Methods and nucleic acids for analyses of cellular proliferative disorders

Abstract

Aspects of the invention provide methods, nucleic acids and kits for detecting, or for detecting and distinguishing between or among liver cell proliferative disorders or for detecting, or for detecting and distinguishing between or among colorectal cell proliferative disorders. Particular aspects disclose and provide genomic sequences the methylation patterns of which have substantial utility for the improved detection of and differentiation between said class of disorders, thereby enabling the improved diagnosis and treatment of patients.

What is claimed is: 
     
         1 . A method for detecting and/or classifying a colorectal carcinoma or pre-cancerous colorectal cell proliferative disorder in a human subject, comprising:
 contacting genomic DNA from a biological sample obtained from a human subject, having a colorectal carcinoma or pre-cancerous colorectal cell proliferative disorder, with at least one agent that provides for determination of a CpG methylation status of the TMEFF2 gene;   determining, based on the contacting, a CpG methylation status of the TMEFF2 gene; and   detecting and/or classifying a colorectal carcinoma or precancerous colorectal cell proliferative disorder in the subject based on increased CpG methylation of the TMEFF2 gene, relative to that of a control sample or standard value.   
     
     
         2 . The method of  claim 1 , wherein the cell proliferative disorder is colorectal cancer. 
     
     
         3 . The method of  claim 1 , wherein the colorectal carcinoma is detected or classified. 
     
     
         4 . The method of  claim 1 , wherein contacting genomic DNA comprises contacting the genomic DNA with at least one reagent, or series of reagents that distinguishes between methylated and non-methylated CpG dinucleotides within at least one target region of the genomic DNA, wherein the target region comprises, or hybridizes under stringent conditions to a sequence of at least 16 contiguous nucleotides of SEQ ID NO: 26, wherein the contiguous nucleotides comprise at least one CpG dinucleotide sequence. 
     
     
         5 . The method of  claim 4 , comprising:
 extracting or otherwise isolating genomic DNA from the biological sample obtained from the subject;   treating the extracted or otherwise isolated genomic DNA, or a fragment thereof with one or more reagents to convert cytosine bases that are unmethylated in the 5-position thereof to uracil or to another base that is detectably dissimilar to cytosine in terms of hybridization properties;   contacting the treated genomic DNA, or the treated fragment thereof, with an amplification enzyme and at least one primer comprising, a contiguous sequence of at least 9 nucleotides that is complementary to, or hybridizes under moderately stringent or stringent conditions to a sequence selected from the group consisting of SEQ ID NOs: 34, 35, 46 and SEQ ID NO: 47, and complements thereof, wherein the treated genomic DNA or the fragment thereof is either amplified to produce at least one amplificate, or is not amplified; and   determining, based on a presence or absence of, or on a property of the amplificate, the methylation state or level of at least one CpG dinucleotide of SEQ ID NO: 26, or an average, or a value reflecting an average methylation state or level of a plurality of CpG dinucleotides of SEQ ID NO: 26.   
     
     
         6 . The method of  claim 4 , wherein treating the genomic DNA, or the fragment thereof, comprises use of a reagent selected from the group consisting of bisulfate, hydrogen sulfite, disulfite, and combinations thereof. 
     
     
         7 . The method of  claim 4 , comprising:
 extracting or otherwise isolating genomic DNA from the biological sample obtained from the subject;   digesting the extracted or otherwise isolated genomic DNA, or a fragment thereof, with at least one methylation sensitive restriction enzyme;   contacting the DNA restriction enzyme digest with an amplification enzyme and at least two primers suitable for the amplification of a sequence comprising at least one CpG dinucleotide of SEQ ID NO: 26; and   determining, based on a presence or absence of an amplificate, the methylation state or level of at least one CpG dinucleotide of SEQ ID NO: 26.   
     
     
         8 . The method of  claim 7 , wherein the presence or absence of an amplificate is determined by means of hybridization to at least one nucleic acid or peptide nucleic acid which is identical, complementary, or hybridizes under stringent or highly stringent conditions to an at least 16 base long segment of SEQ ID NO: 26.