Dna methylation in colorectal and breast cancer diagnostic methods

Abstract

The present invention relates generally to nucleic acid molecules in respect of which changes to DNA methylation levels are indicative of the onset or predisposition to the onset of a neoplasm. More particularly, the present invention is directed to nucleic acid molecules in respect of which changes to DNA methylation levels are indicative of the onset and/or progression of a large intestine or breast neoplasm, such as an adenoma or adenocarcinoma. The DNA methylation status of the present invention is useful in a range of applications including, but not limited to, those relating to the diagnosis and/or monitoring of colorectal or breast neoplasms, such as colorectal or breast adenocarcinomas. Accordingly, in a related aspect the present invention is directed to a method of screening for the onset, predisposition to the onset and/or progression of a neoplasm by screening for modulation in DNA methylation of one or more nucleic acid molecules. The nucleic acid molecules used for diagnostics in the present invention are sequences from LOC 100526820, subsequently named CAHM (colorectal adenocarcinoma hypermethylated).

1 . (canceled) 
     
     
         2 . A method of detecting the onset, or predisposition to the onset, of a large intestine neoplasm in a human subject, said method comprising:
 contacting deoxy ribonucleic acid (DNA) from a human subject with a bisulfite salt, thereby generating bisulfite converted DNA, wherein said DNA from said human subject comprises a nucleic acid having a sequence corresponding to SEQ ID NO: 17, or the complement thereof;   amplifying the bisulfite converted DNA;   measuring the amount of CpG methylation in the bisulfite converted DNA; and   determining whether a greater amount of CpG methylation in the bisulfite converted DNA as compared to a control is present, wherein when a greater amount of CpG methylation in the bisulfite converted DNA as compared to the control is present, the onset, or predisposition to the onset, of a large intestine neoplasm in the human subject is detected.   
     
     
         3 . The method of  claim 2 , wherein the amplifying of the bisulfite converted DNA comprises contacting the bisulfite converted DNA with a primer set comprising forward and reverse primers configured to amplify a region of the bisulfite converted DNA. 
     
     
         4 . The method of  claim 3 , wherein the region of the bisulfite converted DNA comprises the sequence as set forth in SEQ ID NO: 1 or the complement thereof. 
     
     
         5 . The method of  claim 4 , wherein the forward primer has a sequence as set forth in SEQ ID NO: 18, and wherein the reverse primer has a sequence as set forth in SEQ ID NO: 19. 
     
     
         6 . The method of  claim 3 , wherein the region of the bisulfite converted DNA comprises the sequence as set forth in SEQ ID NO: 3 or the complement thereof. 
     
     
         7 . The method of  claim 6 , wherein the forward primer has a sequence as set forth in SEQ ID NO: 13, and wherein the reverse primer has a sequence as set forth in SEQ ID NO: 14. 
     
     
         8 . The method of  claim 3 , wherein the region of the bisulfite converted DNA comprises the sequence as set forth in SEQ ID NO: 4 or the complement thereof. 
     
     
         9 . The method of  claim 8 , wherein the forward primer has a sequence as set forth in SEQ ID NO: 13, and wherein the reverse primer has a sequence as set forth in SEQ ID NO: 15. 
     
     
         10 . The method of  claim 2 , wherein the DNA is free circulating plasma DNA from a blood sample. 
     
     
         11 . The method of  claim 2 , wherein when the onset, or predisposition to the onset, of a large intestine neoplasm is detected in said human subject, one or more of the following procedures is performed on said human subject:
 (a) digital rectal exam;   (b) faecal occult blood test;   (c) sigmoidoscopy or colonoscopy;   (d) double contrast barium enema X-ray;   (e) virtual colonoscopy;   (f) computed axial tomography scan; or   (g) positron emission tomography.   
     
     
         12 . The method of  claim 8 , wherein the measuring of the amount of CpG methylation in the bisulfite converted DNA comprises:
 (i) methylation-specific PCR;   (ii) the MethyLight assay;   (iii) methylation-sensitive single nucleotide primer extension;   (iv) methylated CpG island amplification;   (v) the HeavyMethyl assay;   (vi) Headloop PCR;   (vii) the Helper-dependent chain reaction;   (viii) pyrosequencing; or   (ix) Melting curve analysis.   
     
     
         13 . The method of  claim 2 , wherein the large intestine neoplasm is an adenoma, an adenocarcinoma, or a colorectal neoplasm. 
     
     
         14 . The method of  claim 2 , wherein the control is the amount of methylation of CpG dinucleotides detected in amplified bisulfite converted DNA from a control human subject, which does not have a large intestine neoplasm, wherein DNA from said control human subject prior to bisulfite conversion comprises a nucleic acid having a sequence corresponding to SEQ ID NO: 17. 
     
     
         15 . A kit for assaying biological samples comprising one or more polynucleotides that hybridize to a deoxy ribonucleic acid (DNA) region defined by Hg19 coordinates Chr6: 163834097-163834982 and at least one reagent for detection of gene methylation. 
     
     
         16 . The kit according to  claim 15 , wherein said kit further comprises a compound that selectively mutates a non-methylated cytosine residue. 
     
     
         17 . The kit according to  claim 16 , comprising:
 (i) sodium bisulfite;   (ii) primers that hybridize to a DNA region defined by Hg19 coordinates Chr6: 163834097-163834982; and   (iii) detectably-labelled probes that distinguish between methylated and unmethylated DNA that has been treated with bisulfite.   
     
     
         18 . The kit according to  claim 15 , wherein said kit further comprises one or more control DNA sequences representing methylated or unmethylated forms of said DNA region. 
     
     
         19 . The kit according to  claim 18 , wherein said one or more control DNA sequences have a sequence corresponding to any one of SEQ ID NOs: 5, 6, 7, 8, 9, 10, 11 or 12, or a sequence exhibiting at least 95% identity to said sequences. 
     
     
         20 . The kit according to  claim 15 , wherein said kit comprises one or more amplification primer sets, wherein the primer sets comprise:
 (i) SEQ ID NOs:13 and 14 or a sequence exhibiting at least 95% identity to said sequences;   (ii) SEQ ID NOs:13, 14 and 15 or a sequence exhibiting at least 95% identity to said sequences;   (iii) SEQ ID NOs:18 and 19 or a sequence exhibiting at least 95% identity to said sequences; or   (iv) SEQ ID NOs:20 and 21 or a sequence exhibiting at least 95% identity to said sequences.   
     
     
         21 . An isolated nucleic acid molecule selected from the group consisting of:
 (i) an isolated nucleic acid molecule or complement thereof comprising a nucleotide sequence having at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more identity over the full length of any one of SEQ ID NO:5-12; and   (ii) an isolated nucleic acid molecule or complement thereof comprising a nucleotide sequence corresponding to any one of SEQ ID NO: 5-12.