Methods and compositions for detecting colon cancers

Abstract

This application describes methods and compositions for detecting and treating HLTF-associated neoplasia. Differential methylation of the HLTF nucleotide sequences has been observed in HLTF-associated neoplasia such as colon neoplasia.

1 - 34 . (canceled) 
     
     
         35 . A bisulfite-converted methylated HLTF nucleotide sequence selected from the group consisting of:
 a nucleotide sequence of any one of SEQ ID NOs: 5-8 or a fragment thereof;   a complement of any one of SEQ ID Nos: 5-8 or a fragment thereof; and   a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of any one of SEQ ID NOs: 5-8.   
     
     
         36 . A bisulfite-converted methylated HLTF nucleotide sequence selected from the group consisting of:
 a nucleotide sequence of any one of SEQ ID NOs: 22-25 or a fragment thereof;   a complement of any one of SEQ ID Nos: 22-25 or a fragment thereof; and   a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of any one of SEQ ID NOs: 22-25.   
     
     
         37 . The oligonucleotide primer of  claim 47 , wherein said primer has a sequence selected from the group consisting of SEQ ID NOs: 11-20, 26-38, and 40-43. 
     
     
         38 . A kit for detecting an HLTF-associated neoplasia in a subject, comprising at least two primers of  claim 37 . 
     
     
         39 . The kit of  claim 38 , further comprising a compound to convert a template DNA. 
     
     
         40 . The kit of  claim 39 , wherein the compound is bisulfite. 
     
     
         41 . The kit of  claim 40 , wherein each primer comprises at least a CpG dinucleotide. 
     
     
         42 . A method of converting a nucleic acid sequence at least 95% identical to and one of SEQ ID NOs: 4 and 21 to a bisulfite converted sequence comprising:
 a) providing a nucleotide acid having a nucleotide sequence as set forth in any one of SEQ ID NOs: 4 and 21; and   b) adding a bisulfite compound;   
       whereby the unmethylated cytosine bases of the CpG islands are converted to a different base. 
     
     
         43 . The method of  claim 42 , wherein the unmethylated cytosine is converted to a uracil. 
     
     
         44 . A nucleic acid sequence as prepared by the method of  claim 42 . 
     
     
         45 . An isolated or recombinant methylated HLTF nucleic acid, comprising a nucleotide sequence of any one of SEQ ID NOs: 2-4 and 21, wherein the cytosine of the CpG island is methylated. 
     
     
         46 . An isolated or recombinant HLTF nucleic acid, selected from the group consisting of:
 a nucleotide sequence of any one of SEQ ID NOs: 2-4 and 21 or a fragment thereof;   a complement of any one of SEQ ID Nos: 2-4 and 21;   a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of any one of SEQ ID NOs: 2-4 and 21;   a nucleotide sequence that is at least 98% identical to the nucleotide sequence of any one of SEQ ID NOs: 2-4 and 21;   an nucleotide sequence comprising at least 50 consecutive base pairs of any one SEQ ID Nos; 2-4 and 21;   
       wherein the HLTF nucleotide sequence is differentially methylated in an HLTF-associated disease cell. 
     
     
         47 . An oligonucleotide primer for assessing methylation status in the human HLTF gene, wherein said primer specifically hybridizes under high stringent conditions to a bisulfite-converted template sequence selected from the group consisting of SEQ ID NOs: 5-8, 22-25, and a complement of any one SEQ ID NOs: 22-25. 
     
     
         48 . The primer of  claim 47 , wherein the primer is a methylation-specific PCR primer having a sequence selected from the group consisting of SEQ ID NOs: 11, 12, 15, 17, 18, 26, 27, 30, 32, 33, 36, 37, 40, and 41. 
     
     
         49 . The primer of  claim 48 , wherein said primer is a forward primer selected from the group consisting of SEQ ID NOs: 11, 17, 26, 32, 36, and 40. 
     
     
         50 . The primer of  claim 49 , wherein said primer is a reverse primer selected from the group consisting of SEQ ID NOs: 12, 15, 18, 27, 30, 33, 37, and 41. 
     
     
         51 . The primer of  claim 47 , wherein the primer is an unmethylation-specific PCR primer having a sequence selected from the group consisting of SEQ ID NOs: 13, 14, 16, 19, 20, 28, 29, 31, 34, 35, 37, 38, 42, and 43. 
     
     
         52 . The primer of  claim 51 , wherein the primer is a forward primer selected from the group consisting of SEQ ID NOs: 13, 19, 28, 34, 38, and 42. 
     
     
         53 . The primer of  claim 51 , wherein the primer is a reverse primer selected from the group consisting of SEQ ID NOs: 14, 16, 20, 29, 31, 35, 37, and 43. 
     
     
         54 . The primer of  claim 47 , wherein said primer is further labeled with a detectable marker. 
     
     
         55 . The primer of  claim 54 , wherein said primer is labeled with a fluorescent dye. 
     
     
         56 . The kit of  claim 38 , wherein said kit comprises a forward primer selected from the group consisting of SEQ ID NOs: 11, 17, 26, 32, 36, and 40, and a reverse primer selected from the group consisting of SEQ ID NOs: 12, 15, 18, 27, 30, 33, 37, and 41. 
     
     
         57 . The kit of  claim 56 , wherein said kit further comprises a forward primer selected from the group consisting of SEQ ID NOs: 13, 19, 28, 34, 38, and 42, and a reverse primer selected from the group consisting of SEQ ID NOs: 14, 16, 20, 29, 31, 35, 37, and 43. 
     
     
         58 . An oligonucleotide primer for assessing methylation of the human HLTF gene, wherein the primer has a sequence selected from the group consisting of SEQ ID NOs: 9-10. 
     
     
         59 . A kit for detecting HLTF-associated neoplasia in a subject, comprising at least two primers of  claim 54 .