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 - PubMed (original) (raw)

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

Fan-Ching Shen et al. J Biomed Sci. 2009.

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

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Figures

Figure 1

Figure 1

Representatives of the wild-type, pre-S1, and pre-S2 mutant LHBS gene. The shaded boxes are the regions deleted in the pre-S1 and pre-S2 mutant LHBS. The numbers on the bottom of the gene indicate the pre-S1, S2, and S regions of the LHBS gene in nucleotide order. Nucleotide 1 is the start of the circular genome and the numbers go clockwise. The last nt number is 3221. Here only the S gene, which spans the start of the genome, is shown. The arrow at the top of the diagram indicates the start (nt 1) of the HBV genome.

Figure 2

Figure 2

The Pre-S Gene Chip. The chip (7 mm (H) × 10 mm (W)) contains 42 oligonucleotide probes spanning the pre-S regions. The target region of each probe is indicated in the order of the nucleotides below the chip. The probes with extension numbers (e.g., 8-1, 8-2,... 8-6) are the redundant probes that target the same regions as the primary probes (e.g., 8) do.

Figure 3

Figure 3

Working scheme of Pre-S Gene Chip analysis. The virus DNA is extracted from the patient's blood or liver tissue. The pre-S region is PCR-amplified using PCR-1R and 5'-dig-labeled 1F primers. The PCR products are visualized using agarose gel electrophoresis and ethidium bromide staining. In cases where no PCR products are seen---perhaps because of a low HBV DNA titer---nested PCR using PCR-2R and 5'-dig-labeled 1F primers is done. When only a single PCR product is seen, the DNA product is directly subjected to chip hybridization. However, when two or more different types of pre-S PCR products are seen in agarose gel, the products are first directed to TA cloning. Multiple plasmid clones are then analyzed using colony PCR with M13R and 5'-dig-labeled 13F primers. These PCR products are then analyzed using the Pre-S Gene Chip.

Figure 4

Figure 4

Results of the Pre-S Gene Chip analysis. A: The wild-type LHBS gene. B: The pre-S1 mutant LHBS, with nt 3044-3103 of the HBV genome deleted. The signals on probes 6 (nt 3045-3071) and 7 (nt 3074-3103) are negative. C: The pre-S2 mutant LHBS, with nt 3-57 of the HBV genome deleted. The signals on probes 12 (nt 3-32) and 13 (nt 33-62) are negative.

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