A pre-S gene chip to detect pre-S deletions in hepatitis B virus large surface antigen as a predictive marker for hepatoma risk in chronic hepatitis B virus carriers
© Shen et al; licensee BioMed Central Ltd. 2009
Received: 13 June 2009
Accepted: 15 September 2009
Published: 15 September 2009
Chronic hepatitis B virus (HBV) infection is an important cause of hepatocellular carcinoma (HCC) worldwide. The pre-S1 and -S2 mutant large HBV surface antigen (LHBS), in which the pre-S1 and -S2 regions of the LHBS gene are partially deleted, are highly associated with HBV-related HCC.
The pre-S region of the LHBS gene in two hundred and one HBV-positive serum samples was PCR-amplified and sequenced. A pre-S oligonucleotide gene chip was developed to efficiently detect pre-S deletions in chronic HBV carriers. Twenty serum samples from chronic HBV carriers were analyzed using the chip.
The pre-S deletion rates were relatively low (7%) in the sera of patients with acute HBV infection. They gradually increased in periods of persistent HBV infection: pre-S mutation rates were 37% in chronic HBV carriers, and as high as 60% in HCC patients. The Pre-S Gene Chip offers a highly sensitive and specific method for pre-S deletion detection and is less expensive and more efficient (turnaround time 3 days) than DNA sequencing analysis.
The pre-S1/2 mutants may emerge during the long-term persistence of the HBV genome in carriers and facilitate HCC development. Combined detection of pre-S mutations, other markers of HBV replication, and viral titers, offers a reliable predictive method for HCC risks in chronic HBV carriers.
Chronic hepatitis B virus infection is a major cause of HCC worldwide and its most important cause in Asia [1–6]. HBV infection occurs primarily through blood or body fluid transmission. HBV-related HCC often occurs at the age of 40 or older, suggesting that HBV may persist in carriers for decades before HCC actually develops [6, 7]. Long-term monitoring of chronic HBV carriers is important to help prevent HCC. Furthermore, implementing cancer therapies at early disease stages is beneficial. The HBV markers commonly used to monitor the viral status in chronic HBV carriers are viral DNA titers, HBV surface and core antigens, and hepatitis B envelope (HBe) antigen [7, 8]. Combined detection of these markers reveals the status of virus replication as well as the number of virus particles in host hepatocytes. Active HBV viral replication and high virus titers are associated with the severity of HBV-induced liver inflammation, fibrosis, cirrhosis, and HCC [9, 10].
After pre-S mutant LHBS was discovered, various geographically diverse studies [18–26] screening for pre-S mutations invariably reported that they were prevalent in chronic HBV carriers. In addition, pre-S mutant LHBS, especially the pre-S2 type, is highly correlated with the severity of HBV-related liver diseases, including HCC [20–23, 25]. Therefore, it is important to screen for pre-S deletion mutations in chronic HBV carriers. This type of screening should be done in combination with the detection of other HBV markers, such as viral titers and HBe antigen (Ag), to estimate an HBV carrier's relative risk for HCC.
Identifying pre-S mutations in chronic HBV carriers usually requires multiple experimental procedures, because most pre-S mutants co-exist with wild-type LHBS in blood and hepatocytes; this is probably due to the emergence of the pre-S mutant from the wild-type form. One individual carrier often simultaneously presents multiple pre-S mutant forms that need to be singly cloned and then sequenced. This is both time-consuming and expensive, which makes it difficult to screen large populations of chronic HBV carriers. We developed a convenient and cost-effective oligonucleotide array system for screening pre-S deletions in the LHBS gene. Using this system, we omitted traditional DNA sequencing and shortened the detection procedure from about 7 days to no longer than 3 working days.
Materials and methods
Reagents and participants
The pre-S region of the LHBS gene in two hundred and one HBV-positive serum samples, collected at the National Cheng Kung University Hospital from 2000 through 2002, was PCR-amplified and sequenced . To develop the Pre-S Gene Chip system, 20 HBV-DNA-positive serum samples were obtained from Kung-Chia Young, PhD, at National Cheng Kung University. The pre-S region of the LHBS gene was PCR-amplified and sequenced . The DNA sequences were used to design oligonucleotide probes on the Pre-S Gene Chip. To test the efficacy of the chip, another 20 serum samples from chronic HBV carriers were obtained from the Center for Disease Control, Taipei, Taiwan, and analyzed using the chip. A DNA extraction kit (QIAamp MinElute Virus Spin kit; Qiagen Inc., Valencia, CA) was used to extract the virus DNA in serum. Most of the common chemicals used for chip hybridization were purchased from Sigma-Aldrich Co., St Louis, MO.
The Pre-S Gene Chip
Polymerase chain reaction
The PCR primers used in this study.
HBV genome nt 2818 to nt 2837
HBV genome nt 236 to nt 255
HBV genome nt 180 to nt 199
M13 Forward primer
M13 Reverse primer
In cases where multiple pre-S PCR products were revealed, E. coli TA cloning was done. Three microliters of PCR product was ligated with 50 ng of TA cloning vector (pCR 2.1; Invitrogen) at 14°C overnight. The next day, the ligation product was transformed into an E. coli DH5α strain. The pre-S insert-DNA was then examined using colony PCR. For the DNA sequencing analysis, the colony PCR products were purified using a kit (PCR Clean-Up Kit; Roche Applied Sciences, Indianapolis, IN) and sent to our university's DNA Sequencing Facility for analysis.
Pre-S Gene Chip analysis
The pre-S chip was placed in a 24-well culture plate, soaked in 0.2× standard sodium citrate (SSC: 150 mM NaCl, 15 mM NaH2(C3H5O(COO)3 [pH 7.0]), and then rinsed twice with the same solution. For pre-hybridization, the chip was then gently shaken in microarray hybridization buffer (5× SSC, 1% blocking reagent (Roche), 0.1% N-lauroylsarcosine, 0.02% SDS) at room temperature for 2 h. The chip was then hybridized with 10 μL of heat-denatured PCR product in 300 μL of hybridization buffer at 50°C for 90 min while being gently shaken (60 rpm). After it had been hybridized, the chip was washed four times with 0.1× SSC and then incubated in 1× blocking reagent at room temperature for 1 h. The digoxigenin that had hybridized to the DNA probes on the chip was then recognized by incubating the chip with 0.375 units of anti-digoxigenin-alkaline phosphatase Fab fragments (Roche) for 1 h. After it had been incubated, the chip was washed 3 times with MAB washing buffer (MAB: 0.1 M maleic acid, 0.15 M NaCl, 0.1% Tween 20 [pH 7.5]) for 15 min each. To detect digoxigenin signals, the chip was first soaked in a detection buffer (0.1 M Tris-HCl, 0.1 M NaCl [pH 9.5]) for 5 min and then incubated with two alkaline phosphatase substrates: nitro blue tetrazolium chloride (NBT, 0.375 mg/mL) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP, 0.188 mg/mL) at 37°C for 20 min in the dark. Finally, the chip was washed 3 times with ddH2O and air-dried. Chip images were scanned using an Epson Perfection 1250 scanner .
Using DNA sequencing analysis, we first detected pre-S deletion mutations in the sera of HBV carriers and patients with HBV-related HCC. The pre-S mutation rate was relatively low (7%) in the sera of patients with high HBV titers, which indicates the acute phase of HBV infection (Table 2). The pre-S deletion rate gradually increased during periods of persistent HBV infection: the rate was 37% in HBV carriers with low serum HBV titers, which indicated the chronic phase of HBV infection. In HCC patients with HBV infection, the pre-S deletion rate was as high as 60%, which suggested that pre-S mutants emerge during long-term persistence of the HBV genome in carriers, and that they facilitate the development of HCC.
Pre-S mutations in patients with different serum HBV DNA titers
Serum HBV DNA Titer*
HBV Clones# (n)
Clones with Pre-S Deletions (n)
Summary of the pre-S genotyping results by DNA sequencing and Pre-S Gene Chip analysis in 20 HBV carriers.
DNA Sequencing Result
Pre-S Gene Chip Result
2. del nt 2894 to 3100
2. del nt 2894 to 3013 and nt 3074 to 3103
2. del nt 3040 to 3111
2. del nt 3044 to 3103
del nt 3218 to 8
2. del nt 3084 to 3188
2. del nt 3074 to 3103 and nt 3134 to 3193
3. del nt 3148 to 29
3. del nt 3134 to 32
4. del nt 3138 to 59
4. del nt 3134 to 62
2. del nt 3202 to 36
2. del nt 3194 to 32
del nt. 2913 to 3118 and nt 3217 to 9
del nt 2924 to 2983 and nt 3014 to 3103
2. del nt 3022 to 3205
2. del nt 3014 to 3103 and nt 3134 to 2
3. del nt 3132 to 37
3. del nt 3134 to 32
4. del nt 3057 to 3198
4. del nt 3074 to 3103 and nt 3134 to 3193
5. del nt 3132 to 3182
5. del nt 3134 to 3193
del nt 2854 to 2871
del nt 2854 to 2878
1. del nt 3205 to 3213
2. del nt 34 to 54
2. del nt 33 to 62
2. del nt 3041 to 3126
2. del nt 3044 to 3103
3. del nt 25 to 56
3. del nt 33 to 62
2. del nt 2984 to 3221
2. del nt 2984 to 3013, nt 3044 to 3103, and nt 3134 to 2
2. del nt 3 to 56
2. del nt 3 to 62
del nt 3040 to 3111
del nt. 3044 to 3103
del nt 2948 to 3097 and nt 3112 to 2
del nt 2954 to 3013, nt 3044 to 3103, and nt 3194 to 2
Pre-S mutant Clones ( n )
Cases with Pre-S Mutations ( n ) (%)
In this study, we developed an oligonucleotide array system to detect deletions in the pre-S regions of the HBV LHBS gene in chronic carriers with detectable HBV DNA, the group of carriers at a high risk of HCC . We found that multiple mutation clones often co-exist in a carrier, making genotyping tedious and time-consuming. Cloning individual PCR products is usually necessary to differentially genotype the different pre-S deletion products. We found that doing colony PCR of the individual gene clones, and then directly applying the PCR products to the Pre-S Gene Chip for hybridization and detection, was more time- and cost-effective than traditional DNA sequencing analysis. The Pre-S Gene Chip is made of a 0.7-cm2 nylon membrane and costs substantially less than DNA sequencing analysis, especially for clinical institutions without internal access to a DNA sequencing service. The DNA chip system also simultaneously detects multiple pre-S clones, which makes it convenient for large-scale pre-S mutation screenings in chronic HBV carriers. Using the Pre-S Gene Chip and DNA sequencing in blind tests of 20 cases showed that both methods had close detection rates of pre-S deletions. Therefore, we conclude that the Pre-S Gene Chip delivered comparable results to direct DNA sequencing and is a good screening method for pre-S deletions. A minor disadvantage of the Pre-S Gene Chip system is that it does not appear to be sensitive enough to detect deletions less than 10 nt long: in one blind-tested case, the chip did not identify a 10-nt deletion clone. Nevertheless, considering cost and convenience, the Pre-S Gene Chip is potentially good for large-scale screening of pre-S deletions; perhaps it can be improved.
In this study, we found that pre-S deletion rates were significantly higher in the late phase of chronic HBV infection, which displays lower HBV titers, than in the acute infection phase, which displays high viral titers. In the HBV-induced HCC stage, the pre-S deletion rate was as high as 60%. These findings suggested that pre-S deletions probably occur during long-term persistent HBV infection and eventually become predominant in HBV carriers. Therefore, we hypothesize that pre-S mutants are important in liver carcinogenesis. Our earlier studies [27, 28] found that pre-S mutant LHBS accumulated in ER, where it caused ER and oxidative stress [27, 28]. It also strongly induced DNA damage and mutations, which destabilized the genome . We found that the pre-S2 mutant LHBS induces cyclin A overexpression, cell cycle progression, and cell proliferation, all essential factors for carcinogenesis [29, 30]. These earlier findings showed that pre-S mutant LHBS, especially the pre-S2 type, is involved in carcinogenesis. Identifying the pre-S deletion mutations in chronic HBV carriers is therefore important when screening for persons at high risk for HCC.
In addition to pre-S deletions, a number of HBV markers, such as HBV viral titers and HBeAg, are also believed to be associated with the risk of HCC in chronic HBV carriers [9, 10]. HBV replication causes liver injury and inflammation, which releases cytokines and facilitates the development of fibrosis and liver cell proliferation [7, 8]. In addition, the HBV protein HBX crosstalks with various host factors and behaves as a viral oncoprotein [33, 34]. It transactivates a number of cellular promoters, acting on cis-acting regulatory elements . It also regulates proteasome function and, thus, controls the degradation of cellular and viral proteins . We hypothesize that pre-S mutant LHBS strengthens the detrimental effects of HBX protein by increasing cell proliferation and genomic instability, thereby facilitating hepatocellular carcinogenesis.
We developed the Pre-S Gene Chip system to screen for pre-S deletions in the LHBS gene. The detection sensitivity of the Pre-S Gene Chip for pre-S mutations is close to that of direct DNA sequencing analysis, but it is much more time- and cost-effective. We believe that the Pre-S Gene Chip is feasible for the large-scale screening of pre-S mutations in chronic HBV carriers. Combining the detection of pre-S mutations with other HBV factors, such as HBV viral titers and HBeAg, should offer a reliable predictive method for HCC risk in chronic HBV carriers.
Supported by grants NHRI-EX95-9520BI (to W. Huang) from the National Health Research Institutes, and CDC-DOH93-DC-1128 (to W. Huang) from the Center for Disease Control, Taiwan.
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