# Patent

## Title
Genomic markers of hepatitis B virus associated with hepatocellular carcinoma

## Abstract

The present invention provides methods of predicting a pre-disposition of HBV-infected individuals to develop hepatacellular carcinoma (HCC).

## Background
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
The present patent application is a continuation-in-part of U.S. patent application Ser. No. 10/937,987, filed Sep. 10, 2004, and now is abandoned, which is incorporated by reference in its entirety.


BACKGROUND OF THE INVENTION
Hepatitis B virus (HBV) infects over 300 million people worldwide. For those individuals with high levels of viral replication, chronic active hepatitis with progression to cirrhosis, liver failure and hepatocellular carcinoma (HCC) is common.
The natural progression of chronic HBV infection over a 10 to 20 year period leads to cirrhosis in 20-to-50% of patients and progression of HBV infection to hepatocellular carcinoma has been well documented. There have been no studies that have determined sub-populations of hepatitis B virus that are most likely to cause hepatocellular carcinoma, thus to date all hepatitis B virus have been considered of equal risk of hepatocarcarcinogesis.
It is important to note that the survival for patients diagnosed with hepatocellular carcinoma is only 0.9 to 12.8 months from initial diagnosis (Takahashi et al., American Journal of Gastroenterology 88:240-243 (1993)). Treatment of hepatocellular carcinoma with chemotherapeutic agents has not proven effective and only 10% of patients will benefit from surgery due to extensive tumor invasion of the liver (Trinchet et al., Presse Medicine 23:831-833 (1994)). Given the aggressive nature of primary hepatocellular carcinoma, the only viable treatment alternative to surgery is liver transplantation (Pichlmayr et al., Hepatology 20:33S-40S (1994)).
BRIEF SUMMARY OF THE INVENTION
The present invention provides for methods of determining a pre-disposition of an individual infected with hepatitis B virus (HBV) to develop hepatocellular carcinoma (HCC). In some embodiments, the methods comprise:
(a) determining nucleotides in the genome of HBV isolated from the individual at positions corresponding to nucleotides 31, 53, 312, 799, 961, 1165, 1499, 1613, 1762, 1764, 1899, 2170, 2441, 2525, and/or 2712 of SEQ ID NO:1; and
(b) comparing the determined nucleotides to nucleotides associated with a pre-disposition to cause HCC, wherein the nucleotides associated with a pre-disposition to cause HCC comprise: 31C, 53C, 312C, 799G, 961G, 1165T, 1499G, 1613A, 1762T, 1764A, 1899A, 2170C, 2170G, 2441C, 2525C, 2712C, 2712A, and/or 2712G.
In some embodiments, the methods comprise:
(a) determining nucleotides in the genome of a genotype B HBV isolated from the individual at positions corresponding to nucleotides 1165, 1762, 1764, 2525 or 2712 of SEQ ID NO:1; and
(b) comparing the determined nucleotides to nucleotides associated with a pre-disposition to cause HCC, wherein the nucleotides associated with a pre-disposition to cause HCC comprise: 1165T, 1762T, 1764A, 2525C, 2712C, 2712A, or 2712G.
In some embodiments, the methods comprise:
(a) determining nucleotides in the genome of a genotype B HBV isolated from the individual at positions corresponding to nucleotides 1165, 1762, 1764, 2525 and 2712 of SEQ ID NO:1; and
(b) comparing the determined nucleotides to nucleotides associated with a pre-disposition to cause HCC, wherein the nucleotides associated with a pre-disposition to cause HCC in genotype B comprise:
1762T and 1764A and 2712A; or
1762T and 1764A and 2712C; or
1762T and 1764A and 2712G; or
1762T and 1764A and 2712T and 2525C; or
1762A and 1764G and 1165T.
In some embodiments, the method comprises determining the genotype of the HBV from the individual.
In some embodiments, the determining step comprises nucleotide sequencing the HBV genome flanking the nucleotides at positions corresponding to nucleotides 1165, 1762, 1764, 2525 and 2712 of SEQ ID NO:1.
In some embodiments, the determining step comprises amplifying at least a portion of the HBV genome to produce one or more amplification products comprising the nucleotides at the positions corresponding to nucleotides 1165, 1762, 1764, 2525 and 2712 of SEQ ID NO:1. In some embodiments, the method comprises contacting the one or more amplification products with one or more probes that hybridize to HCC-associated nucleotides:
1762T and 1764A and 2712A; or
1762T and 1764A and 2712C or;
1762T and 1764A and 2712G; or
1762T and 1764A and 2712T and 2525C; or
1762A and 1764G and 1165T;
under conditions to allow for hybridization of a probe to an amplification product only if the amplification product comprises a complementary nucleotide at the position of the HCC-associated nucleotide. In some embodiments, the hybridization is performed as a line probe assay.
In some embodiments, the method comprises:
(a) determining nucleotides in the genome of a genotype C HBV isolated from the individual at positions corresponding to nucleotides 31, 53, 312, 799, 961, 1499, 1613, 1899, 2170, or 2441; and
(b) comparing the determined nucleotides to nucleotides associated with a pre-disposition to cause HCC, wherein the nucleotides associated with a pre-disposition to cause HCC comprise: 31C, 53C, 312C, 799G, 961G, 1499G, 1613A, 1899A, 2170C, 2170G, or 2441C.
In some embodiments, the method comprises
a) determining the subtype of a genotype C HBV from the individual, wherein:
subtype C1 comprises nucleotides 2733A, 1856C, 1009T and 2892T,
subtype C2 comprises nucleotides 2733C, 1856T, 1009T and 2892T, and
subtype C3 comprises nucleotides 2733C, 1856C, 1009C and 2892T;
b1) if the HBV is genotype C1, determining the nucleotides at positions corresponding to nucleotides 31, 53 and 1499 of SEQ ID NO:1; or
b2) if the HBV is genotype C2, determining the nucleotides at positions corresponding to nucleotides 799, 2441 and 2170 of SEQ ID NO:1; and
b3) if the HBV is genotype C3, determining the nucleotides at positions corresponding to nucleotides 312, 961, 1613, 1899 of SEQ ID NO:1; and
c) comparing the determined nucleotides to nucleotides at the positions associated with a pre-disposition to cause HCC, wherein the nucleotides associated with a pre-disposition to cause HCC in subtype C1 comprise:
31C; and/or
53C; and/or
1499G; and
the nucleotides associated with a pre-disposition to cause HCC in subtype C2 comprise:
2170C; and/or
2170G; and/or
2441C; and/or
799G; and
the nucleotides associated with a pre-disposition to cause HCC in subtype C3 comprise:
312C; and/or
961G; and/or
1613A; and/or
1899A
In some embodiments, the determining step comprises nucleotide sequencing the HBV genome flanking the nucleotides at positions corresponding to nucleotides 31, 53, and 1499 of SEQ ID NO:1. In some embodiments, the determining step comprises nucleotide sequencing the HBV genome flanking the nucleotides at positions corresponding to nucleotides 799, 2441, and 2170 of SEQ ID NO:1. In some embodiments, the determining step comprises amplifying at least a portion of the HBV genome to produce one or more amplification products comprising the nucleotides at the positions corresponding to nucleotides 31, 53, and 1499 of SEQ ID NO:1. In some embodiments, the determining step comprises nucleotide sequencing the HBV genome flanking the nucleotides at positions corresponding to nucleotides 312, 961, 1613, and 1899 of SEQ ID NO:1
In some embodiments, the determining step comprises amplifying at least a portion of the HBV genome to produce one or more amplification products comprising the nucleotides at the positions corresponding to nucleotides 799, 2441, and 2170 of SEQ ID NO:1. In some embodiments, the determining step comprises amplifying at least a portion of the HBV genome to produce one or more amplification products comprising the nucleotides at the positions corresponding to nucleotides 312, 961, 1613, and 1899 of SEQ ID NO:1.
In some embodiments, the method comprises contacting the one or more amplification products with one or more probes that hybridize to HCC-associated nucleotides:
31C; and/or
53C; and/or
1499G;
under conditions to allow for hybridization of a probe to an amplification product only if the amplification product comprises a complementary nucleotide at the position of the HCC-associated nucleotide.
In some embodiments, the hybridization is performed as a line probe assay.
In some embodiments, the method comprises contacting the one or more amplification products with probes that hybridize to HCC-associated nucleotides:
2170G; and/or
2441C; and/or
799G;
under conditions to allow for hybridization of the probes to the amplification product only if the amplification product comprises a complementary nucleotide at the position of the HCC-associated nucleotide. In some embodiments, the hybridization is performed as a line assay.
In some embodiments, the method comprises contacting the one or more amplification products with probes that hybridize to HCC-associated nucleotides:
312C; and/or
961G; and/or
1613A; and/or
1899A;
under conditions to allow for hybridization of the probes to the amplification product only if the amplification product comprises a complementary nucleotide at the position of the HCC-associated nucleotide. In some embodiments, the hybridization is performed as a line assay.
In some embodiments, the method further comprises determining the genotype of the HBV from the individual.
In some embodiments, the method comprises:
determining the genotype of the HBV, wherein genotype B comprises 2733C, 1856C, 1009T and 2892T, genotype C1 comprises 2733A, 1856C, T1099T and 2892T, genotype C2 comprises 2733C, 1856T, 1009T and 2892T and genotype C3 comprises 2733C, 1856C, 1009C and 2892T;
determining nucleotides 1165, 1762, 1764, 2525 and 2712 of the HBV genome if the HBV is genotype B; and/or
determining nucleotides 31 and/or 53 and/or 1499 of the HBV genome if the HBV is C1; and/or
determining nucleotides 2170 and/or 2441 and/or 799 of the HBV genome if the HBV is C2; and/or
determining nucleotides 312 and/or 961 and/or 1613 and/or 1899 of the HBV genome if the HBV is C3; and
comparing the determined nucleotides to nucleotides associated with a pre-disposition to cause HCC,
wherein nucleotides associated with a pre-disposition to cause HCC in genotype B comprise:
1762T and 1764A and 2712A; or
1762T and 1764A and 2712C or;
1762T and 1764A and 2712G; or
1762T and 1764A and 2712T and 2525C; or
1762A and 1764G and 1165T;
wherein nucleotides associated with a pre-disposition to cause HCC in genotype C1 comprise:
31C; and/or
53C; and/or
1499G; and
wherein nucleotides associated with a pre-disposition to cause HCC in genotype C2 comprise:
2170C; and/or
2170G; and/or
2441C; and/or
799G;
wherein nucleotides associated with a pre-disposition to cause HCC in genotype C3 comprise:
312C; and/or
961G; and/or
1613A; and/or
1899A;
thereby determining the pre-disposition of the individual to develop HCC.
The present invention also provides kits for detecting HBV isolates that are associated with the development hepatocellular carcinoma (HCC).
In some embodiments, the kits comprise: one or more probe which, when contacted to an HBV genome, selectively hybridizes to the genome if the genome comprises at least one of the following nucleotides: 31C, 53C, 312C, 799G, 961G, 1165T, 1499G, 1613A, 1762T, 1762A, 1764A, 1764G, 1899A, 2441C, 2170C, 2170G, 2712A, 2712C, 2712G; or 2525C.
In some embodiments, the probe is linked to a solid support.
In some embodiments, the probe selectively hybridizes to:
1762T and 1764A and 2712A; and/or
1762T and 1764A and 2712C; and/or;
1762T and 1764A and 2712G; and/or
1762T and 1764A and 2712T and 2525C; and/or
1762A and 1764G and 1165T.
In some embodiments, the probe selectively hybridizes to:
31C; and/or
53C; and/or
1499G.
In some embodiments, the probe selectively hybridizes to:
2170C; and/or
2170G; and/or
2441C; and/or
799G.
In some embodiments, the probe selectively hybridizes to:
312C; and/or
961G; and/or
1613A; and/or
1899A.
In some embodiments, the kits further comprise primers for amplification of at least a portion of the HBV genome.
The present invention also provides a computer readable medium for determining whether an HBV sequence is likely to result in the development of HCC. In some embodiments, the computer readable form comprises:

    
    
        a) code for receiving information describing: nucleotides at positions corresponding to nucleotides 31, 53, 312, 799, 961, 1165, 1499, 1613, 1762, 1764, 1899, 2170, 2441, 2525, or 2712 of SEQ ID NO:1;
        b) code for comparing the nucleotides received in a) to nucleotides associated with a pre-disposition to cause HCC; and
        c) code for providing a determination of the pre-disposition of the HBV to cause HCC,

wherein nucleotides associated with a pre-disposition to cause HCC comprise: 31C, 53C, 312C, 799G, 961G, 1165T, 1499G, 1613A, 1762T, 1764A, 1899A, 2170C, 2170G, 2441C, 2525C, 2712C, 2712A, or 2712G.

    
    


DEFINITIONS
A probe “selectively hybridizes” to a viral genome comprising a particular nucleotide when the probe hybridizes to the genome when the particular nucleotide (at the specified position) is present, but does not hybridize if the nucleotide at the specified position is different or absent. Conditions to allow for hybridization of a probe to a particular DNA molecule only if a complementary nucleotide is present in a particular target DNA are generally “stringent hybridization conditions.”
The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences, or at least to no other sequences at which a particular position is anything but one particular nucleotide. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions for Southern hybridization are generally those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective hybridization, a positive signal is at least two times background, optionally 10 times background hybridization, i.e., hybridization to another nucleotide sequence with a different nucleotide at the position of interest. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes.
“Determining nucleotides in the genome of HBV at positions corresponding to” particular nucleotides of a reference sequence (e.g., SEQ ID NO:1) refers to identifying a position in an isolated HBV genome that occurs in a position that is the equivalent of the particular position in the reference sequence. The variants identified in the present invention are not limited to predicting sequence pre-disposition of variants of SEQ ID NO:1, but instead apply to any HBV strain carrying particular corresponding nucleotides. Thus, when the genome of an HBV isolate differs from SEQ ID NO:1 (e.g., by changes in nucleotides or addition or deletion of nucleotides), it may be that a particular nucleotide associated with the development of HCC will not be in exactly the same position as it is in SEQ ID NO:1. For example, the nucleotide corresponding to nucleotide 31C of SEQ ID NO:1 may occur at position 32 of a particular HBV strain due to a one nucleotide insertion at an earlier position in the strain's genome. Nevertheless, position 32 of the HBV strain would correspond to position 31 of SEQ ID NO:1, which can be readily illustrated in an alignment of the two sequences. As described herein, the corresponding nucleotide in the genome of an HBV isolate can be determined using an alignment algorithm such as BLAST.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the locations of various primers used for amplification of HBV and the resulting amplified fragments relative to the HBV genome, represented as a line at the bottom of the figure.
FIGS. 2A and 2B illustrate the genome (SEQ ID NO:1) of an exemplary HBV genotype B isolate comprising highlighted nucleotides associated with the development of HCC.
FIGS. 3A and 3B illustrate the genome (SEQ ID NO:2) of an exemplary HBV genotype C1 isolate comprising highlighted nucleotides associated with the development of HCC.
FIGS. 4A and 4B illustrate the genome (SEQ ID NO:3) of an exemplary HBV genotype C2 isolate comprising highlighted nucleotides associated with the development of HCC.



DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
The present invention is based on the discovery that certain sequence variants of HBV are associated with the development of hepatocellular carcinoma (HCC) in individuals infected with HBV. Specifically, the presence of the following nucleotides in an HBV genome is associated with the development of HCC: 31C, 53C, 312C, 799G, 961G, 1165T, 1499G, 1613A, 1762T, 1764A, 1899A, 2170C, 2170G, 2441C, 2525C, 2712C, 2712A, or 2712G. Accordingly, the invention provides for methods of determining whether an individual infected with HBV has a predisposition for HCC by detecting the nucleotide sequence of the HBV variant infecting the individual. The method also provides for kits comprising reagents to detect any of the specific variants associated with HCC and computer readable forms for applying the methods of the invention.
II. Detecting HBV Variants Associated with HCC
Any number of methods may be used to determine the nucleotides at the positions corresponding to nucleotides at positions 31, 53, 312, 799, 961, 1165, 1499, 1613, 1762, 1764, 1899, 2170, 2441, 2525, and/or 2712 of SEQ ID NO:1 and/or other positions as described herein.
In some embodiments, nucleotide sequencing is used to determine the nucleotides at particular positions of the HBV genome. Without intending to limit the invention, examples of nucleotide sequencing include chain termination sequencing. See, e.g., Sanger et al. Proc. Nat. Acad. Sci. USA 74:5463-5467 (1977); Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)). Sequencing may be performed following amplification of the HBV genome or a fragment thereof. Direct sequencing of PCR generated amplicons by selectively incorporating boronated nuclease resistant nucleotides into the amplicons during PCR and digestion of the amplicons with a nuclease to produce sized template fragments may also be performed (Porter et al., Nucleic Acids Research 25(8):1611-1617 (1997)). Alternatively, microfluidic techniques such as those described in U.S. patent Publication No. 2003/0215862 may be used. See also U.S. patent Publication No. 2003/0152996 describing alternate sequencing methods.
Specific probes that bind to nucleotides at particular positions in the HBV genome may also be used to detect nucleotides in the HBV genome. Probes that detect the particular nucleotides associated with HCC may be used in a reverse hybridization assay format using immobilized oligonucleotide probes present at distinct locations on a solid support. More particularly, the Line Probe Assay (LiPA) may be used. The LiPA is a reverse hybridization assay using oligonucleotide probes immobilized as parallel lines on a solid support strip. See, e.g., PCT Publication No. WO 94/12670. In this assay, specific oligonucleotides may be immobilized at known locations on membrane strips and hybridized under strictly controlled conditions with the labeled PCR product. Different probes may be designed such that each probe on the strip comprises an HBV nucleotide sequence, or complement thereof, but contains a different nucleotide at a particular position. Amplifying an HBV genome, or fragment thereof, and hybridizing the amplification product to one or more probes specific for a particular variant will result in complete or at least preferential hybridization of one of the probes to the product, thereby indicating which nucleotide at the particular position is contained in the amplified genome. Hybridization conditions using this assay are generally set at a high stringency such that only one probe binds to the amplification product. Exemplary conditions may include, e.g., standard hybridization and washing conditions (e.g., 1×SSC buffer containing 0.1% sodium dodecyl sulfate at 62° C.).
Amplification of HBV
The HBV genome or a portion thereof may be amplified before the nucleotides at positions associated with HCC are determined. An “amplification” refers to any chemical, including enzymatic, reaction that results in increased copies of a template nucleic acid sequence. Amplification reactions include polymerase chain reaction (PCR) and ligase chain reaction (LCR) (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)), strand displacement amplification (SDA) (Walker, et al. Nucleic Acids Res. 20(7):1691-6 (1992); Walker PCR Methods Appl 3(1):1-6 (1993)), transcription-mediated amplification (Phyffer, et al., J. Clin. Microbiol. 34:834-841 (1996); Vuorinen, et al. , J. Clin. Microbiol. 33:1856-1859 (1995)), nucleic acid sequence-based amplification (NASBA) (Compton, Nature 350(6313):91-2 (1991), rolling circle amplification (RCA) (Lisby, Mol. Biotechnol. 12(1):75-99 (1999)); Hatch et al., Genet. Anal. 15(2):35-40 (1999)) and branched DNA signal amplification (bDNA) (see, e.g., Iqbal et al., Mol. Cell Probes 13(4):315-320 (1999)).
Amplified portions of the HBV genome (optionally labeled) may be hybridized to DNA comprising one or more HCC-associated nucleotides, or a complement thereof, thereby allowing for determination of the identity of nucleotides at a nucleotide position of interest. Alternatively, the probes may detect non-HCC-associated nucleotides, thereby allowing for detection of HCC-associated HBV variants by detecting a lack of hybridization.
In some embodiments, the amplified fragment of the genome will comprise more than one HCC-associated nucleotide. Thus, in some embodiments, the fragment will comprise any combination of positions corresponding to nucleotides at positions 31, 53, 312, 799, 961, 1165, 1499, 1613, 1762, 1764, 1899, 2170, 2441, 2525, and/or 2712 of SEQ ID NO:1. In some embodiments, the fragment will comprise positions corresponding to nucleotides 1165, 1762, 1764, 2525 and 2712 of SEQ ID NO:1. In some embodiments, the fragment will comprise positions corresponding to nucleotides 31, 53, 312, 799, 961, 1499, 1613, 1899, 2170, and 2441 of SEQ ID NO:1.
In some cases, more than one fragment of HBV is amplified. In these cases, the sum of all fragments amplified may comprise any combination of positions corresponding to nucleotides at positions 31, 53, 312, 799, 961, 1165, 1499, 1613, 1762, 1764, 1899, 2170, 2441, 2525, and/or 2712 of SEQ ID NO:1. For example, one fragment may comprise positions 31, 53, 312, 799, 961, 1165, 1499, 1613, 1762, 1764 and a second fragment may comprise positions 1899, 2170, 2441, 2525, or 2712. In some embodiments, the sum of all amplified fragments will comprise positions corresponding to nucleotides 1165, 1762, 1764, 2525 and 2712 an SEQ ID NO:1. In some embodiments, the sum of all amplified fragments will comprise positions corresponding to nucleotides 31, 53, 312, 799, 961, 1499, 1613, 1899, 2170, and 2441.
In some embodiments, amplification and detection methods are used in combination, and sometimes in the same reaction vessel, to detect HBV polynucleotides using detectably-labeled probes that distinguish between HCC-associated nucleotides and nucleotides not associated with HCC. Binding of a probe to its complementary hybridization sequence allows the user to quantify the accumulation of a particular sequence without necessarily removing the contents from the reaction vessel. In general, any type of label that allows for the detection and differentiation of different probes can be used according to the methods of the invention.
Accumulation of amplified product can be quantified by any method known to those in the art. For instance, fluorescence from a probe can be detected by measurement of light at a particular frequency. Similarly, the accumulation of various chemical products created via an enzymatic reaction linked to the probe can be measured, for instance, by measuring absorbance of light at a particular wavelength. In other embodiments, amplification reactions can be quantified directly by blotting them onto a solid support and hybridizing with a detectably-labeled nucleic acid probe. Once unbound probe is washed away, the amount of probe can be quantified by measuring radioactivity as is known to those of skill in the art. Other variations of this technique employ the use of chemiluminescence to detect hybridization events.
Measurement of amplification products can be performed after the reaction has been completed or can be measured in “real time” (i.e., as the reaction occurs). If measurement of accumulated amplified product is performed after amplification is complete, then detection reagents (e.g. probes) can be added after the amplification reaction. Alternatively, probes can be added to the reaction prior or during the amplification reaction, thus allowing for measurement of the amplified products either after completion of amplification or in real time. Real time measurements can be particularly useful because they allow for measurement at any given cycle of the reaction and thus provide more information about accumulation of products throughout the reaction. For measurement of amplification product in real time, fluorescent probes are often used.
One amplification assay utilizing a FRET pair to detect an amplification product is the “TaqMan®” assay described in Gelfand et al. U.S. Pat. No. 5,210,015, and Livak et al. U.S. Pat. No. 5,538,848. The probe is a single-stranded oligonucleotide labeled with a FRET pair. In a TaqMan® assay, a DNA polymerase releases single or multiple nucleotides by cleavage of the oligonucleotide probe when it is hybridized to a target strand. That release provides a way to separate the quencher label and the fluorophore label of the FRET pair.
Another type of nucleic acid hybridization probe assay utilizing FRET pairs is described in Tyagi et al. U.S. Pat. No. 5,925,517, which utilizes labeled oligonucleotide probes, which are referred to as “molecular beacons.” See Tyagi, S. and Kramer, F. R., Nature Biotechnology 14: 303-308 (1996). A molecular beacon probe is an oligonucleotide whose end regions hybridize with one another in the absence of target but are separated if the central portion of the probe hybridizes to its target sequence. The rigidity of the probe-target hybrid precludes the simultaneous existence of both the probe-target hybrid and the intramolecular hybrid formed by the end regions. Consequently, the probe undergoes a conformational change in which the smaller hybrid formed by the end regions disassociates, and the end regions are separated from each other by the rigid probe-target hybrid. For molecular beacon probes, a central target-recognition sequence is flanked by arms that hybridize to one another when the probe is not hybridized to a target strand, forming a “hairpin” structure, in which the target-recognition sequence (which is commonly referred to as the “probe sequence”) is in the single-stranded loop of the hairpin structure, and the arm sequences form a double-stranded stem hybrid. When the probe hybridizes to a target, that is, when the target-recognition sequence hybridizes to a complementary target sequence, a relatively rigid helix is formed, causing the stem hybrid to unwind and forcing the arms apart.
One of skill will recognize that a large number of different fluorophores can be used to label probes useful in the invention. Some fluorophores useful in the methods and composition of the invention include: fluorescein, fluorescein isothiocyanate (FITC), carboxy tetrachloro fluorescein (TET),NHS-fluorescein, 5 and/or 6-carboxy fluorescein (FAM), 5- (or 6-) iodoacetamidofluorescein, 5-{[2(and 3)-5-(Acetylmercapto)-succinyl]amino}fluorescein (SAMSA-fluorescein), and other fluorscein derivatives, rhodamine, Lissamine rhodamine B sulfonyl chloride, Texas red sulfonyl chloride, 5 and/or 6 carboxy rhodarnine (ROX) and other rhodamine derivatives, coumarin, 7-amino-methyl-coumarin, 7-Amino-4-methylcoumarin-3-acetic acid (AMCA), and other coumarin derivatives, BODIPYTM fluorophores, Cascade BlueTM fluorophores such as 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt, Lucifer yellow fluorophores such as 3,6-Disulfonate-4-amino-naphthalimide, phycobiliproteins derivatives, Alexa fluor dyes (available from Molecular Probes, Eugene, Oreg.) and other fluorophores known to those of skill in the art. For a general listing of useful fluorophores, see Hermanson, G. T., BIOCONJUGATE TECHNIQUES (Academic Press, San Diego, 1996). Thus, each probe used in a reaction may fluoresce at a different wavelength and can be individually detected without interference from the other probes. This is useful, for example, if probes that detect different nucleotides at a particular position are used in a reaction. Thus, for example, one wavelength may indicate binding of a probe that detects 31T while a probe comprising a label with a different wavelength will detect 31C.
Preparing HBV from a Test Sample
The presence or amount of HBV nucleic acids in a test sample can be determined by amplifying the target regions within the HBV gene. Thus, any liquid or solid material believed to comprise HBV nucleic acids can be an appropriate sample. Preferred sample tissues include plasma, serum, whole blood, blood cells, lymphatic fluid, cerebral spinal fluid, synovial fluid and others.
As used herein, the term “test sample” refers to any liquid or solid material believed to comprise HBV nucleic acids. A test sample may be obtained from a biological source, such as cells in culture or a tissue sample from an animal, e.g., a human. Sample tissues of the instant invention may include, but are not limited to, plasma, serum, whole blood, blood cells, lymphatic fluid, cerebrospinal fluid, synovial fluid, urine, saliva, and skin or other organs (e.g. liver biopsy material).
Such sample will often be taken from patients suspected of having HBV infection, or having any of the wide spectrum of liver diseases related to HBV infection.
Nucleic acids representing the HBV gene of interest may be extracted from tissue samples. Various commercial nucleic acid purification kits, such as QIAmp 96 Virus BioRobot Kit and Qiagen's BioRobot 9604 are known to the skilled artisan, and used to isolate HBV nucleic acids from samples.
III. Determination of HBV Genotype
The present methods may also involve a determination of the genotype of HBV in an individual. For example, particular nucleotide variants identified herein may have a stronger predisposition to cause HCC if the variants are found in one genotype than in another. In this context, “genotype” refers to the at least 8 genotypes of HBV (genotypes A, B, C, D, E, F, G, and H) deduced from genome comparisons and designated genotypes A to H. See, e.g., Westland C. Hepatology 36: 2-8 (2002); Borchani-Chabchoub I, et al., Microbes Infect 2: 607-12 (2000); Grandjacques C, et al., J Hepatol 33: 430-9 (2000); Kato H, et al., J Virol Methods 98: 153-9 (2001); Ashton-Rickardt P G, et al., J Med Virol 29: 204-14 (1989). Thus, by detecting nucleotides at particular positions identified to occur only in a specific genotype, one may determine the genotype of HBV. Of course, other methods such as serological methods may also be used.
In some embodiments, the presence or absence of the B or C genotype of HBV will be determined. In some embodiments, genotype B comprises 2733C, 1856C, 1009T and 2892T. Further, the subtype of genotype may also be determined. For example, in some embodiments, subtype C1 is characterized by 2733A, 1856C, T1099T and 2892T. In some embodiments, subtype C2 is identified by 2733C, 1856T, 1009T and 2892T. In some embodiments, subtype C3 is identified by 2733C, 1856C, 1009C and 2892T. The details are showed in the table below:















2733
1856
1009
2892







B
C
C
T
T


C1
A
C
T
T


C2
C
T
T
T


C3
C
C
C
T









Detection of the nucleotides associated with a particular genotype may be detected by any method useful for detecting nucleotide sequences, including all of those described herein (e.g., amplification, nucleotide sequencing and/or probes, etc.).
IV. Comparing Nucleotides of HBV with Nucleotides Associated with HCC
Nucleotide sequence information regarding an isolate from an individual may be compared to nucleotides associated with HCC by any method.
Where a nucleotide sequence of the isolate is determined, the sequence may be aligned with SEQ ID NO:1 or another HBV genomic sequence to determine the position of the specific nucleotides of interest. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).
An example of algorithm that is suitable for aligning sequences and determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 may be used, with the parameters described herein, to determine an optimal alignment. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands and it is generally useful to turn off the complexity filter.
Positions of nucleotides of interest are provided throughout this application with reference to the first C of the first EcoR1 cleavage site (GAACTCC) that generally occur in the HBV genome. The first “C” is position 1 of SEQ ID NO:1. Thus, following alignment of a sequence of interest with SEQ ID NO:1, a particular nucleotide of the sequence of interest may be assigned a position relative to the corresponding position in the alignment with SEQ ID NO:1.
The presence of any of the following nucleotides is indicative of a pre-disposition for HCC: 31C, 53C, 312C, 799G, 961G, 1165T, 1499G, 1613A, 1762T, 1764A, 1899A, 2170C, 2170G, 2441C, 2525C, 2712C, 2712A, or 2712G. While those of skill in the art will recognize that any number of algorithms may be useful for predicting a predisposition for developing HCC, as described in the Example, particularly good sensitivity and specificity may be obtained using the following algorithm:
For genotype B HBV, the presence of:
1762T and 1764A and 2712A; or
1762T and 1764A and 2712C or;
1762T and 1764A and 2712G; or
1762T and 1764A and 2712T and 2525C; or
1762A and 1764G and 1165T, indicates a pre-disposition for HCC.
For genotype C1 HBV, the presence of:
31C; and/or
53C; and/or
1499G, indicates a pre-disposition for HCC.
For genotype C2 HBV, the presence of:
2170C; and/or
2170G; and/or
2441C; and/or
799G, indicates a pre-disposition for HCC.
For genotype C3 HBV, the presence of:
312C; and/or
961G; and/or
1613A; and/or
1899A, indicates a pre-disposition for HCC.
In some embodiments of the invention, it is useful to apply the above-listed algorithms in a computer readable form. The code for performing any of the functions described herein can be executed by the digital computers and may be stored on any suitable computer readable media. Examples of computer readable media include magnetic, electronic, or optical disks, tapes, sticks, chips, etc. The code for performing any of the functions described herein may also be written in any suitable computer programming language including, for example, Fortran, C, C++, etc. The graphical user interfaces and functions underlying the graphical user interfaces can be created using an object oriented programming language such as Java.
V. Benefits of Identifying Individuals Pre-Disposed for HCC
The conventional methods of surveillance for HCC are testing an infected person's serum alfa-fetoprotein levels (see, e.g., Liaw Y F et al., Gastroenterology, 30:263-267 (1986); Colombo M. et al., N. Engl. J. Med., 325:675-680 (1991); Oka H. et al., Hepatology 12:680-687 (1990) or by subjecting the person to abdominal ultrasound scanning. Another method for diagnosis of HCC is detecting des-gamma-carboxy prothrombin (Chan C Y et al. J Hepatol. 13:21-24 (1991); Weitz I C et al., Hepatology 18:990-997 (1993)). Another marker for HCC is TGF-1β. See, e.g., U.S. patent Publication No.2004/0121414.
However, without information regarding which patients may be pre-disposed for HCC, it is necessary to screen every person infected with HBV on a regular basis to catch HCC as early as possible. Unfortunately, given the large number of people infected with HBV, as well as the finite resources available to screen individuals, it is impossible to perform all of the necessary screens. The present invention addresses this problem, by indicating which individuals should have intense surveillance for the initial signs of HCC and which individuals do not require such intense surveillance. Thus, the present invention provides for detecting those individuals carrying HBV that is pre-disposed to cause HCC and then further testing those individuals on a regular basis for the presence of HCC and optionally, only rarely or never testing those individuals lacking HCC-associated HBV variants.
VI. Kits
Kits comprising the components needed in the methods (typically in an unmixed form) and kit components (packaging materials, instructions for using the components and/or the methods, one or more containers (reaction tubes, columns, etc.)) for holding the components are a feature of the present invention. Kits of the present invention may contain reagents for detecting any one or more of the following nucleotide variants in an HBV genome: 31C, 53C, 312C, 799G, 961G, 1165T, 1499G, 1613A, 1762T, 1764A, 1899A, 2170C, 2170G, 2441C, 2525C, 2712C, 2712A, and/or 2712G. For example, the kits of the invention may comprise combinations of primers and/or probes as described herein for the detection of nucleotide variants associated with HCC. Optionally, the kits may contain reagents for amplification, including but not limited to, thermostable polymerases such as Taq polymerase, nucleotides, buffers, etc.
EXAMPLE
Our goal was to discover genetic markers of HCC cases from HBV DNA sequences. In other words, we built up a classification model based on HBV DNA to predict cancer. Several classification models including Naive Bayes, Decision Tree, Neural Networks, and Rule Learning Using Evolutionary Algorithm, have been applied to classify the DNA datasets. The experimental results showed that the Rule Learning Using Evolutionary Algorithm has the best performance. In this section, we present the results of applying the Rule Learning Using Evolutionary Algorithm to classify the HBV DNA data in liver cancer (HCC) and normal cases.
Experimental Methodology
For each experiment, 90% of samples are selected randomly as the training set and the remains 10% samples form the testing set. For each dataset, the experiment is repeated for 10 times.
In medical diagnosis and disease predication problems, the algorithm or model performance is not only judged by accuracy, but also sensitivity and specificity. Sensitivity is generally more important than specificity and accuracy in medical diagnoses because doctors and patients prefer not to miss any patients with diseases. Extra diagnosis and tests can be performed to confirm their prediction and remove initial false positives.
We evaluated our model in all these three measurements.



  
    Accuracy
    =
    
      
        
          True
          ⁢
          
            
          
          ⁢
          Positive
        
        +
        
          True
          ⁢
          
            
          
          ⁢
          Negative
        
      
      
        
          True
          ⁢
          
            
          
          ⁢
          Positive
        
        +
        
          True
          ⁢
          
            
          
          ⁢
          Negative
        
      
    
  




  
    Sensitivity
    =
    
      
        True
        ⁢
        
          
        
        ⁢
        Positive
      
      
        
          True
          ⁢
          
            
          
          ⁢
          Positive
        
        +
        
          False
          ⁢
          
            
          
          ⁢
          Negative
        
      
    
  




  
    Specificity
    =
    
      
        True
        ⁢
        
          
        
        ⁢
        Negative
      
      
        
          True
          ⁢
          
            
          
          ⁢
          Negative
        
        +
        
          False
          ⁢
          
            
          
          ⁢
          Positive
        
      
    
  



The true positive is the number of all the patients with the disease and a positive test result, whereas the true negative is the number of all the patients without the disease and a negative test result. The false positive is the number of all the patients without the disease but a positive test result, whereas the false negative is the number of all the patients with the disease but a negative test result. In medical diagnosis, a false negative is the most undesirable case.
Results
Data Description
Genotype B and genotype C data were separated for analysis. The proportion of patients in each genotype or C subtypes is shown in Table 2. “CON” refers to “control,” i.e., no HCC.













TABLE 2







Datasets
CON
HCC
Total
%






















B
49
37
86
43.8776



C1
10
16
26
13.2653



C2
18
22
40
20.4082



C3
19
25
44
22.4490



Total
96
100
196
100










Genotype B

Table 3 shows the details of the markers for HBV genotype B.







TABLE 3







HBV markers for HCC of genotype B









Markers
Normal value
HCC-related value





1762, 1764
AG
TA


1165
C
T


2712
T
C (A, G)


2525
A, T
C









The classification rules based on the applied data cleansing process for genotype B are as follows: If 1762A and 1764G and 1165T are present in genotype B, then HCC is likely to occur. If 1762T and 1764A and 2712A, 2712C or 2712G are present in genotype B, then HCC is likely to occur. If 1762T and 1764A and 2712T and 2525C are present in genotype B, then HCC is likely to occur.
The experimental results for the genotype B dataset are shown in Table 4.







TABLE 4







Results of genotype B HBV dataset to predict HCC











Results
Training set (STD)
Testing set (STD)







Sensitivity
0.75029 (0.05361)
0.75 (0.16667)



Specificity
  0.68 (0.06215)
0.66 (0.13499)



Accuracy
 0.7093 (0.02615)
0.70 (0.07499)










C1 Subgroup

Table 5 shows the details of the markers for C1 subgroup.







TABLE 5







HCC related markers for C1 subgroup









Markers
Normal value
HCC-related value












31
T
C


53
T
C


1499
A
G









The classification rules based on the applied data cleansing process for C1 subgroup are as follows: If 31C or 53C or 1499G are present in genotype C1, then HCC is likely to occur.
The Experimental results for the C1 subgroup are showed in Table 6.







TABLE 6







Results of genotype C1 HBV dataset to predict HCC











Results
Training set (STD)
Testing set (STD)







Sensitivity
0.80769 (0.04054)
0.75 (0.26252)



Specificity
 0.7875 (0.06038)
 0.7 (0.48305)



Accuracy
  0.8 (0.03012)
0.7333 (0.21082) 










C2 Subgroup

Table 7 shows the details of the markers for C2 subgroup.







TABLE 7







HCC related markers for C2 subgroup









Markers
Normal value
HCC-related value












2170
T
C, G


2441
T
C


799
A
G









The classification rules based on the applied data cleansing process for C2 subgroup are as follows: If 2170C or 2170G or 2441C or 799G are present in genotype C2, then HCC is likely to occur.
The Experimental results on the C2 subgroup are showed in Table 8.







TABLE 8







Results of C2 genotype dataset to predict HCC











Results
Training set (STD)
Testing set (STD)







Sensitivity
 0.84706 (0.06323)
0.85 (0.24152)



Specificity
0.97857 (0.0345)
  1 (0.00000)



Accuracy
0.90645 (0.0355)
0.925 (0.12076) 










The classification rules based on the applied data cleansing process for C3 subgroup are as follows: If C312 or G961 or A1613 or A1899 are present in genotype C3, then HCC is likely to occur.
The Experimental results on the C3 subgroup are showed in Table 9.







TABLE 9







Results of C3 genotype dataset to predict HCC











Results
Training set (STD)
Testing set (STD)







Sensitivity
0.75 (0.0044)
0.77 (0.22)



Specificity
0.81 (0.0040)
0.80 (0.26)



Accuracy
0.77 (0.0024)
0.78 (0.18)










Patients and Methods

Patients

Residual serum samples of one hundred chronic hepatitis B patients suffering from hepatocellular carcinoma (HCC) and one hundred age-matched control patients who had chronic hepatitis B but without hepatocellular carcinoma were studied. Consecutive patients with confirmed diagnosis of HCC who had positive HBsAg attending the Joint Hepatoma Clinic, Prince of Wales Hospital from July 1999 to 2001 were included. Confirmed diagnosis of HCC is defined by either histology or radiological evidence of a hepatic mass with a serum alpha-fetoprotein (AFP) of 500 μg/l or more. Patients who had positive anti-HCV or history of alcoholism were excluded. Informed consent to provide serum sample for experimental study were routinely obtained from patients in Joint Hepatoma Clinic. Relevant clinical information of enrolled patients was collected retrospectively.
Age-matched control patients were identified from the cohort of chronic hepatitis B patients prospectively follow-up in the Hepatitis Clinic since December 1997. Patients who had other possible causes of hepatitis or liver cirrhosis including autoimmune liver disease, primary biliary cirrhosis, Wilson's disease and hemochromatosis were also excluded. At initial presentation, abdomen ultrasounds were performed to exclude any pre-existing HCC. Patients were prospectively followed up every 6 monthly, or more frequently if clinically indicated, with monitoring of liver biochemistry, HBeAg and anti-HBe status as well as alfa-fetoprotein levels. Abdomenal ultrasounds, computerized tomography, hepatic angiogram and/or liver biopsy were performed whenever alfa-fetoprotein level was higher than 50 μg/l or on a rising trend over 20 μg/l to confirm the diagnosis of HCC. For patients with normal alfa-fetoprotein levels, ultrasound abdomen was performed every 1-2 yearly.
Laboratory Method
Extraction Of DNA
Serum viral DNA was extracted using QIAamp DNA Blood Mini Kit (Qiagen, CA, USA) according to the manufacturer's instructions.
Amplification of HBV DNA
To obtain the full-length HBV DNA sequence, a long distance semi-nested PCR was performed to amplify three overlapping fragments (A, B and C). Relative positions of these PCR fragments to the map of HBV genome are shown in FIG. 1 and the nucleotide sequences of the PCR primers can be found in Table 1.








TABLE 1







The sequences of primers used for amplifying



and sequencing the HBV DNA












Nucleotide





Name
sequence (5′→3′)
Nt positions
Direction










Primers used for PCR (SEQ ID NOS:4-12)











P1
TTTTTCACCTCTGCCTAATCA
1821-1841
sense



P2
CCCTAGAAAATTGAGAGAAGTC
262-283
antisense


p3a
CCACTGCATGGCCTGAGGATG
3193-3213
antisense


P4
GCCTCATTTTGTGGGTCACCATA
2801-2824
sense


P5
TTCTTTGACATACTTTCCA
979-997
antisense


P6a
TTGGGGTGGAGCCCTCAGGCT
3070-3090
sense


P7a
TTGGCCAAAATTCGCAGTC
300-318
sense


P8a
CCCCACTGTTTGGCTTTCAG
714-734
sense


P9a
GTTGATAAGATAGGGGCATTTGG
2299-2325
antisense



TGG


 







Primers used for sequencing (SEQ ID NOS:13-21)











S1
CTCCGGAACATTGTTCACCT
2031-2050
sense



S2
AAGGTGGGAAACTTTACTGGGC
2469-2490
sense


S3
GCTGACGCAACCCCCACTGG
1186-1205
sense


S4
TCGCATGGAGACCACCGTGA
1604-1623
sense


S5
GGCAAAAACGAGAGTAACTC
1940-1959
antisense


S6
GGGTCGTCCGCGGGATTCAG
1441-1460
antisense


S7
GACATACTTTCCAATCAATAGG
970-991
antisense


S8
GAAGATGAGGCATAGCAGCAGG
411-433
antisense


S9
CATGCTGTAGCTCTTGTTCC
2831-2850
antisense





aThese primers were also used for sequencing.






Fragment A

When amplifying fragment A, 5 μl of the extracted DNA was subjected to PCR in the presence of 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl, 200 μM of each dNTP, 1.25 units Taq DNA polymerase (Amersham Biosciences), 1.5 units pfu DNA polymerase (Promega), and 10 pmol of each P1 primer and P2 primer in a final volume of 50 μl. PCR was carried out under a 5-min initial denaturation at 95° C., followed by 10 cycles of amplification (94° C., 36 sec; 60° C, 36 sec; 72° C, 2.5 min) and then 30 cycles of amplification (94° C., 36 sec; 50° C, 36 sec; 72° C, 2.5 min) and 7-min final extension at 72° C.
The PCR product was further amplified in a semi-nested PCR. One microliter of the product was subjected to PCR in the presence of 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl, 200 μM of each dNTP, 2.5 units Taq DNA polymerase (Amersham Biosciences) and 10 pmol of each P1 primer and P3 primer in a final volume of 50 μl. PCR was carried out under a 5-min initial denaturation at 95° C., followed by 10 cycles of amplification (94° C., 36 sec; 60° C., 36 sec; 72° C., 2 min) and then 30 cycles of amplification (94° C., 36 sec; 52° C., 36 sec; 72° C., 2 min) and a 7-min final extension at 72° C. Finally, quality and quantity of the PCR product was examined on a 1.0% agarose/EtBr gel run in 1× TBE buffer.
Fragment B
When amplifying fragment B, 5 μl of the extracted DNA was subjected to PCR in the presence of 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl, 200 μM of each dNTP, 1.25 units Taq DNA polymerase (Amersham Biosciences), 1.5 units pfu DNA polymerase (Promega), and 10 pmol of each P4 primer and P5 primer in a final volume of 50 μl. PCR was carried out under a 5-min initial denaturation at 95° C., followed by 10 cycles of amplification (94° C., 36 sec; 60° C., 36 sec; 72° C., 90 sec) and then 30 cycles of amplification (94° C., 36 sec; 50° C., 36 sec; 72° C., 90 sec) and a 7-min final extension at 72° C.
The PCR product was further amplified in a semi-nested PCR. One microliter of the product was subjected to PCR in the presence of 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl, 200 μM of each dNTP, 2.5 units Taq DNA polymerase (Amersham Biosciences) and 10 pmol of each P5 primer and P6 primer in a final volume of 50 μl. PCR was carried out under a 5-min initial denaturation at 95° C., followed by 10 cycles of amplification (94° C, 36 sec; 60° C, 36 sec; 72° C, 90 sec) and then 30 cycles of amplification (94° C., 36 sec; 52° C., 36 sec; 72° C., 90 sec) and a 7-min final extension at 72° C. Finally, quality and quantity of the PCR product was examined on a 1.0% agarose/EtBr gel run in 1× TBE buffer.
Fragment C
When amplifying fragment C, 5 μl of the extracted DNA was subjected to PCR in the presence of 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl, 200 μM of each dNTP, 1.25 units Taq DNA polymerase (Amersham Biosciences), 1.5 units pfu DNA polymerase (Promega), and 10 pmol of each P7 primer and P9 primer in a final volume of 50 μl. PCR was carried out under a 5-min initial denaturation at 95° C., followed by 10 cycles of amplification (94° C., 36 sec; 60° C., 36 sec; 72° C., 2 min and 15 sec) and then 30 cycles of amplification (94° C., 36 sec; 50° C., 36 sec; 72° C., 2 min and 15 sec) and a 7-min final extension at 72° C.
The PCR product was further amplified in a semi-nested PCR. One microliter of the product was subjected to PCR in the presence of 50 mM KCl, 1.5 mM MgCl2, 10 mM Tris-HCl, 200 μM of each dNTP, 2.5 units Taq DNA polymerase (Amersham Biosciences) and 10 pmol of each P8 primer and P9 primer in a final volume of 50 μl. PCR was carried out under a 5-min initial denaturation at 95° C., followed by 10 cycles of amplification (94° C., 36 sec; 60° C., 36 sec; 72° C., 1 min and 50 sec) and then 30 cycles of amplification (94° C., 36 sec; 52° C., 36 sec; 72° C., 1 min and 50 sec) and a 7-min final extension at 72° C. Finally, quality and quantity of the PCR product was examined on a 1.0% agarose/EtBr gel run in 1× TBE buffer.
DNA Sequencing
All semi-nested PCR products (plus and minus strands) were directly sequenced with the Cycling Sequencing Kit DYEnamic ET Dye terminator for MegaBACE (Amersham Biosciences).
Primers for the sequencing of three HBV DNA fragments (primers sequences are listed in Table 1):

    Fragment A: S1, S2, P3, S9
    Fragment B: P6, P7, S7, S8
    Fragment C: P8, S3, S4, P9, S5, S6


One microliter of unpurified PCR product was used as the DNA template for cycle sequencing. It was subjected to sequencing reaction in the presence of 8 μl of DYEnamic ET reagent premix and 10 pmol primer in a final volume of 20 μl. Sequencing reaction mix was subjected to a 2 min initial denaturation at 95° C., followed by 30 cycles at 95° C., 25 sec; 52° C., 30 sec; 60° C.; 60 sec.
The sequencing products were purified by post reaction clean up using ethanol precipitation. In each reaction tube, 2 μl of 7.5M ammonium acetate and 2.5 volumes (55 μl) of 100% ethanol were added so that the final concentration of ethanol was 70%. Then it was subjected to centrifugation at 4,000 rpm for 30 min at 14° C. Afterwards, the supernatant was drawn off by performing a brief inverted spin (1 min at 500 rpm). The DNA pellet was washed by 100 μl of 70% ethanol. Then, it was subjected to centrifugation at 4,000 rpm for 15 min at 14° C. and the supernatant was drawn off by performing a brief inverted spin (1 min at 500 rpm). Then the DNA pellet was allowed to air dry and was resuspended in 10 μl of loading buffer (70% formamide and 1 mM EDTA). The samples were stored at 4° C. before gel electrophoresis analysis using the MegaBACE 1000 DNA sequencer.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

## Claims

1. A method of determining a pre-disposition of an individual infected with hepatitis B virus (HBV) to develop hepatocellular carcinoma (HCC), the method comprising:
determining nucleotides in the genome of an HBV isolated from the individual at positions corresponding to nucleotides 31, 53, 1165, 1499, 1899, 2170, 2441, 2525, and 2712 of SEQ ID NO:1; and
determining that the HBV genome does have 31C, 53C, 1165T, 1499G, 1899A, 2170C, 2170G, 2441C, 2525C, 2712C, 2712A, and/or 2712G, wherein if the HBV has one of the nucleotides, the individual has a pre-disposition to develop HCC.

2. The method of claim 1, the method further comprising
determining nucleotides in the genome of a genotype B HBV isolated from the individual at positions corresponding to nucleotides 1762 or 1764 of SEQ ID NO:1; and
comparing the determined nucleotides to nucleotides associated with a pre-disposition to cause HCC, wherein the nucleotides associated with a pre-disposition to cause HCC comprise: 1762T or 1764A.

3. The method of claim 1, the method further comprising
determining nucleotides in the genome of a genotype B HBV isolated from the individual at positions corresponding to nucleotides 1762 or 1764 of SEQ ID NO:1; and
comparing the determined nucleotides to nucleotides associated with a pre-disposition to cause HCC, wherein the nucleotides associated with a pre-disposition to cause HCC in genotype B comprise:
1762T and 1764A and 2712A; or
1762T and 1764A and 2712C; or
1762T and 1764A and 2712G; or
1762T and 1764A and 2712T and 2525C; or
1762A and 1764G and 1165T.

4. The method of claim 3, wherein the determining step comprises nucleotide sequencing the HBV genome flanking the nucleotides at positions corresponding to nucleotides 1165, 2525 and 2712 of SEQ ID NO:1.

5. The method of claim 3, wherein the determining step comprises amplifying at least a portion of the HBV genome to produce one or more amplification products comprising the nucleotides at the positions corresponding to nucleotides 1165, 2525 and 2712 of SEQ ID NO:1.

6. The method of claim 5, comprising contacting the one or more amplification products with one or more probes that hybridize to HCC-associated nucleotides:
2712A; or
2712C or;
2712G; or
2712T and 2525C; or
1165T;
under conditions to allow for hybridization of a probe to an amplification product only if the amplification product comprises a complementary nucleotide at the position of the HCC-associated nucleotide.

7. The method of claim 6, wherein the hybridization is performed as a line probe assay.

8. The method of claim 3, further comprising determining the genotype of the HBV from the individual.

9. The method of claim 1, the method comprising
determining nucleotides in the genome of a genotype C HBV isolated from the individual at positions corresponding to nucleotides 31, 53, 1499, 2170, and 2441; and
comparing the determined nucleotides to nucleotides associated with a pre-disposition to cause HCC, wherein the nucleotides associated with a pre-disposition to cause HCC comprise: 31C, 53C, 1499G, 2170C, 2170G, or 2441C.

10. The method of claim 9, the method comprising
a) determining the subtype of a genotype C HBV from the individual, wherein:
subtype C1 comprises nucleotides 2733A, 1856C, 1009T and 2892T, subtype C2 comprises nucleotides 2733C, 1856T, 1009T and 2892T, and subtype C3 comprises nucleotides 2733C, 1856C, 1009C and 2892T;
b1) if the HBV is genotype C1, determining the nucleotides at positions corresponding to nucleotides 31, 53 and 1499 of SEQ ID NO:1; or
b2) if the HBV is genotype C2, determining the nucleotides at positions corresponding to nucleotides 2441 and 2170 of SEQ ID NO:1; and
b3) if the HBV is genotype C3, determining the nucleotides at positions corresponding to nucleotides 1899 of SEQ ID NO:1; and
c) comparing the determined nucleotides to nucleotides at the positions associated with a pre-disposition to cause HCC, wherein the nucleotides associated with a pre-disposition to cause HCC in subtype C1 comprise:
31C; and/or
53C; and/or
1499G; and
the nucleotides associated with a pre-disposition to cause HCC in subtype C2 comprise:
2170C; and/or
2170G; and/or
2441C; and
the nucleotides associated with a pre-disposition to cause HCC in subtype C3 comprise:
1899A.

11. The method of claim 10, wherein the determining step comprises nucleotide sequencing the HBV genome flanking the nucleotides at positions corresponding to nucleotides 31, 53, and 1499 of SEQ ID NO:1.

12. The method of claim 10, wherein the determining step comprises nucleotide sequencing the HBV genome flanking the nucleotides at positions corresponding to nucleotides 2441, and 2170 of SEQ ID NO:1.

13. The method of claim 10, wherein the determining step comprises nucleotide sequencing the HBV genome flanking the nucleotides at positions corresponding to nucleotides 1899 of SEQ ID NO:1.

14. The method of claim 10, wherein the determining step comprises amplifying at least a portion of the HBV genome to produce one or more amplification products comprising the nucleotides at the positions corresponding to nucleotides 31, 53, and 1499 of SEQ ID NO:1.

15. The method of claim 10, wherein the determining step comprises amplifying at least a portion of the HBV genome to produce one or more amplification products comprising the nucleotides at the positions corresponding to nucleotides 2441, and 2170 of SEQ ID NO:1.

16. The method of claim 10, wherein the determining step comprises amplifying at least a portion of the HBV genome to produce one or more amplification products comprising the nucleotides at the positions corresponding to nucleotides 1899 of SEQ ID NO:1.

17. The method of claim 14, comprising contacting the one or more amplification products with one or more probes that hybridize to HCC-associated nucleotides:
31C; and/or
53C; and/or
1499G;
under conditions to allow for hybridization of a probe to an amplification product only if the amplification product comprises a complementary nucleotide at the position of the HCC-associated nucleotide.

18. The method of claim 17, wherein the hybridization is performed as a line probe assay.

19. The method of claim 14, comprising contacting the one or more amplification products with probes that hybridize to HCC-associated nucleotides:
2170C; and/or
2170G; and/or
2441C;
under conditions to allow for hybridization of the probes to the amplification product only if the amplification product comprises a complementary nucleotide at the position of the HCC-associated nucleotide.

20. The method of claim 19, wherein the hybridization is performed as a line assay.

21. The method of claim 14, comprising contacting the one or more amplification products with probes that hybridize to HCC-associated nucleotides:
1899A;
under conditions to allow for hybridization of the probes to the amplification product only if the amplification product comprises a complementary nucleotide at the position of the HCC-associated nucleotide.

22. The method of claim 21, wherein the hybridization is performed as a line assay.

23. The method of claim 10, further comprising determining the genotype of the HBV from the individual.