRETbased RT-PCR for detection of the preS1 deletion shows promise for the earlier prediction of the risk of liver disease progression in chronic HBV subjects.

Background
The preS1 start codon deletion proved to be prevalent in chronic hepatitis B virus (HBV) subjects with genotype C using our previous molecular epidemiologic study based on a direct sequencing protocol. However, its clinical implications have rarely been investigated. In this study, the authors developed a molecular diagnostic method to detect this mutation and gained insight into its clinical implications.

Research frontiers
This study developed a novel fluorescence resonance energy transfer (FRET)based real-time polymerase chain reaction (RT-PCR) method to detect the preS1 start codon deletion and applied it to 2 Korean chronic HBV patient cohorts.

Innovations and breakthroughs
For the first time, the authors proved that the HBV genotype C preS1 deletion might significantly contribute to disease progression in chronic HBV subjects by extending the duration of Hepatitis B virus e antigen (HBeAg) seropositive status and increasing HBV replications. This study provides novel insight into the understanding of the higher infectivity and virulence of genotype C compared with other genotypes.

Applications
The FRET-based RT-PCR method for detection of the preS1 deletion developed in this study might hold potential for the early prediction of the risk of liver disease progression in chronic HBV subjects.

Terminology
The FRET-based RT-PCR could be used for molecular epidemiologic purposes. It can permit not only the simultaneous identification of coexisting quasispecies but also the direct identification of target mutations from primary specimens, such as serum through melting curve analyses of the amplification product.

Peer-review
For the first time, the authors discovered the clinical implications of the preS1 start codon deletion via a molecular epidemiologic approach using FRET-based RT-PCR, which might significantly contribute to disease progression in chronic HBV subjects infected with genotype C by extending the duration of HBeAg seropositive status and increasing HBV replications. The results are interesting and may explain the higher infectivity and virulence observed in genotype C compared with other genotypes.