Introduction

Currently, Hepatitis B Virus (HBV) infection is a global health problem, and more than 350 million people around the world are chronic carriers of this virus (1). Because of the spontaneous error rate of viral reverse transcriptase, the hepatitis B virus (HBV) genome evolves during the course of infection under the antiviral pressure of the host immunity (2). These HBV variants could display alterations in epitopes important to the host immune recognition, resulting in enhanced virulence with increased replication of HBV, resistance to antiviral therapies, or facilitated cell attachment and penetration (3). HBV genome has 4 open reading frames (ORFs), including envelope genes coding region (pre-s₁, pre-s₂ and s), pre-core (pc) and core (c), polymerase (p), x gene coding region (4). The HBV x gene (HBx) is a 16–18 kDa protein, a transcriptional transactivator and can positively regulate the transcription of a wide variety of viral and cellular promoters (5). The observation which reported that HBx transactivates

several cellular genes regulating cell growth suggests that HBx may induce uncontrolled cell proliferation (6). HBx is a multi-functional protein that plays a role in the development of hepatocellular carcinoma (HCC). The basal-core promoter (BCP) is the minimal essential promoter sequence accurately mapped from nucleotide 1742 to 1849 of HBV complete genome and overlaps with the x gene region (nucleotide 1374-1836). Since the x gene overlaps with the BCP region, mutations in the x region will also have specific effects on BCP region (7). T-A mutations in BCP are frequent in all genotypes while other mutations seem to be more related to specific genotypes (8). Pre-core (PC) variants are more common among patients with genotype D (65-75%) than genotype A (9-18%) (9,10). Isolates with transversion at A1762T together with G1764A mutations in the BCP are often present in hepatitis B carriers with chronic hepatitis, fulminant hepatitis, HCC and less often in inactive carriers and immune suppressed patients (11).

The most frequently observed mutation in the PC region is a G-A change at nucleotide 1896 resulting in a stop codon and hence, leading to premature termination of the PC/Core protein construction, which is the precursor of HBeAg (12). However, this mutation is located within the epsilon (ε) structure, a highly conserved stem-loop essential for initiation of encapsidation within the viral replication cycle. In order to stabilize the ε structure, the nucleotide at position 1896 is paired with the nucleotide at position 1858, which naturally is a thymidine (T) in genotypes B, D, E, and G and a cytosine (C) in genotype A (13). In an experimental study, the 1762 and 1764 mutations reduced the transcription of pre-core mRNA by interfering with the binding of transcription factors, thus, supporting the assumption that these mutations down-regulate HBeAg synthesis (14). Moreover, it was also frequently found in both HBeAg-positive and HBeAg-negative chronic hepatitis (15). Iran is considered as a country with low endemicity of chronic HBV infection (16). Our previous study in Golestan province showed higher prevalence of HBV infection and HBV S gene mutation comparing to other parts of Iran (17, 18). In this study, the PC and BCP mutations in chronic HBV patients in Golestan province of Iran was investigated.

 Materials and Methods

Sample selection

A total of 120 patients with proven chronic HBV infection for more than two years, were chosen according to clinical and paraclinical evidence from Golestan province, Iran from the year 2008 to 2012.

None of the patients were vaccinated for HBV or had antiviral therapy; all patients had negative results for antibodies against hepatitis C, hepatitis D and Human Immunodeficiency Virus. The research project received the confirmation of the Ethics Committee and all the patients signed the testimonial letter. Blood samples were collected in the EDTA 5% anti-coagulant and plasma was separated for further examination.

 HBV infection marker detection

The HBV serological markers (HBeAg) were tested using commercially available enzyme-linked immune sorbent assay kits (DIALAB GmbH, Germany).

 DNA extraction

HBV-DNA was extracted from 200 μl of each plasma sample using QIAamp DNA Mini Kit (QIAGEN, Hamburg, Germany) following the manufacturer instruction and extracted DNA was stored at -20^°C for PCR process.

Primer selection

Suitable primers were selected to achieve optimum PCR sensitivity and the BCP and PC region amplification. Following primers listed in Table 1 yielded 390 bp amplification according to nucleotide position 1689 to 2078.

PCR and DNA amplification

Amplification mixture contained: 100 ng of extracted DNA 1X PCR buffer, 1.5 mM MgCl₂, 0.2 mM dNTP, 2.5 U Taq DNA polymerase (QIAGEN, Hamburg, Germany) and 0.4 pmol/μl of each primer in a total volume of 50 μl with distilled water. The PCR profile started with an initial 5 min denaturation at 95^°C, followed by 30 cycles of amplification including denaturation for 1 min at 95^°C, primer annealing for 1 min at 55^°C and extension for 2 min at 72^°C, with a final extension at 72^°C for 5 min. The 390 bp amplification products were analyzed by gel electrophoresison 1% agarose gel stained with ethidium bromide to determine HBV-DNA positive and negative samples.

 DNA sequencing and mutation analysis

The positive PCR products were subjected to automated sequencing (Macrogen Inc., Korea).          Then, nucleotide sequences were aligned with standard hepatitis B sequence, [Accession number: AB033559] from Gene Bank database, for mutation detection and analysis (20). All data that were collected from the study were analyzed using SPSS.16 software.

One of the samples with critical mutations in BCP and PC region was registered in NCBI under the accession number: KC928094

 Results

The mean age of patients was 36.8 year, 74% of them were male and 26% were female. All of the patients were HBsAg positive and 100 out of 120 (83.3%) patients were HBeAg negative. Isolates belonged to genotype D subgenotype D₁, subtype ayw₂ according to our previous study (18). Comparison of our nucleotide sequences with reference sequence showed high rates of mutations with a frequency of 97.5% and 99.2% in BCP and PC regions, respectively. In BCP region A1762T and A1762G mutations were detected in 25 (20.83%) and 5 (4.16%) of patients, respectively. The most important mutations detected in BCP were the 8 bp nucleotide deletion from 1763 to 1770 which            were observed in 7 (5.83%) patients. In the case of

mutations in PC region, G1896A was detected as the most prevalent mutation in 44 (36.66%) patients leading creation of stop codon at the 28^th amino acid of PC region in 39 (32.50%) of patients which 41 (93.18%) of them were HBeAg negative. Frame shift mutation was found in 78 patients (65%) in BCP region, but this rate of mutation in PC region was seen only in 8 patients (6.6%).

 HBeAg positivity and BCP and PC mutations

Our results showed that among 31 cases with A1762T and 44 cases with G1764A point mutations in BCP region of HBV, 24 (77.4%) and 34 (77.27%) were found in HBeAg negative patients, respectively. In the case of mutations in PC region, G1896A was detected as the most prevalent mutation in 44 patients from which 41 (93.18%) were HBeAg negative (Table 2).

 Point mutations in basal-core promoter (BCP) region

Our findings demonstrated the occurrence of 59 point mutations including 21 (35.6%) cases as missense mutation (with amino acid altering) and 38 (64.4%) cases as silent mutation (without amino acid altering) in BCP region. These mutations occurred in area of BCP which have overlap with X region of HBV (HBx). Distribution of single, double, triple, quadruple, and more than quadruple point mutations in the BCP region were 5 (4.2%), 4 (3.3%), 9 (7.5%), 6 (5%) and 8 (6.7), respectively. Mutations at T1753G and T1753C of BCP region were seen in 10 (8.33%) and 12 (10%) cases, respectively, which caused I127C amino acid mutation in 27 (22.5%) of patients. A1762T and A1762G mutations were detected in 25 (20.83%) and 5 (4.16%) of patients, respectively, which affected the amino acid L130M in 10 (8.33%) patients. G1764A, G1764T and G1764C substitutions were seen in 24 (20%), 16 (13.33%) and 4 (3.33%) cases, respectively, which caused V131I change in 9 (7.5%) patients (Table 3).

The most important mutations detected in BCP were the 8 bp nucleotide deletion from 1763 to 1770 which was observed in 7 (5.83%) patients. Figure 1 shows the distribution and frequency of amino acid substitutions in BCP and x region of HBV in chronic patients.

 Point mutations in precore (PC) region

Among 78 point mutations in PC region, 26 (33.3%) occurred as silent mutation and 52 (66.7%) as missense mutation. In the case of point mutation distribution, 49 (40.8%), 21 (17.5%), 20 (16.66%) and 21 (17.4) cases showed double, triple, quadruple and more than quadruple mutations in the sequenced region of PC region, respectively. Substitution at G1896A was seen in 44 (36.66%) leading to the creation of stop codon in the 28^th amino acid of PC region in 39 (32.50%) of patients. The most prevalent mutations in PC region                       at the level of amino acid substitution, were A64S  and

 Table 3. The rate of basal-core promoter (BCP) and pre-core (PC) mutations in chronic patients and their HBeAg result

Figure 1. Frequency and distribution of amino acid substitution in X gene

 Discussion

HBV core promoter overlaps partially with HBx coding sequence, so, the nucleotide mutations in core promoter induce HBV X protein substitution (21). Since HBV genotypes are not uniformly distributed around the world, pre-core mutants also have a particular distribution, being more frequent in some regions such as Asia and the Mediterranean basin, where genotypes B, C, or D are predominant, and rarely in North America and Europe, where genotype A is commonly found (22). This study was designed to determine the BCP and CP mutations in patients infected with HBV genotype D and its distribution in HBeAg negative cases using sequencing and alignment with standard sequence.

As it was shown in the results, genotype D was dominant type of HBV in our study and overall prevalence of BCP and PC mutation was 99.2% and 97.5%, respectively. It has been shown that the prevalence of PC and BCP mutations depends on HBV

genotypes, and these mutations are related to fulminant and severe hepatitis and hepatocellular carcinoma (23-26).

The high rate of mutations in PC and BCP region of HBV genotype D patients in compare with other genotypes has been reported from different studies in Brazil (22, 27, 28) and Netherland (29). In contrast with our data a report from UAE revealed low rate of mutation in precore (58.0%) and Basal Core Promoter (25.3%) regions (30).

The G1896A mutation in PC has been found to be the most common mutation in chronic HBeAg-negative patients (31). This mutation creates a stop codon that prevents translation of the pre-core protein and terminates the production of HBeAg. However, these patients continue to synthesize HBV DNA at sufficient levels to continuously damage liver and progress toward cirrhosis (32). Median prevalence of the 1896 PC mutation in HBeAg-negative adults was reported to be 50% in Asia, 92% in the Mediterranean, and 24% in America (33). The frequency of the 1896 PC mutation varies geographically and primarily depends on             the genotype of HBV (34, 35). The present study showed substitution at G1896A in 44 (36.66%) patients from which, 41 (93.18%) were HBeAg negative. In

Figure 2. Frequency and distribution of amino acid substitution in PC region