HBx M130K and V131I (T-A) mutations in HBV genotype F during a follow-up study in chronic carriers

Background Around 400 million people worldwide are chronically infected with Hepatitis B virus (HBV). An estimated 10% of these chronic patients develop progressive liver damage including cirrhosis and Hepatocellular Carcinoma (HCC). The HBx gene encodes a protein of 154 amino acids which is a transactivator and has been associated with HBV pathogenesis. A change in the amino acid sequences at positions 130 and 131 in the HBV-X protein (M130K and V131I) produced by T-A point mutations at the nucleic acids level has been associated with severe liver damage and HCC in patients from China and Africa. Further, such changes have been proposed as a prognostic marker for progressive liver damage and HCC. The purpose of this study was to determine if T-A mutations are present in HBV chronic carriers with genotype F (the major genotype in Costa Rica) and further, if these mutations are associated with HBV disease progression in Costa Rica HBV patients from 1972 to 1985. Results Serum samples from 50 HBV positive individuals were amplified and directly sequenced, 48 belonged to genotype F, 1 from genotype D and another was classified as D or E. T-;A mutations were absent in 17 acute patients who recovered, but was present in 12 of 29 chronic carrier samples (42.8%), in one sample the T-A mutations were detected as early as 29 days after clinical onset of disease. In 17 carriers with available liver biopsies, T-;A mutations were found in 8 sera of 13 (61.5%) classified as moderate or severe, and none in 4 biopsies with mild liver damage. However, it was not possible to demonstrate a statistical association between the presence of T-A mutations and moderate/severe liver damage, using a Fischer exact test, 1 tail, p = 0.05. In 4 patients HCC was diagnosed, and 2 of them presented the T-A mutations in their sera. Conclusion T-A mutations were found in HBV genotype F in chronic carriers but not in patients who recovered from acute infection. These mutations could be developing early during infection although the possibility of infection with the mutant virus could not be excluded. More studies are necessary to establish if the T-A mutation can be used as a prognostic marker for severity of liver disease in patients infected with HBV.


Background
The hepatitis B virus (HBV) is a small double stranded DNA virus that produces a chronic infection in 2-10% of adults and in approximately 90% of infected infants. Approximately 10% of these chronic patients develop progressive liver damage including cirrhosis and Hepatocellular Carcinoma (HCC) [1]. The mechanism by which HBV progression to liver cirrhosis and/or HCC occurs is not clear, however many studies suggest that the X protein (HBx) is related to this process. HBx has been associated with a variety of biological functions. As a transcriptional transactivator, it can regulate transcription of a wide diversity of viral and cellular promoters [2,3]. HBx overlaps with regions of crucial importance for viral replication such as: the direct repeat sequences DR1 and DR2, the preC/C gene promoter and the enhancer II region. There are controversial results about the consequence of mutations in this region and its relationship with pathogenesis. A study carried out in Korea determined that mutations in the core promoter have little effect on viral load and the HBeAg status [4]. In contrast, another study points out that changes in HBx especially in the core promoter region may alter HBV gene expression [5]. Among other alterations observed in the HBx gene are deletions and one of the most common is the 8 bp deletion between nucleotides 1763-1770 [6], which has been described to decrease the virus replication [7,8]. These deletions in HBx as in other HBV genes have also been related to development of cirrhosis in long term renal transplant patients [9].
Natural mutations in the HBx gene have been related to progression to chronic disease as a consequence of the rescission of anti proliferative and apoptotic effects, which might produce uncontrolled growth and contribute to multistep hepatocarcinogenesis [10].
A double point mutation with a transversion nucleotide from adenine to thymine at nucleotide 1762, K130M with a transition from adenine to guanine at position 1764 V131I (T-A mutations), has been found more frequently in patients with hepatic tumors than in asymptomatic chronic patients from China [11,12] and Africa [13]. In East Asia where genotype C is the most common genotype, it has been reported that the T-A mutation occurs more frequently in relation to this genotype [14]. HBV is classified worldwide into eight genotypes designated A to H, with a specific geographical distribution [15][16][17]. Genotype F has been described as the HBV genotype of the Amerindians. In Central America a study determined 79% of samples belong to genotype F [18] and in Costa Rica genotype F is the most common, while the overall prevalence of HBV is considered low (0.5 -1%).
From 1972 to 1985 a study on the natural history of HBV was done in San Ramón and Palmares, two adjacent Costa Rican counties [19]. In this study 488 cases of HBV were diagnosed, 80% with an age range between 5 and 40 years. In the group ≤ 5 years old 33% became chronic carriers and in the group > 5 years only 4.7% did. The 77.7% cases were primary HBV infections and the rest were due to household contacts. The purpose of this study was to analyze the presence of T-A mutations in the HBx gene for this population; the time which at they occur and if they are related to hepatic injure. Furthermore, the presence of other mutations in this gene were also observed

PCR detection rate
Of the 77 selected samples, 18 were from group A, 14 from group B and 45 from group C; overall, 50 samples (64.9%) could be amplified and sequenced. Of these fifty, 17 (94.4%) were from group A (recovered patients), 12 (85.7%) from group B (paired samples -known onset), and 21(46.6%) from group C (chronic patient with unknown onset). The sensitivity of the nested PCR was 8000 copies/ml. Table 1 shows the mutation rate of T-A in HBx for M130K and V131I amongst the three study groups. The T-A mutations were not present in any of the 17 sequences from * One of these two sample had V131I mutation in the first sample and TGA mutations later after a five year interval. One sample presented a deletion in that position ** One sample presented a deletion in the T-A position group A, where the average days in which samples were taken was 17 days ranging from 3-33 days. Of 8 chronic patients in group B, the T-A mutations were identified in 5 (62.5%) of the sequenced samples and V131I alone was detected in two. In one of the patients, T-A mutations were detected at day 29 after clinical onset. Four patients were not considered in the distribution of T-A mutations, since the follow-up samples could not be amplified. From group C the T-A mutations were detected in 7 of the 21 sequenced samples, and V131I alone in 3 samples.

Biopsy results and T-A mutations distribution
Of the 29 chronic carrier samples from groups B and C sequenced during the chronic phase, 18 patients had a liver biopsy characterized using the Knodell Index (KI). Five (26%) patients had a KI ≤ 2 points, (mild liver lesions with fatty deposits), 9 (47%) had KI between 3 and 4 points (moderate lesions) and 4 (21%) had a KI > 4 points (severe lesions). These are shown in fig. 1a, 1b and 1c respectively.
Of the 5 carriers with biopsy classified as KI ≤ 2, one sample had an 8 bp deletion that included the T-A mutations site and another sample the V131I mutation alone. In the group with KI > 2 points (moderate/severe) T-A mutations were present in 8 (61.5%) of the sequenced samples ( Table 2). According with statistics of the Costa Rican National Tumor Registry (NTR), four patients included in this study died from HCC during the last 2 decades and two of these had the presence of T-A mutations. To confirm that these deletions were not a PCR artifact, the samples were further digested by SspI. Of the four samples presenting the deletion only 2 were corrob-orated by SspI, both samples (467 and 6516) were reamplified from the PCR1 product.

Mutations observed in HBV acute infected patients that recovered versus chronic carriers
The percentage of the most frequent polymorphism found in the study as well as the consensus sequences of each of the population selected for statistical analysis are shown in Table 4. Group A presented more amino acid or nucleotide variability than the other groups, however, in acute phase samples from group B, 50% of these had common mutations at position 12 (T12A).

Samples Genotype
Of the total 50 samples sequenced ; 48 belonged to genotype F, one sample belonged to genotype D subtype adw, and the other to subtype ay, which was classified as genotype E by a web-based genotyping tool and as D by phylogenetic tree analysis (data not shown).

Discusion
T-A mutations were not found in any of 17 samples from HBV patients who had recovered; a similar result had been obtained in a study with self-limited acute hepatitis [20]. However, another study showed T-A mutations during the acute phase in one out of 11 from genotype A, none of the 5 patients from genotype B and 4 out of 27 from genotype C [21]. The significance of this finding needs to be further studied.
T-A mutations were found in 12 (41.3%) of 29 samples from chronic carriers. In one carrier the mutations were detected 29 days after onset, with the probability that this carrier could have been directly infected with HBV containing the T-A mutations. In the 23 acute phase samples, T-A mutations were not detected and therefore the possibility to have an initial infection with T-A in other populations appears to be low. However, Kobayashi et al, has shown in their study a higher prevalence of the T-A mutations in chronic patients during the acute phase than in acute self limited HBV infection in patients infected with genotypes C, A and B [21].
In chronic carriers, with a liver biopsy classified as moderate or severe, T-A mutations were present in 61.5% (8/13) and none in 4 biopsies classified as mild. However this result was not statistically significant based on the Fisher exact test, 1 tail, p = 0.05, probably due to the small sample size in the groups. Other studies have shown a better correlation between the presence of T-A mutations and patients with fulminant hepatitis, severe exacerbation [20] or liver cirrhosis [22] especially with genotypes A or C when compared with asymptomatic carriers [12][13][14]. In agreement with the literature T-A mutations seem to Sample deletions treated with Ssp I restriction enzymes appear more frequently in genotypes C [23,24] and A [13] than D [25,26] or B [27].
In this study, V131I also occurred alone in 5 samples (17%) of the 29 chronic patients; this event has been commonly reported by others [6,14,25,27,28]; nevertheless M130K alone is very unusual. It has been described in 1 of 12 fulminant hepatitis patients [20] and in 1 genotype B strain [27]. In one of the paired samples from this study and in another from reference [6], the V131I mutation appears in time before the methionine change at position 130.
In a Korean study T-A mutations were found in 32% (13/ 41) of HBV carriers, and a triple mutation G1714A, C1718T, A1721G was found in 27% (11/41) patients [4]. In our study wild type (wt) HBV strain nucleotide were found in the 1714 and 1718 positions, but the mutation A1721G was found in genotype F samples and not in two samples with other genotypes. Again, T-A mutations are common in all genotypes while other mutations seem to be more related to specific genotypes.
No association could be established between the presence of T-A mutations and HBeAg status (Table 3), similar to other published data [4,24]. Of the four samples with the 8 bp deletion only (467 and 6516), two were re-amplified from the PCR1 product and corroborated by enzyme restriction digestion, which demonstrates that the deletion was not a PCR artifact. This 8 bp deletion in the T-A site has been reported previously [6,8,9,27,29] and it has been associated with a low viral load [7,8,29]. Different clones isolated from several patients showed a heterogeneous population of strains including T-A mutations, wt strains as well as the 8 bp deletion. This could be a possible reason why we observed different results in amplified samples of the initial PCR products with an 8 deletion than in the reanalyzed two samples where the deletion was not detected.
An interesting fact is that these deletions alter the X open reading frame, changing K130N and introducing an iso-leucine in the 131 site and a stop codon in the position135.
The polymorphic differences observed between the sequence of acute HBV recovered patients and chronic carriers are related to the genetic diversity of strain more than the study group classification (A,B,C). All sequences isolated in this study belong to genotype F with the exception of 2. Using blast searches sequences from genotype F can be divided in AY090455 -1889 NIC sequences similar to those which are related to South American sequences and the AY090456-1980HCR sequences similar to those which are related to Central America sequences. The polymorphism observed in the nucleotides as well as the amino acids in these groups may be due to a variability present in the group related to the South American sequences.
Many efforts have been made in order to clarify the role of viral variants in the pathogenesis of HBV infection; and still there is no final consensus. T-A mutations have been proposed as possible prognostic markers for liver disease progression [14] however, more studies are needed to elucidate the role of the T-A mutations and its relation to HBV diversity and disease outcome.

Conclusion
According to our results, T-A mutations were frequently observed in HBV chronic carriers, but were not found in acute recovered patients.
T-A mutations are frequent in all genotypes while other mutations seem to be more related to specific genotypes.
T-A mutations may appear early during HBV infection although the possibility of initial infection cannot be excluded.

Study population
Samples were obtained from a study of HBV in San Ramón and Palmares, Costa Rica areas outside of the capital city, San José, between 1972-1985 [19]. Based on  with at least 3 years difference between the samples. Group C included 45 chronic patients with unknown date of onset. Twenty-nine patients had liver biopsy results, 4 from group B and 25 from group C.
The samples from all groups were negative by anti HAV IgM or anti-HCV [31] and were kept frozen.
This project was approved by the Ethical Committee of the Universidad of Costa Rica.

Biopsy classification Pathology
The inflammatory activity of Knodell in Chronic Persistent Hepatitis (CPH) between 1 and 2 points, is represented by a uniform and diffuse cobblestone arrangement of swollen hepatocytes, with compressed sinusoids; some of which show Hadziyannis cells containing abundant HBsAg.
Lobular Chronic Hepatitis (LCH) is between 2 and 6 points with an intact lobular architecture, perivenular cell swelling, focal hepatocytolysis and a variable degree of inflammatory activity [32]. Further, these lesions are characterized by focal necrosis, abnormal hepatocytes and scattered passive fibrous interportal bridges.
In this study the Knodell Index (KI) was used as follows: ≤ 2 points was considered mild liver lesion, 3 and 4 moderate and > 4 as severe liver damage.
Dilutions of 1:10 of a commercial CPG ® DNA plasmid with 10 5 copies/µl of the total HBV genome were prepared and used as control as well as to determine the limit detection (sensitivity) of the PCR system.

Sequencing conditions
Nested PCR product (616 bp) was run on 1% agarose gels and the expected band was cut and purified by a Qiagen column system following manufacturer's instructions.
Primers were diluted to a concentration of 3 pM in TE buffer. All reagents were used according to manufacturer's instructions. The first denaturation step was at 94°C for 2:30 min followed by 35 cycles of 0:30 min at 94°C, 0:30 min at 50°C, 1 min at 70°C and a final extension step at 72°C for 7 min. Finally, 1.5 µl of each sample was run in a polyacrylamide gel at 1500 volts for 90 min.
A consensus sequence of the genotype F strain (NCBI AB036919, AB036905, X75658) was used as our wild type sequence.

Genotype sequencing
The HBx gene sequences were compared with homologue sequences obtained from the GeneBank data base using the BLAST program [33]. The genotype was determined using a web-based genotyping tool for viral sequences [34]. The subtype of some of the samples was determined previously by specific antibodies available in our laboratory.

Restriction Enzyme digestion
In order to corroborate an 8 bp deletion observed in some sequences, a restriction enzyme SspI was used (New England, BioLabs INC, ). As a positive control a sample from HIV having the same recognition site was used and a HBx sample with the wild type sequence was employed as a negative control. Ten µl of each purified product from the nested PCR were dispensed into two different vials of 200 µl. In one vial 1 µl of SspI enzyme (5000 units/ml), 2 µl of enzyme buffer (New England, BioLabs INC, ) and 7 µl of water were added; while in the other vial the enzyme was omitted. All samples were heated at 37°C for 90 minutes and run in a 3% agarose gel. Results were visualized with ethidium bromide.

Statistical analysis
The Fisher's exact test was used to evaluate the relationship between two discrete and dichotomy variables. The t test, for independent samples, was used to analyze continuous variables when it was necessary. A new dichotomy variable for hepatic damage was built into biopsy results and using data from the Costa Rican National Tumor Registry (NTR); by division into "mild damage" and "moderate/severe damage". The relative risk (RR) was calculated with a 95% confidence interval. All analyzes were done with the JMP 4 software version 4.0.4 A BUSINESS UNIT OF SAS Copyright © 1989 -2001 SAS Institute Inc. (all rights reserved) and Epiinfo software CDC.