Genomic markers of hepatitis B virus associated with hepatocellular carcinomas

Abstract

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

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 HBV isolated from the individual at positions corresponding to nucleotides 31, 53, 799, 1165, 1499, 1762, 1764, 2170, 2441, 2525, and/or 2712 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: 31C, 53C, 799G, 1165T, 1499G, 1762T, 1764A, 2170C, 2170G, 2441C, 2525C, 2712C, 2712A, and/or 2712G.    
     
     
         2 . The method of  claim 1 , the method comprising 
 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    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.    
     
     
         3 . The method of  claim 3 , the method comprising 
 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    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, 1762, 1764, 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, 1762, 1764, 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: 
 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.    
     
     
         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, 799, 1499, 2170, or 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, 799G, 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 2783G and 2733A,    subtype C2 comprises nucleotides 2783G, 2733C and 3033A, and    subtype C3 comprises 2783G, 2733C and 3033C;    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    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.    
     
     
         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 799, 2441, and 2170 of SEQ ID NO:1.  
     
     
         13 . 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.  
     
     
         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 799, 2441, and 2170 of SEQ ID NO:1.  
     
     
         15 . The method of  claim 13 , 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.    
     
     
         16 . The method of  claim 15 , wherein the hybridization is performed as a line probe assay.  
     
     
         17 . The method of  claim 13 , comprising contacting the one or more amplification products with probes that hybridize to HCC-associated nucleotides: 
 2170C; and/or    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.    
     
     
         18 . The method of  claim 17 , wherein the hybridization is performed as a line assay.  
     
     
         19 . The method of  claim 10 , further comprising determining the genotype of the HBV from the individual.  
     
     
         20 . The method of  claim 1 , the method comprising 
 determining the genotype of the HBV, wherein genotype B comprises 2783A, wherein genotype C1 comprises 2783G and 2733A, genotype C2 comprises 2783G, 2733C and 3033A and genotype C3 comprises 2783G, 2733C and 3033C;    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    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;    thereby determining the pre-disposition of the individual to develop HCC.    
     
     
         21 . A kit for detecting HBV isolates that are associated with the development hepatocellular carcinoma (HCC), comprising 
 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, 799G, 1165T, 1499G, 1762T, 1762A, 1764A, 1764G, 2441C, 2170C, 2170G, 2712A; 2712C, 2712G; or 2525C.    
     
     
         22 . The kit of  claim 21 , wherein the probe is linked to a solid support.  
     
     
         23 . The kit of  claim 21 , wherein 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.    
     
     
         24 . The kit of  claim 21 , wherein the probe selectively hybridizes to: 
 31C; and    53C; and    1499G.    
     
     
         25 . The kit of  claim 21 , wherein the probe selectively hybridizes to: 
 2170C; and/or    2170G; and/or    2441C; and/or    799G.    
     
     
         26 . The kit of  claim 21 , further comprising primers for amplification of at least a portion of the HBV genome.  
     
     
         27 . A computer readable medium comprising, 
 a) code for receiving information describing: 
 nucleotides at positions corresponding to nucleotides 31, 53, 799, 1165, 1499, 1762, 1764, 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, 799G, 1165T, 1499G, 1762T, 1764A, 2170C, 2170G, 2441C, 2525C, 2712C, 2712A, or 2712G.