Patent Publication Number: US-2022213559-A1

Title: Diagnostic gene marker panel for colorectal cancer

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation application of U.S. application Ser. No. 14/400,313, filed Nov. 10, 2014, which is a U.S. National Phase Application of PCT International Application Number PCT/AU2013/000481, filed May 10, 2013, designating the United States of America and published in the English language, which is an International Application of and claims the benefit of priority to U.S. Provisional Application No. 61/646,174, filed May 11, 2012, the disclosures of which are hereby expressly incorporated by reference in their entireties. 
    
    
     REFERENCE TO SEQUENCE LISTING 
     A Sequence Listing submitted as an ASCII text file via EFS-Web is hereby incorporated by reference in accordance with 35 U.S.C. § 1.52(e). 
     FIELD 
     The present invention relates generally to a method of screening for the onset, predisposition to the onset and/or progression of a neoplasm. More particularly, the present invention relates to a method of screening for the onset, predisposition to the onset and/or progression of a neoplasm by screening for changes to the methylation levels of a panel of gene markers. The method 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 neoplasms, such as colorectal adenocarcinosis. 
     BACKGROUND 
     Colorectal cancer includes cancerous growths in the colon, rectum and appendix. With 655,000 deaths worldwide per year, it is the fourth most common form of cancer in the United States and the third leading cause of cancer-related death in the Western world. Colorectal cancers arise from adenomatous polyps in the colon. These mushroom-shaped growths are usually benign, but some develop into cancer over time. Localized colon cancer is usually diagnosed through colonoscopy. 
     Invasive cancers that are confined within the wall of the colon (stages I and II) are curable with surgery. If untreated, they spread to regional lymph nodes (stage III), where up to 73% are curable by surgery and chemotherapy. Cancer that metastasizes to distant sites (stage IV) is usually not curable, although chemotherapy can extend survival, and in rare cases, surgery and chemotherapy together have seen patients through to a cure (Markowitz and Bertagnolli, 2009,  N. Engl. J. Med.  361(25): 2449-60). Radiation is used with rectal cancer. 
     Colorectal cancer is preceded by adenomas. Adenomas are benign tumors, or neoplasms, of epithelial origin which are derived from glandular tissue or exhibit clearly defined glandular structures. Some adenomas show recognizable tissue elements, such as fibrous tissue (fibroadenomas) and epithelial structure, while others, such as bronchial adenomas, produce active compounds that might give rise to clinical syndromes. 
     Adenomas may progress to become an invasive neoplasm and are then termed adenocarcinomas. Accordingly, adenocarcinomas are defined as malignant epithelial tumors arising from glandular structures, which are constituent parts of many organs of the body. The term adenocarcinoma is also applied to tumors showing a glandular growth pattern. These tumors may be sub-classified according to the substances that they produce, for example mucus secreting and serous adenocarcinomas, or to the microscopic arrangement of their cells into patterns, for example papillary and follicular adenocarcinomas. These carcinomas may be solid or cystic (cystadenocarcinomas). Each organ may produce tumors showing a variety of histological types, for example the ovary may produce both mucinous and cystadenocarcinoma. 
     Adenomas in different organs behave differently. In general, the overall chance of carcinoma being present within an adenoma (i.e. a focus of cancer having developed within a benign lesion) is approximately 5%. However, this is related to size of an adenoma. For instance, in the large bowel (colon and rectum specifically) occurrence of a cancer within an adenoma is rare in adenomas of less than 1 centimeter. Such a development is estimated at 40 to 50% in adenomas which are greater than 4 centimeters and show certain histopathological change such as villous change, or high-grade dysplasia. Adenomas with higher degrees of dysplasia have a higher incidence of carcinoma. In any given colorectal adenoma, the predictors of the presence of cancer now or the future occurrence of cancer in the organ include size (especially greater than 9mm) degree of change from tubular to villous morphology, presence of high-grade dysplasia and the morphological change described as “serrated adenoma”. In any given individual, the additional features of increasing age, familial occurrence of colorectal adenoma or cancer, male gender or multiplicity of adenomas, predict a future increased risk for cancer in the organ—so-called risk factors for cancer. Except for the presence of adenomas and its size, none of these is objectively defined and all those other than number and size are subject to observer error and to confusion as to precise definition of the feature in question. Because such factors can be difficult to assess and define, their value as predictors of current or future risk for cancer is imprecise. 
     Once a sporadic adenoma has developed, the chance of a new adenoma occurring is approximately 30% within 26 months. 
     The symptoms of colorectal cancer depend on the location of tumor in the bowel, and whether it has metastasized. Unfortunately, many of the symptoms may occur in other diseases as well, and hence symptoms may not be conclusively diagnostic of colorectal cancer. 
     Local symptoms are more likely if the tumor is located closer to the anus. There may be a change in bowel habit (new-onset constipation or diarrhea in the absence of another cause), a feeling of incomplete defecation and reduction in diameter of stools. Tenesmus and change in stool shape are both characteristic of rectal cancer. Lower gastrointestinal bleeding, including the passage of bright red blood in the stool, may indicate colorectal cancer, as may the increased presence of mucus. Melena, black stool with a tarry appearance, normally occurs in upper gastrointestinal bleeding (such as from a duodenal ulcer), but is sometimes encountered in colorectal cancer when the disease is located in the beginning of the large bowl. 
     A tumor that is large enough to fill the entire lumen of the bowel may cause bowel obstruction. This situation is characterized by constipation, abdominal pain, abdominal distension and vomiting. This occasionally leads to the obstructed and distended bowel perforating and causing peritonitis. 
     Certain local effects of colorectal cancer occur when the disease has become more advanced. A large tumor is more likely to be noticed on feeling the abdomen, and it may be noticed by a doctor on physical examination. The disease may invade other organs, and may cause blood or air in the urine or vaginal discharge. 
     If a tumor has caused chronic occult bleeding, iron deficiency anemia may occur. This may be experienced as fatigue, palpitations and noticed as pallor. Colorectal cancer may also lead to weight loss, generally due to a decreased appetite. 
     More unusual constitutional symptoms are an unexplained fever and one of several paraneoplastic syndromes. The most common paraneoplastic syndrome is thrombosis, usually deep vein thrombosis. 
     Colorectal cancer most commonly spreads to the liver. This may go unnoticed, but large deposits in the liver may cause jaundice and abdominal pain (due to stretching of the capsule). If the tumor deposit obstructs the bile duct, the jaundice may be accompanied by other features of biliary obstruction, such as pale stools. 
     Colorectal cancer can take many years to develop and early detection of colorectal cancer greatly improves the prognosis. Even modest efforts to implement colorectal cancer screening methods can result in a drop in cancer deaths. Despite this, colorectal cancer screening rates remain low. There are currently several different tests available for this purpose: 
     Digital rectal exam: The doctor inserts a lubricated, gloved finger into the rectum to feel for abnormal areas. It only detects tumors large enough to be felt in the distal part of the rectum but is useful as an initial screening test. 
     Fecal occult blood test: a test for blood in the stool. Two types of tests can be used for detecting occult blood in stools i.e. guaiac based (chemical test) and immunochemical. The sensitivity of immunochemical testing is superior to that of chemical testing without an unacceptable reduction in specificity (Weitzel J N (December 1999). “Genetic cancer risk assessment. Putting it all together”.  Cancer  86 (11 Suppl): 2483-92). 
     Endoscopy: 
     Sigmoidoscopy: A lit probe (sigmoidoscope) is inserted into the rectum and lower colon to check for polyps and other abnormalities. 
     Colonoscopy: A lit probe called a colonoscope is inserted into the rectum and the entire colon to look for polyps and other abnormalities that may be caused by cancer. A colonoscopy has the advantage that if polyps are found during the procedure they can be removed immediately. Tissue can also be taken for biopsy. 
     Double contrast barium enema (DCBE): First, an overnight preparation is taken to cleanse the colon. An enema containing barium sulfate is administered, then air is insufflated into the colon, distending it. The result is a thin layer of barium over the inner lining of the colon which is visible on X-ray films. A cancer or a precancerous polyp can be detected this way. This technique can miss the (less common) flat polyp. 
     Virtual colonoscopy replaces X-ray films in the double contrast barium enema (above) with a special computed tomography scan and requires special workstation software in order for the radiologist to interpret. This technique is approaching colonoscopy in sensitivity for polyps. However, any polyps found must still be removed by standard colonoscopy. 
     Standard computed axial tomography is an x-ray method that can be used to determine the degree of spread of cancer, but is not sensitive enough to use for screening. Some cancers are found in CAT scans performed for other reasons. 
     Blood tests: Measurement of the patient&#39;s blood for elevated levels of certain proteins can give an indication of tumor load. In particular, high levels of carcinoembryonic antigen (CEA) in the blood can indicate metastasis of adenocarcinoma. While these tests are frequently false positive or false negative, and are not recommended for screening, they can be useful to assess disease recurrence. CA19-9 and CA 242 biomarkers can indicate e-selectin related metastatic risks, help follow therapeutic progress, and assess disease recurrence. Recently, an assay for detection in plasma of methylated sequences of the Septin 9 gene has also become available to assist in diagnosis of colorectal cancer. 
     Positron emission tomography (PET) is a 3-dimensional scanning technology where a radioactive sugar is injected into the patient, the sugar collects in tissues with high metabolic activity, and an image is formed by measuring the emission of radiation from the sugar. Because cancer cells often have very high metabolic rates, this can be used to differentiate benign and malignant tumors. PET is not used for screening and does not (yet) have a place in routine workup of colorectal cancer cases. 
     Stool DNA testing is an emerging technology in screening for colorectal cancer. Premalignant adenomas and cancers shed DNA markers from their cells which are not degraded during the digestive process and remain stable in the stool. Capture, followed by PCR amplifies the DNA to detectable levels for assay. 
     High C-Reactive Protein Levels as Risk Marker 
     Despite the existence of these tests, diagnosis remains problematic. Most of the more sensitive tests are quite invasive and expensive and therefore uptake by patients is low. There is therefore an ongoing need to develop simpler and more informative diagnostic protocols or aids to diagnosis that enable one to direct colonoscopy at people more likely to have developed adenomas or carcinomas. A simple and accurate screening test would enable much more widely applicable screening systems to be set up. 
     To this end, more recently there have been identified genetic markers which are modulated, in terms of their expression levels, in individuals who have developed a neoplasm of the large intestine. Some of these markers are upregulated in terms of their level of expression, while others are downregulated. However, a feature common to most of these markers, and the use of genetic marker expression levels in general as a diagnostic, is that they often exhibit only moderate levels of sensitivity and specificity. The development of diagnostic protocols which provide high levels of sensitivity and specificity is highly sought after. 
     In work leading up to the present invention, it has been unexpectedly determined that the gene markers BCAT1, IKZF1 IRF4, GRASP and CAHM, although each individually being one of a number of gene markers known to exhibit utility in terms of diagnosing colorectal neoplasia development, have in fact been determined to collectively enable a significantly higher level of sensitivity or specificity to be achieved than where any one of these gene markers is analyzed either alone or together with other unrelated gene markers. More specifically, screening for an increase in the level of methylation of any two or more of these five specific markers can be designed so as to achieve a level of specificity or sensitivity not previously achievable. 
     Bearing in mind the large number of gene markers which, in differential gene expression analysis studies, have been shown to exhibit modulated expression levels in neoplasia, the identification that five specific markers can in fact collectively provide improved diagnostic outcomes relative either to these markers individually or to other groups of markers is both unexpected and unpredictable. 
     SUMMARY 
     Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. 
     As used herein, the term “derived from” shall be taken to indicate that a particular integer or group of integers has originated from the species specified, but has not necessarily been obtained directly from the specified source. Further, as used herein the singular forms of “a”, “and” and “the” include plural referents unless the context clearly dictates otherwise. 
     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. 
     The subject specification contains nucleotide sequence information prepared using the program PatentIn Version 3.5, presented herein after the bibliography. Each nucleotide sequence is identified in the sequence listing by the numeric indicator &lt;210&gt; followed by the sequence identifier (e.g. &lt;210&gt;1, &lt;210&gt;2, etc.). The length, type of sequence (DNA, etc.) and source organism for each sequence is indicated by information provided in the numeric indicator fields &lt;211&gt;, &lt;212&gt; and &lt;213&gt;, respectively. Nucleotide sequences referred to in the specification are identified by the indicator SEQ ID NO: followed by the sequence identifier (e.g. SEQ ID NO:1, SEQ ID NO:2, etc.). The sequence identifier referred to in the specification correlates to the information provided in numeric indicator field &lt;400&gt; in the sequence listing, which is followed by the sequence identifier (e.g. &lt;400&gt;1, &lt;400&gt;2, etc.). That is SEQ ID NO:1 as detailed in the specification correlates to the sequence indicated as &lt;400&gt;1 in the sequence listing. 
     One aspect of the present invention is directed to a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of a DNA region selected from:
     (i) the region, including 2 kb upstream of the transcription start site, defined by any two or more of Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798   (3) chr6:391739..411443;   (4) chr12:52400748..52409671; and   (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of any two or more of:
       (1) BCAT1 (2) IKZF1 (3) IRF4 (4) GRASP and (5) CAHM
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In another aspect said method is directed to identifying biological samples in which any one of said DNA regions exhibits a higher level of methylation. 
     In still another aspect said method is directed to identifying biological samples in which two or more of said DNA regions exhibit a higher level of methylation. 
     In yet another aspect there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393; and   (2) chr7:50344378...50472798;   and optionally one or more of (3) chr6:391739..411443, (4) chr12:52400748..52409671 and (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1; and   (2) IKZF1;   and optionally one or more of (3) IRF4, (4) GRASP and (5) CAHM
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In one embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798; and   (3) chr6:391739..411443; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1, (2) IKZFI and (3) IRF4.   
       

     In another embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798 and   (4) chr12:52400748..52409671; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1, (2) IKZFI; and (4) GRASP.   
       

     In still another embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798 and   (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCATI, (2) 1KZF1 and (5) CAHM   
       

     In still yet another embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798   (3) chr6:391739..411443; and   (4) chr12:52400748..52409671; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1, (2) IKZF1, (3) IRF4 and (4) GRASP.   
       

     In yet still another embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798   (3) chr6:391739..411443; and   (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1, (2) IKZF1, (3) IRF4 and (5) CAHM   
       

     In a further embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798   (4) chr12:52400748..52409671 and   (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1, (2) 1KZF1, (4) GRASP and (5) CAHM   
       

     In yet another further embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798   (3) chr6:391739..411443   (4) chr12:52400748..52409671 and   (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1, (2) IKZF1, (3) IRF4, (4) GRASP and (5) CAHM.   
       

     In another embodiment there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393; and   (5) chr6:163834097..163834982;   and optionally one or more of (2) chr7:50344378...50472798, (3) chr6:391739..411443; and (4) chr12:52400748..52409671; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1; and   (5) CAHM;   and optionally one or more of (2) IKZF1, (3) IRF4 and (4) GRASP
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In still another embodiment there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (2) chr7:50344378..50472798; and   (5) chr6:163834097..163834982;   and optionally one or more of (1) chr12:24962958..25102393, (3) chr6:391739..411443 and (4) chr12:52400748..52409671; or   
       (ii) the gene region, including 2 kb upstream of:
       (2) IKZF 1; and   (5) CAHM;   and optionally one or more of (1) BCAT1, (3) IRF4 and (4) GRASP
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In a further embodiment there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393; and   (3) chr6:391739..411443;   and optionally one or more of (2) chr7:50344378...50472798, (4) chr12:52400748..52409671 and (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1; and   (3) IRF4;   and optionally one or more of (2) IKZF 1, (4) GRASP and (5) CAHM
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In yet another further embodiment there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393; and   (4) chr12:52400748..52409671;   and optionally one or more of (2) chr7:50344378...50472798, (3) chr6:391739..411443 and (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1; and   (4) GRASP;   and optionally one or more of (2) IKZF1, (3) IRF4 and (5) CAHM
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In still another further embodiment there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (2) chr7:50344378..50472798; and   (3) chr6:391739..411443;   and optionally one or more of (1) chr12:24962958..25102393, (4) chr12:52400748..52409671 and (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (2) IKZF 1; and   (3) IRF4;   and optionally one or more of (1) BCAT1, (4) GRASP and (5) CAHM
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In yet still another embodiment there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (2) chr7:50344378..50472798; and   (4) chr12:52400748..52409671;   and optionally one or more of (1) chr12:24962958..25102393, (3) chr6:391739..411443 and (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (2) IKZF 1; and   (4) GRASP;   and optionally one or more of (1) BCAT1, (3) IRF4 and (5) CAHM
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In still yet another embodiment there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (3) chr6:391739..411443; and   (4) chr12:52400748..52409671;   and optionally one or more of (1) chr12:24962958..25102393, (2) chr7:50344378...50472798 and (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (3) IRF4; and   (4) GRASP;   and optionally one or more of (1) BCAT1, (2) IKZF1 and (5) CAHM
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In another embodiment there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (3) chr6:391739..411443; and   (5) chr6:163834097..163834982;   and optionally one or more of (1) chr12:24962958..25102393, (2) chr7:50344378...50472798 and (4) chr12:52400748..52409671; or   
       (ii) the gene region, including 2 kb upstream of:
       (3) IRF4; and   (5) CAHM;   and optionally one or more of (1) BCAT1, (2) IKZF1 and (4) GRASP
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     The subregions which have been determined to exhibit particular utility are listed below with reference to the gene and chromosomal region within which they are found:
     (1) BCAT subregions chr12:25101992-25102093 (SEQ ID NO:1 or corresponding minus strand) and chr12:25101909-25101995 (SEQ ID NO:2 or corresponding minus strand)   (2) IKZF1 subregions: chr7:50343867-50343961 (SEQ ID NO:3 or corresponding minus strand) and chr7:50343804-5033895 (SEQ ID NO:4 or corresponding minus strand)   (3) IRF4 subregions chr6:392036-392145 (SEQ ID NO:5 or corresponding minus strand)   (4) GRASP subregions: chr12:52399672-52399922, chr12:52400821-52401051 (SEQ ID NO:6 or corresponding minus strand), chr12:52401407-52401664 (SEQ ID NO:7 or corresponding minus strand) chr12:52400866-52400973 and Chr12:52401107-52401664.   (5) CAHM subregions: chr6:163834295-163834500 (SEQ ID NO:8 or corresponding minus strand), chr6:163834621-163834906, chr6:163834393-163834455 and chr6:163834393-163834519.   

     To the extent that the method of the present invention includes analyzing the methylation of GRASP, the subject residues are: 
                                            chr12:52399713   chr12:52399731   chr12:52399749   chr12:52399783       chr12:52399796   chr12:52399808   chr12:52399823   chr12:52399835       chr12:52399891                   chr12:52400847   chr12:52400850   chr12:52400859   chr12:52400866       chr12:52400869   chr12:52400873   chr12:52400881   chr12:52400886       chr12:52400893   chr12:52400895   chr12:52400899   chr12:52400902       chr12:52400907   chr12:52400913   chr12:52400919   chr12:52400932       chr12:52400938   chr12:52400958   chr12:52400962   chr12:52400971       chr12:52400973   chr12:52400976   chr12:52400998   chr12:52401008       chr12:52401010   chr12:52401012   chr12:52401016   chr12:52401019       chr12:52401025   chr12:52401041   chr12:52401044   chr12:52401053       chr12:52401060   chr12:52401064   chr12:52401092   chr12:52401118       chr12:52401438   chr12:52401448   chr12:52401460   chr12:52401465       chr12:52401474   chr12:52401477   chr12:52401479   chr12:52401483       chr12:52401504   chr12:52401514   chr12:52401523   chr12:52401540       chr12:52401553   chr12:52401576   chr12:52401588   chr12:52401595       chr12:52401599   chr12:52401604   chr12:52401606   chr12:52401634       chr12:52401640   chr12:52401644   chr12:52401659           chr12:52401160   chr12:52401165   chr12:52401174   chr12:52401177       chr12:52401179   chr12:52401183   chr12:52401204   chr12:52401215       chr12:52401223   chr12:52401240   chr12:52401253   chr12:52401288       chr12:52401295   chr12:52401299   chr12:52401304   chr12:52401334       chr12:52401340   chr12:52401344   chr12:52401359                    
or a corresponding cytosine at position n+1 on the opposite DNA strand.
 
     To the extent that the method of the present invention includes analyzing CAHM, the subject residues are: 
                                        chr6: 163834330   chr6: 163834332   chr6: 163834357       chr6: 163834373   chr6: 163834384   chr6: 163834390       chr6: 163834392   chr6: 163834406   chr6: 163834412       chr6: 163834419   chr6: 163834443   chr6: 163834448       chr6: 163834452   chr6: 163834464   chr6: 163834483       chr6: 163834653   chr6: 163834660   chr6: 163834672       chr6: 163834675   chr6: 163834678   chr6: 163834681       chr6: 163834815   chr6: 163834824   chr6: 163834835       chr6:163834840   chr6:163834853   chr6:163834855       chr6:163834858   chr6:163834863   chr6:163834869       chr6:163834872                    
or a corresponding cytosine at position n+1 on the opposite DNA strand.
 
     To the extent that the method of the present invention includes analyzing IKZF1, the subject residues are: 
                                                    chr7: 50343869   chr7:50343872   chr7:50343883           chr7:50343889   chr7:50343890   chr7: 50343897           chr7: 50343907   chr7:50343909   chr7:50343914           chr7:50343934   chr7:50343939   chr7:50343950           chr7:50343959   chr7:50343805   chr7: 50343822           chr7: 50343824   chr7:50343826   chr7: 50343829           chr7:50343831   chr7:50343833   chr7:50343838           chr7: 50343847   chr7:50343850   chr7:50343858           chr7: 50343864   chr7:50343869   chr7: 50343872           chr7: 50343890                        
or a corresponding cytosine at position n+1 on the opposite DNA strand.
 
     To the extent that the method of the present invention includes analyzing IRF4, the subject residues are: 
                                                    chr6:392036   chr6:392047   chr6:392049           chr6:392057   chr6:392060   chr6:392066           chr6:392080   chr6:392094   chr6:392102           chr6:392131                        
or a corresponding cytosine at position n+1 on the opposite DNA strand.
 
     Another aspect of the present invention is directed to a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the level of expression of a DNA region selected from:
     (i) the region, including 2 kb upstream of the transcription start site, defined by any two or more of Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798   (3) chr6:391739..411443; or   (4) chr12:52400748..52409671; and   (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of any two or more of:
       (1) BCAT1 (2) IKZF1 (3) IRF4 (4) GRASP and (5) CAHM
 
in a biological sample from said individual wherein a lower level of expression of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a neoplastic state.
   
       

    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  depicts the genomic sequence of GRASP. 
         FIG. 2  depicts the genomic sequence of CAHM. 
         FIG. 3  depicts the genomic sequence of IRF4. 
         FIG. 4  depicts the genomic sequence of BCAT1. 
         FIG. 5  depicts the genomic sequence of IKZF 1. 
     
    
    
     DETAILED DESCRIPTION 
     The present invention is predicated, in part, on the determination that the change in methylation pattern of a panel of gene markers can provide a higher level of either diagnostic specificity or diagnostic sensitivity when tested collectively than if any one of these markers is tested either in isolation or together with markers other than those specified herein. This finding has therefore now facilitated the development of an improved test for diagnosing, prognosing or monitoring neoplasms of the large intestine based on assessing the methylation of two or more of the gene markers BCAT1, IKZF1, CAHM, GRASP and IRF4. 
     In accordance with the present invention, it has been determined that certain specific panels of genes are modulated, in terms of differential changes to their levels of methylation, depending on whether or not the cell in issue is neoplastic or not. It should be understood that the genes in issue are described herein both by reference to their name and their chromosomal coordinates. The chromosomal coordinates are consistent with the human genome database version Hg19 which was released in February 2009 (herein referred to as “Hg19 coordinates”). 
     Accordingly, one aspect of the present invention is directed to a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of a DNA region selected from:
     (i) the region, including 2 kb upstream of the transcription start site, defined by any two or more of Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798   (3) chr6:391739..411443;   (4) chr12:52400748..52409671; and   (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of any two or more of:
       (1) BCAT1 (2) IKZF1 (3) IRF4 (4) GRASP and (5) CAHM
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In one embodiment, said method is directed to identifying biological samples in which any one of said DNA regions exhibits a higher level of methylation. 
     In another embodiment, said method is directed to identifying biological samples in which two or more of said DNA regions exhibit a higher level of methylation. 
     Without limiting the present invention to any one theory or mode of action, the panel of DNA regions (markers) specified herein provide not only an improved diagnostic outcome relative to prior art methods but, in addition, enable the development of a screening method which can be designed to focus on providing either a high level of diagnostic specificity or a high level of diagnostic sensitivity. As would be understood by the person of skill in the art, in the context of diagnostics “sensitivity” defines the proportion of positive results which are correctly identified, that is, the percentage of individuals correctly identified as having the disease in issue. “Specificity”, however, defines the proportion of negative results which are correctly identified, that is, the percentage of individuals correctly identified as not having the disease in issue. 
     In the context of the present invention it has been determined that screening a patient sample for the methylation status of the specified panel of markers can be designed to provide either a diagnostic result which exhibits a high level of specificity or a diagnostic result which exhibits a high level of sensitivity. 
     Where a high level of sensitivity is sought, the screening method is designed to identify samples in which any one of the markers of the panel exhibits increased methylation relative to control levels. That is, not all of the markers are required to exhibit hypermethylation in order to define the result as positive. Where a higher level of sensitivity is sought, the level of specificity is inherently reduced. However, if it is desired to pursue a higher level of specificity (which may reduce the level of sensitivity) then the method is designed to identify samples in which two or more of the panel of DNA regions exhibit increased methylation. 
     Accordingly, to the extent that the present invention is directed to embodiments of the method in which “any one of” the DNA regions (markers) of the specified panel exhibits a higher level of methylation, these embodiments are designed to achieve results exhibiting increased sensitivity based on any one of the markers in the panel being hypermethylated. It should be understood that it need not be the same marker which is hypermethylated in each sample. Rather, it is simply that one of the markers which forms part of the panel is hypermethylated. It should also be understood that in relation to some samples, two or more of the markers of the panel may be hypermethylated. These samples should be understood to nevertheless fall within the scope of this embodiment of the invention since this embodiment is not excluding the situation where multiple markers are hypermethylated but is merely including within its scope all those samples where as few as one marker are hypermethylated. Overall these data will provide increased sensitivity but reduced specificity. 
     To the extent that the present invention is directed to embodiments of the method in which “two or more” of the DNA regions (markers) of the specified panel exhibit a higher level of methylation, these embodiments are designed to achieve a higher level of specificity. This is achieved by virtue of the fact that at least two, if not more, of the specified panel of markers are determined to be hypermethylated. 
     Reference to “large intestine” should be understood as a reference to a cell derived from one of the eight anatomical regions of the large intestine, which regions commence after the terminal region of the ileum, these being: 
     (i) the cecum; 
     (ii) the ascending colon; 
     (iii) the transverse colon; 
     (iv) the descending colon; 
     (v) the sigmoid colon; 
     (vi) the rectum; 
     (vii) the splenic flexure; and 
     (viii) the hepatic flexure. 
     Reference to “neoplasm” should be understood as a reference to a lesion, tumor or other encapsulated or unencapsulated mass or other form of growth which comprises neoplastic cells. A “neoplastic cell” should be understood as a reference to a cell exhibiting abnormal growth. The term “growth” should be understood in its broadest sense and includes reference to proliferation. In this regard, an example of abnormal cell growth is the uncontrolled proliferation of a cell. Another example is failed apoptosis in a cell, thus prolonging its usual life span. The neoplastic cell may be a benign cell or a malignant cell. In a preferred embodiment, the subject neoplasm is an adenoma or an adenocarcinoma. Without limiting the present invention to any one theory or mode of action, an adenoma is generally a benign tumor of epithelial origin which is either derived from epithelial tissue or exhibits clearly defined epithelial structures. These structures may take on a glandular appearance. It can comprise a malignant cell population within the adenoma, such as occurs with the progression of a benign adenoma or benign neoplastic legion to a malignant adenocarcinoma. 
     Preferably, said neoplastic cell is an adenoma or adenocarcinoma and even more preferably a colorectal adenoma or adenocarcinoma. 
     Reference to “DNA region” should be understood as a reference to a specific section of genomic DNA. These DNA regions are specified either by reference to a gene name or a set of chromosomal coordinates. Both the gene names and the chromosomal coordinates would be well known to, and understood by, the person of skill in the art. As detailed hereinbefore, the chromosomal coordinates correspond to the Hg19 version of the genome. In general, a gene can be routinely identified by reference to its name, via which both its sequences and chromosomal location can be routinely obtained, or by reference to its chromosomal coordinates, via which both the gene name and its sequence can also be routinely obtained. 
     Reference to each of the genes/DNA regions detailed above should be understood as a reference to all forms of these molecules and to fragments or variants thereof. As would be appreciated by the person of skill in the art, some genes are known to exhibit allelic variation between individuals or single nucleotide polymorphisms. SNPs encompass insertions and deletions of varying size and simple sequence repeats, such as dinucleotide and trinucleotide repeats. Variants include nucleic acid sequences from the same region sharing at least 90%, 95%, 98%, 99% sequence identity i.e. having one or more deletions, additions, substitutions, inverted sequences etc. relative to the DNA regions described herein. Accordingly, the present invention should be understood to extend to such variants which, in terms of the present diagnostic applications, achieve the same outcome despite the fact that minor genetic variations between the actual nucleic acid sequences may exist between individuals. The present invention should therefore be understood to extend to all forms of DNA which arise from any other mutation, polymorphic or allelic variation. 
     It should be understood that the “individual” who is the subject of testing may be any human or non-human mammal. Examples of non-human mammals includes primates, livestock animals (e.g. horses, cattle, sheep, pigs, donkeys), laboratory test animals (e.g. mice, rats, rabbits, guinea pigs), companion animals (e.g. dogs, cats) and captive wild animals (e.g. deer, foxes). 
     Preferably the mammal is a human. 
     The panel of genes which has been identified demonstrates increased methylation in large intestine neoplastic cells relative to corresponding non-neoplastic cells. This increased methylation is, in some cases, localized to a few specific CpG sites within the DNA region while in other cases it is observed across a wide range of CpG sites. However, although specific regions from all genes exhibit increased methylation and are therefore useful diagnostic markers, it is the analysis of these specific markers as a panel which has provided an independent and very significant improvement over the sensitivity and specificity could be obtainable over these markers are analyzed individually. This improvement is also significant even when considered against the sensitivity and specificity obtainable using other markers known to exhibit increased methylation as a marker of the onset of a large intestine neoplasm. Accordingly, the method of the present invention has now provided a means of achieving a higher level of sensitivity and specificity than has been achievable to date. 
     In one aspect there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393; and   (2) chr7:50344378...50472798;   and optionally one or more of (3) chr6:391739..411443, (4) chr12:52400748..52409671 and (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1; and   (2) IKZF1;   and optionally one or more of (3) IRF4, (4) GRASP and (5) CAHM
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In one embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798; and   (3) chr6:391739..411443; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1, (2) IKZF1 and (3) IRF4.   
       

     In another embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798 and   (4) chr12:52400748..52409671; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1, (2) IKZF1; and (4) GRASP.   
       

     In still another embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798 and   (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1, (2) IKZF1 and (5) CAHM   
       

     In still yet another embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798   (3) chr6:391739..411443; and   (4) chr12:52400748..52409671; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1, (2) IKZF1, (3) IRF4 and (4) GRASP.   
       

     In yet still another embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798   (3) chr6:391739..411443; and   (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1, (2) IKZF1, (3) IRF4 and (5) CAHM   
       

     In a further embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798   (4) chr12:52400748..52409671 and   (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1, (2) IKZF1, (4) GRASP and (5) CAHM   
       

     In yet another further embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798   (3) chr6:391739..411443   (4) chr12:52400748..52409671 and   (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1, (2) IKZF1, (3) IRF4, (4) GRASP and (5) CAHM   
       

     In another embodiment there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393; and   (5) chr6:163834097..163834982;   and optionally one or more of (2) chr7:50344378...50472798, (3) chr6:391739..411443; and (4) chr12:52400748..52409671; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1; and   (5) CAHM;   and optionally one or more of (2) IKZF 1, (3) IRF4 and (4) GRASP
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In still another embodiment there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (2) chr7:50344378..50472798; and   (5) chr6:163834097..163834982;   and optionally one or more of (1) chr12:24962958..25102393, (3) chr6:391739..411443 and (4) chr12:52400748..52409671; or   
       (ii) the gene region, including 2 kb upstream of:
       (2) IKZF 1; and   (5) CAHM;   and optionally one or more of (1) BCAT1, (3) IRF4 and (4) GRASP
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In a further embodiment there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393; and   (3) chr6:391739..411443;   and optionally one or more of (2) chr7:50344378...50472798, (4) chr12:52400748..52409671 and (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1; and   (3) IRF4;   and optionally one or more of (2) IKZF 1, (4) GRASP and (5) CAHM
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In yet another further embodiment there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393; and   (4) chr12:52400748..52409671;   and optionally one or more of (2) chr7:50344378...50472798, (3) chr6:391739..411443 and (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1; and   (4) GRASP;   and optionally one or more of (2) IKZF1, (3) IRF4 and (5) CAHM
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In still another further embodiment there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (2) chr7:50344378..50472798; and   (3) chr6:391739..411443;   and optionally one or more of (1) chr12:24962958..25102393, (4) chr12:52400748..52409671 and (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (2) IKZF 1; and   (3) IRF4;   and optionally one or more of (1) BCAT1, (4) GRASP and (5) CAHM
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In yet still another embodiment there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (2) chr7:50344378..50472798; and   (4) chr12:52400748..52409671;   and optionally one or more of (1) chr12:24962958..25102393, (3) chr6:391739..411443 and (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (2) IKZF 1; and   (4) GRASP;   and optionally one or more of (1) BCAT1, (3) IRF4 and (5) CAHM
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In still yet another embodiment there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (3) chr6:391739..411443; and   (4) chr12:52400748..52409671;   and optionally one or more of (1) chr12:24962958..25102393, (2) chr7:50344378...50472798 and (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (3) IRF4; and   (4) GRASP;   and optionally one or more of (1) BCAT1, (2) IKZF1 and (5) CAHM
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In another embodiment there is provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (3) chr6:391739..411443; and   (5) chr6:163834097..163834982;   and optionally one or more of (1) chr12:24962958..25102393, (2) chr7:50344378...50472798 and (4) chr12:52400748..52409671; or   
       (ii) the gene region, including 2 kb upstream of:
       (3) IRF4; and   (5) CAHM;   and optionally one or more of (1) BCAT1, (2) IKZF1 and (4) GRASP
 
in a biological sample from said individual wherein a higher level of methylation of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a large intestine neoplastic state.
   
       

     In accordance with these aspects and embodiments, in yet another embodiment said control level is a non-neoplastic level. 
     In one embodiment, said method is directed to identifying biological samples in which any one of said DNA regions exhibits a higher level of methylation. 
     In another embodiment, said method is directed to identifying biological samples in which two or more of said DNA regions exhibits a higher level of methylation. 
     Still further, in another embodiment, said large intestine tissue is preferably colorectal tissue. 
     In still another embodiment, the neoplasm is malignant, such as a carcinoma. 
     In a further embodiment, the neoplasm is non-malignant, such as an adenoma. 
     In terms of screening for the methylation of these gene regions, it should be understood that the assays can be designed to screen either the specific regions listed herein (which correspond to the “plus” strand of the gene) or the complementary “minus” strand. It is well within the skill of the person in the art to choose which strand to analyze and to target that strand based on the chromosomal coordinates provided herein. In some circumstances, assays may be established to screen both strands. 
     It should be understood that one may screen for the specified panel of markers exclusively or one may elect to additionally screen for other markers, such as other DNA hypermethylation markers, other RNA expression level markers or other protein markers. These other markers may, for example, provide additional information in relation to the health status of the patient in issue. 
     Without limiting the present invention to any one theory or mode of action, although measuring the methylation levels across these DNA regions is diagnostic of a large intestine neoplastic condition, it has been determined that discrete subregions are particularly useful in this regard since these subregions contain a high density of CpG dinucleotides which are frequently hypermethylated in large intestine neoplasias, such as colorectal cancers. This finding renders these subregions a particularly useful target for analysis since it both simplifies the screening process due to a shorter more clearly defined region of DNA requiring analysis and, further, the fact that the results from these regions will provide a significantly more definitive result in relation to the presence, or not, of hypermethylation than would be obtained if analysis was performed across the DNA region as a whole. This finding therefore both simplifies the screening process and increases the sensitivity and specificity of large intestine neoplasia diagnosis. 
     The subregions which have been determined to exhibit particular utility are listed below with reference to the gene and chromosomal region within which they are found:
     (1) BCAT subregions chr12:25101992-25102093 (SEQ ID NO:1 or corresponding minus strand) and chr12:25101909-25101995 (SEQ ID NO:2 or corresponding minus strand)   (2) IKZF1 subregions: chr7:50343867-50343961 (SEQ ID NO:3 or corresponding minus strand) and chr7:50343804-5033895 (SEQ ID NO:4 or corresponding minus strand)   (3) IRF4 subregions chr6:392036-392145 (SEQ ID NO:5 or corresponding minus strand)   (4) GRASP subregions: chr12:52399672-52399922, chr12:52400821-52401051 (SEQ ID NO:6 or corresponding minus strand), chr12:52401407-52401664 (SEQ ID NO:7 or corresponding minus strand) chr12:52400866-52400973 and Chr12:52401107-52401664.   (5) CAHM subregions: chr6:163834295-163834500 (SEQ ID NO:8), chr6:163834621-163834906, chr6:163834393-163834455 and chr6:163834393-163834519.   

     Without limiting the present invention to any one theory or mode of action, the skilled person may screen one or more subregions for each gene marker. 
     In one embodiment, the methylation marker subregions tested for each selected gene marker are:
         (1) The BCAT subregion defined by SEQ ID NO:1 or SEQ ID NO:2 or corresponding minus strand;   (2) The IKZF1 subregion defined by SEQ ID NO:3 or SEQ ID NO:4 or corresponding minus strand;   (3) The IRF4 subregion defined by SEQ ID NO:5 or corresponding minus strand;   (4) The GRASP subregion defined by SEQ ID NO:6 or 7 or corresponding minus strands; and   (5) The CAHM subregion defined by SEQ ID NO:8 or corresponding minus strand.       

     Without limiting the present invention to any one theory or mode of action, DNA methylation is universal in bacteria, plants, and animals. DNA methylation is a type of chemical modification of DNA that is stable over rounds of cell division but does not involve changes in the underlying DNA sequence of the organism. Chromatin and DNA modifications are two important features of epigenetics and play a role in the process of cellular differentiation, allowing cells to stably maintain different characteristics despite containing the same genomic material. In eukaryotic organisms DNA methylation occurs only at the number 5 carbon of the cytosine pyrimidine ring. In mammals, DNA methylation occurs mostly at the number 5 carbon of the cytosine of a CpG dinucleotide. CpG dinucleotides comprise approximately 1% human genome. 
     70-80% of all CpGs are methylated. CpGs may be grouped in clusters called “CpG islands” that are present in the 5′ regulatory regions of many genes and are frequently unmethylated. In many disease processes such as cancer, gene promoters and/or CpG islands acquire abnormal hypermethylation, which is associated with heritable transcriptional silencing. DNA methylation may impact the transcription of genes in two ways. First, the methylation of DNA may itself physically impede the binding of transcriptional proteins to the gene, thus blocking transcription. Second, methylated DNA may be bound by proteins known as Methyl-CpG-binding domain proteins (MBDs). MBD proteins then recruit additional proteins to the locus, such as histone deacetylases and other chromatin remodeling proteins that can modify histones, thereby forming compact, inactive chromatin termed silent chromatin. This link between DNA methylation and chromatin structure is very important. In particular, loss of Methyl-CpG-binding Protein 2 (MeCP2) has been implicated in Rett syndrome and Methyl-CpG binding domain protein 2 (MBD2) mediates the transcriptional silencing of hypermethylated genes in cancer. 
     In humans, the process of DNA methylation is carried out by three enzymes, DNA methyltransferase 1, 3a and 3b (DNMT1, DNMT3a, DNMT3b). It is thought that DNMT3a and DNMT3b are the de novo methyltransferases that set up DNA methylation patterns early in development. DNMT1 is the proposed maintenance methyltransferase that is responsible for copying DNA methylation patterns to the daughter strands during DNA replication. DNMT3L is a protein that is homologous to the other DNMT3s but has no catalytic activity. Instead, DNMT3L assists the de novo methyltransferases by increasing their ability to bind to DNA and stimulating their activity. Finally, DNMT2 has been identified as an “enigmatic” DNA methyltransferase homolog, containing all 10 sequence motifs common to all DNA methyltransferases; however, DNMT2 may not methylate DNA but instead has been shown to methylate a small RNA. 
     “Methylation status” should therefore be understood as a reference to the presence, absence and/or quantity of methylation at a particular nucleotide, or nucleotides, within a DNA region. The methylation status of a particular DNA sequence (e.g. DNA region as described herein) can indicate the methylation state of every base in the sequence or can indicate the methylation state of a subset of the base pairs (e.g., of cytosines or the methylation state of one or more specific restriction enzyme recognition sequences) within the sequence, or can indicate information regarding regional methylation density within the sequence without providing precise information of where in the sequence the methylation occurs. The methylation status can optionally be represented or indicated by a “methylation value.” A methylation value can be generated, for example, by quantifying the amount of intact DNA present following restriction digestion with a methylation dependent restriction enzyme. In this example, if a particular sequence in the DNA is quantified using quantitative PCR, an amount of template DNA approximately equal to a mock treated control indicates the sequence is not highly methylated whereas an amount of template substantially less than occurs in the mock treated sample indicates the presence of methylated DNA at the sequence. Accordingly, a value, i.e., a methylation value, for example from the above-described example, represents the methylation status and can thus be used as a quantitative indicator of the methylation status. This is of particular use when it is desirable to compare the methylation status of a sequence in a sample to a threshold value. 
     The method of the present invention is predicated on the comparison of the level of methylation of specific DNA regions of a biological sample with the control methylation levels of these DNA regions. The “control level” is the “normal level”, which is the level of methylation of the DNA region of a corresponding large intestine cell or cellular population which is not neoplastic or in another biological sample from which DNA may be isolated for assay. 
     The normal (or “non-neoplastic”) methylation level may be determined using non-neoplastic tissues derived from the same individual who is the subject of testing. However, it would be appreciated that this may be quite invasive for the individual concerned and it is therefore likely to be more convenient to analyze the test results relative to a standard result which reflects individual or collective results obtained from individuals other than the patient in issue. This latter form of analysis is in fact the preferred method of analysis since it enables the design of kits which require the collection and analysis of a single biological sample, being a test sample of interest. The standard results which provide the normal methylation level may be calculated by any suitable means which would be well known to the person of skill in the art. For example, a population of normal tissues can be assessed in terms of the level of methylation of the genes of the present invention, thereby providing a standard value or range of values against which all future test samples are analyzed. It should also be understood that the normal level may be determined from the subjects of a specific cohort and for use with respect to test samples derived from that cohort. Accordingly, there may be determined a number of standard values or ranges which correspond to cohorts which differ in respect of characteristics such as age, gender, ethnicity or health status. Said “normal level” may be a discrete level or a range of levels. An increase in the methylation level of the subject genes relative to normal levels is indicative of the tissue being neoplastic. 
     The term “methylation” shall be taken to mean the presence of a methyl group added by the action of a DNA methyl transferase enzyme to a cytosine base or bases in a region of nucleic acid, e.g. genomic DNA. As described herein, there are several methods known to those skilled in the art for determining the level or degree of methylation of nucleic acid. 
     By “higher level” is meant that there are a higher number of methylated CpG dinucleotides in the subject diagnosed than in a control sample, that is, either the proportion of DNA molecules methylated at a particular CpG site is higher or there are a higher number of separate CpG sites methylated in the subject. It should be understood that the terms “enhanced” and “increased” are used interchangeably with the term “higher”. 
     In relation to detecting a “higher level” of methylation, it should be understood that in some situations the normal/control level will in fact correspond to the absence of any detectable methylation while the neoplastic level will correspond to the presence of methylation, per se. In this situation the diagnostic method is relatively simple since one need only screen for the mere presence of methylation (i.e. a qualitative assessment only), rather than assessing the methylation levels relative to a control level of methylation, which analysis necessarily involves a measure of quantification. Without limiting the present invention in any way, it is observed in blood-derived samples, for example, that in the context of some markers the change in methylation of that marker upon the onset of neoplasia is a shift from undetectable levels of methylation to the presence of detectable methylation. In these situations a relatively simple qualitative assessment is enabled where one need only screen a test sample to determine the presence or not of methylation. In the context of the definitions provided herein, reference to “higher level” encompasses both a relative increase in the level of methylation of a marker or the onset of methylation where previously none was evident. As detailed hereinbefore, the control level may be newly assessed for each patient or there may be a standard result against which all test samples are assessed. Where it is known that methylation is not present on the marker of interest, one need only screen for the presence or not of methylation since the control level is the absence of methylation and the “higher level” is thereby the presence of any amount of methylation. 
     The present invention is not to be limited by a precise number of methylated residues that are considered to be diagnostic of neoplasia in a subject, because some variation between patient samples will occur. The present invention is also not limited by positioning of the methylated residue. 
     Nevertheless, a number of specific cytosine residues which undergo hypermethylation within these subregions have also been identified. In another embodiment, therefore, a screening method can be employed which is specifically directed to assessing the methylation status of one or more of either these residues or the corresponding cytosine at position n+1 on the opposite DNA strand. 
     To this end, detailed in Table 1 are the cytosine residues which have been identified in this regard. It should be appreciated by the person of skill in the art that these individual residues are numbered by reference to Hg19, which also corresponds to the numbering of the specific subregions listed hereinbefore and which can be further identified when the coordinate numbering for each subregion is applied to the corresponding subregion sequences which are provided in the sequence listing. It should be understood that these residues have been identified in the context of the subregion DNA. However, there are other residues which are hypermethylated outside the subregions themselves but within the larger DNA region from which the subregions derive. Accordingly, these specified residues represent a particularly useful subset of individual cytosine residues which undergo hypermethylation within the context of the DNA regions and subregions herein disclosed. These individual residues are grouped below according to the DNA region within which they occur. These DNA regions are identified by reference to both the Hg19 chromosomal coordinates and the gene region name. 
     To the extent that the method of the present invention includes analyzing the methylation of GRASP, the subject residues are: 
                                            chr12:52399713   chr12:52399731   chr12:52399749   chr12:52399783       chr12:52399796   chr12:52399808   chr12:52399823   chr12:52399835       chr12:52399891                   chr12:52400847   chr12:52400850   chr12:52400859   chr12:52400866       chr12:52400869   chr12:52400873   chr12:52400881   chr12:52400886       chr12:52400893   chr12:52400895   chr12:52400899   chr12:52400902       chr12:52400907   chr12:52400913   chr12:52400919   chr12:52400932       chr12:52400938   chr12:52400958   chr12:52400962   chr12:52400971       chr12:52400973   chr12:52400976   chr12:52400998   chr12:52401008       chr12:52401010   chr12:52401012   chr12:52401016   chr12:52401019       chr12:52401025   chr12:52401041   chr12:52401044   chr12:52401053       chr12:52401060   chr12:52401064   chr12:52401092   chr12:52401118       chr12:52401438   chr12:52401448   chr12:52401460   chr12:52401465       chr12:52401474   chr12:52401477   chr12:52401479   chr12:52401483       chr12:52401504   chr12:52401514   chr12:52401523   chr12:52401540       chr12:52401553   chr12:52401576   chr12:52401588   chr12:52401595       chr12:52401599   chr12:52401604   chr12:52401606   chr12:52401634       chr12:52401640   chr12:52401644   chr12:52401659           chr12:52401160   chr12:52401165   chr12:52401174   chr12:52401177       chr12:52401179   chr12:52401183   chr12:52401204   chr12:52401215       chr12:52401223   chr12:52401240   chr12:52401253   chr12:52401288       chr12:52401295   chr12:52401299   chr12:52401304   chr12:52401334       chr12:52401340   chr12:52401344   chr12:52401359                    
or a corresponding cytosine at position n+1 on the opposite DNA strand.
 
     To the extent that the method of the present invention includes analyzing CAHM, the subject residues are: 
                                                    chr6: 163834330   chr6:163834332   chr6: 163834357           chr6: 163834373   chr6:163834384   chr6: 163834390           chr6: 163834392   chr6:163834406   chr6: 163834412           chr6: 163834419   chr6:163834443   chr6: 163834448           chr6: 163834452   chr6:163834464   chr6: 163834483           chr6: 163834653   chr6:163834660   chr6: 163834672           chr6: 163834675   chr6:163834678   chr6: 163834681           chr6: 163834815   chr6:163834824   chr6: 163834835           chr6: 163834840   chr6:163834853   chr6: 163834855           chr6: 163834858   chr6:163834863   chr6: 163834869           chr6: 163834872                        
or a corresponding cytosine at position n+1 on the opposite DNA strand.
 
     To the extent that the method of the present invention includes analyzing IKZF 1, the subject residues are: 
                                                    chr7: 50343869   chr7: 50343872   chr7:50343883           chr7:50343889   chr7:50343890   chr7: 50343897           chr7: 50343907   chr7:50343909   chr7:50343914           chr7:50343934   chr7:50343939   chr7:50343950           chr7:50343959   chr7:50343805   chr7: 50343822           chr7: 50343824   chr7:50343826   chr7: 50343829           chr7:50343831   chr7:50343833   chr7:50343838           chr7: 50343847   chr7:50343850   chr7:50343858           chr7: 50343864   chr7:50343869   chr7: 50343872           chr7: 50343890                        
or a corresponding cytosine at position n+1 on the opposite DNA strand.
 
     To the extent that the method of the present invention includes analyzing IRF4, the subject residues are: 
                                                    chr6:392036   chr6:392047   chr6:392049           chr6:392057   chr6:392060   chr6:392066           chr6:392080   chr6:392094   chr6:392102           chr6:392131                        
or a corresponding cytosine at position n+1 on the opposite DNA strand.
 
     The detection method of the present invention can be performed on any suitable biological sample. To this end, reference to a “biological sample” should be understood as a reference to any sample of biological material derived from an animal such as, but not limited to, cellular material, biofluids (e.g. blood), feces, tissue biopsy specimens, surgical specimens or fluid which has been introduced into the body of an animal and subsequently removed (such as, for example, the solution retrieved from an enema wash). The biological sample which is tested according to the method of the present invention may be tested directly or may require some form of treatment prior to testing. For example, a biopsy or surgical sample may require homogenization prior to testing or it may require sectioning for in situ testing of the qualitative expression levels of individual genes. Alternatively, a cell sample may require permeabilization prior to testing. Further, to the extent that the biological sample is not in liquid form, (if such form is required for testing) it may require the addition of a reagent, such as a buffer, to mobilize the sample. 
     To the extent that the DNA region of interest is present in a biological sample, the biological sample may be directly tested or else all or some of the nucleic acid present in the biological sample may be isolated prior to testing. In yet another example, the sample may be partially purified or otherwise enriched prior to analysis. For example, to the extent that a biological sample comprises a very diverse cell population, it may be desirable to enrich for a sub-population of particular interest. It is within the scope of the present invention for the target cell population or molecules derived therefrom to be treated prior to testing, for example, inactivation of live virus. It should also be understood that the biological sample may be freshly harvested or it may have been stored (for example by freezing) prior to testing or otherwise treated prior to testing (such as by undergoing culturing). 
     The choice of what type of sample is most suitable for testing in accordance with the method disclosed herein will be dependent on the nature of the situation. Preferably, said sample is a fecal (stool) sample, enema wash, surgical resection, tissue biopsy or blood sample (e.g. whole blood, serum or plasma). 
     More preferably, said biological sample is a blood sample, biopsy sample or stool sample. 
     As detailed hereinbefore, the present invention is designed to screen for a neoplastic cell or cellular population, which is located in the large intestine. Accordingly, reference to “cell or cellular population” should be understood as a reference to an individual cell or a group of cells. Said group of cells may be a diffuse population of cells, a cell suspension, an encapsulated population of cells or a population of cells which take the form of tissue. 
     Reference to the “onset” of a neoplasm, such as adenoma or adenocarcinoma, should be understood as a reference to one or more cells of that individual exhibiting dysplasia. In this regard, the adenoma or adenocarcinoma may be well developed in that a mass of dysplastic cells has developed. Alternatively, the adenoma or adenocarcinoma may be at a very early stage in that only relatively few abnormal cell divisions have occurred at the time of diagnosis. The present invention also extends to the assessment of an individual&#39;s predisposition to the development of a neoplasm, such as an adenoma or adenocarcinoma. Without limiting the present invention in any way, changed methylation levels may be indicative of that individual&#39;s predisposition to developing a neoplasia, such as the future development of an adenoma or adenocarcinoma or another adenoma or adenocarcinoma. 
     Although the preferred method is to assess methylation levels for the purpose of diagnosing neoplasia development or predisposition thereto, the detection of converse changes in the levels of said methylation may be desired under certain circumstances, for example, to monitor the effectiveness of therapeutic or prophylactic treatment directed to modulating a neoplastic condition, such as adenoma or adenocarcinoma development. For example, where elevated levels of methylation indicate that an individual has developed a condition characterized by adenoma or adenocarcinoma development, screening for a decrease in the levels of methylation subsequently to the onset of a therapeutic treatment regime may be utilized to indicate successful clearance of the neoplastic cells. In another example, one can use this method to test the tissue at the margins of a tumor resection in order to determine whether the full margin of the tumor has been removed. 
     The present method can therefore be used in the diagnosis, prognosis, classification, prediction of disease risk, detection of recurrence of disease, selection of treatment of a number of types of neoplasias and monitoring of neoplasias. A cancer at any stage of progression can be detected, such as primary, metastatic, and recurrent cancers. 
     The present invention provides methods for determining whether a mammal (e.g., a human) has a neoplasia of the large intestine, whether a biological sample taken from a mammal contains neoplastic cells or DNA derived from neoplastic cells, estimating the risk or likelihood of a mammal developing a neoplasm, monitoring the efficacy of anti-cancer treatment, or selecting the appropriate anti-cancer treatment in a mammal with cancer. Such methods are based on the determination that neoplastic cells have a different methylation status than normal cells in the DNA regions described herein. Accordingly, by determining whether or not a cell contains differentially methylated sequences in the DNA regions as described herein, it is possible to determine that a cell is neoplastic. 
     The method of the invention can be used to evaluate individuals known or suspected to have a neoplasia or as a routine clinical test, i.e., in an individual not necessarily suspected to have a neoplasia. Further diagnostic assays can be performed to confirm the status of neoplasia in the individual. 
     Further, the present methods may be used to assess the efficacy of a course of treatment. For example, the efficacy of an anti-cancer treatment can be assessed by monitoring DNA methylation of the sequences described herein over time in a mammal having cancer. For example, a reduction or absence of methylation in any of the diagnostic sequences of the invention in a biological sample taken from a mammal following a treatment, compared to a level in a sample taken from the mammal before, or earlier in, the treatment, indicates efficacious treatment. 
     The method of the present invention is therefore useful as a one-time test or as an on-going monitor of those individuals thought to be at risk of neoplasia development or as a monitor of the effectiveness of therapeutic or prophylactic treatment regimens directed to inhibiting or otherwise slowing neoplasia development. In these situations, mapping the modulation of methylation levels in any one or more classes of biological samples is a valuable indicator of the status of an individual or the effectiveness of a therapeutic or prophylactic regime which is currently in use. Accordingly, the method of the present invention should be understood to extend to monitoring for increases or decreases in methylation levels in an individual relative to their normal level (as hereinbefore defined), or relative to one or more earlier methylation levels determined from a biological sample of said individual. 
     The methods for detecting neoplasia can comprise the detection of one or more other cancer-associated polynucleotide or polypeptides sequences. Accordingly, detection of methylation by the method of the invention can be used either alone, or in combination with other screening methods for the diagnosis or prognosis of neoplasia. 
     Any method for detecting DNA methylation can be used in the methods of the present invention. A number of methods are available for detection of differentially methylated DNA at specific loci in either primary tissue samples or in patient samples such as blood, urine, stool or saliva (reviewed in Kristensen and Hansen  Clin Chem.  55:1471-83, 2009; Ammerpohl et al.  Biochim Biophys Acta.  1790:847-62, 2009; Shames et al.  Cancer Lett.  251:187-98, 2007; Clark et al.  Nat Protoc.  1:2353-64, 2006). For analysis of the proportion or extent of DNA methylation in a target gene, DNA is normally treated with sodium bisulfite and regions of interest amplified using primers and PCR conditions that will amplify independently of the methylation status of the DNA. The methylation of the overall amplicon or individual CpG sites can then be assessed by sequencing, including pyrosequencing, restriction enzyme digestion (COBRA) or by melting curve analysis. Alternatively ligation-based methods for analysis of methylation at specific CpG sites may be used. Detection of aberrantly methylated DNA released from tumors and into bodily fluids is being developed as a means of cancer diagnosis. Here, in the case of hypermethylated sequences, it is necessary to use sensitive methods that allow the selective amplification of the methylated DNA sequence from a background of normal cellular DNA that is unmethylated. Such methods based on bisulfite-treated DNA, for example; include methylation selective PCR (MSP), Heavymethyl PCR, Headloop PCR and Helper-dependent chain reaction (PCT/AU2008/001475). 
     Briefly, in some embodiments, methods for detecting methylation include randomly shearing or randomly fragmenting the genomic DNA, cutting the DNA with a methylation-dependent or methylation-sensitive restriction enzyme and subsequently selectively identifying and/or analyzing the cut or uncut DNA. Selective identification can include, for example, separating cut and uncut DNA (e.g., by size) and quantifying a sequence of interest that was cut or, alternatively, that was not cut. See, e.g., U.S. Pat. No. 7,186,512. Alternatively, the method can encompass amplifying intact DNA after restriction enzyme digestion, thereby only amplifying DNA that was not cleaved by the restriction enzyme in the area amplified. See, e.g., U.S. patent application Ser. Nos. 10/971,986; 11/071,013; and 10/971,339. In some embodiments, amplification can be performed using primers that are gene specific. Alternatively, adaptors can be added to the ends of the randomly fragmented DNA, the DNA can be digested with a methylation-dependent or methylation-sensitive restriction enzyme, intact DNA can be amplified using primers that hybridize to the adaptor sequences. In this case, a second step can be performed to determine the presence, absence or quantity of a particular gene in an amplified pool of DNA. In some embodiments, the DNA is amplified using real-time, quantitative PCR. 
     In some embodiments, the methods comprise quantifying the average methylation density in a target sequence within a population of genomic DNA. In some embodiments, the method comprises contacting genomic DNA with a methylation-dependent restriction enzyme or methylation-sensitive restriction enzyme under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved; quantifying intact copies of the locus; and comparing the quantity of amplified product to a control value representing the quantity of methylation of control DNA, thereby quantifying the average methylation density in the locus compared to the methylation density of the control DNA. 
     The quantity of methylation of a locus of DNA can be determined by providing a sample of genomic DNA comprising the locus, cleaving the DNA with a restriction enzyme that is either methylation-sensitive or methylation-dependent, and then quantifying the amount of intact DNA or quantifying the amount of cut DNA at the DNA locus of interest. The amount of intact or cut DNA will depend on the initial amount of genomic DNA containing the locus, the amount of methylation in the locus, and the number (i.e., the fraction) of nucleotides in the locus that are methylated in the genomic DNA. The amount of methylation in a DNA locus can be determined by comparing the quantity of intact DNA or cut DNA to a control value representing the quantity of intact DNA or cut DNA in a similarly-treated DNA sample. The control value can represent a known or predicted number of methylated nucleotides. Alternatively, the control value can represent the quantity of intact or cut DNA from the same locus in another (e.g., normal, non-diseased) cell or a second locus. 
     By using at least one methylation-sensitive or methylation-dependent restriction enzyme under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved and subsequently quantifying the remaining intact copies and comparing the quantity to a control, average methylation density of a locus can be determined. A methylation-sensitive enzyme is one which cuts DNA if its recognition sequence is unmethylated while a methylation-dependent enzyme cuts DNA if its recognition sequence is methylated. If the methylation-sensitive restriction enzyme is contacted to copies of a DNA locus under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved, then the remaining intact DNA will be directly proportional to the methylation density, and thus may be compared to a control to determine the relative methylation density of the locus in the sample. Similarly, if a methylation-dependent restriction enzyme is contacted to copies of a DNA locus under conditions that allow for at least some copies of potential restriction enzyme cleavage sites in the locus to remain uncleaved, then the remaining intact DNA will be inversely proportional to the methylation density, and thus may be compared to a control to determine the relative methylation density of the locus in the sample. Such assays are disclosed in, e.g., U.S. patent application Ser. No. 10/971,986. 
     Kits for the above methods can include, e.g., one or more of methylation-dependent restriction enzymes, methylation-sensitive restriction enzymes, amplification (e.g., PCR) reagents, probes and/or primers. 
     Quantitative amplification methods (e.g., quantitative PCR or quantitative linear amplification) can be used to quantify the amount of intact DNA within a locus flanked by amplification primers following restriction digestion. Methods of quantitative amplification are disclosed in, e.g., U.S. Pat. Nos. 6,180,349; 6,033,854; and 5,972,602, as well as in, e.g., Gibson et al.,  Genome Research  6:995-1001 (1996); DeGraves, et al.,  Biotechniques  34(1):106-10, 112-5 (2003); Deiman B, et al.,  Mol. Biotechnol.  20(2):163-79 (2002). Amplifications may be monitored in “real time.” 
     Additional methods for detecting DNA methylation can involve genomic sequencing before and after treatment of the DNA with bisulfite. See, e.g., Frommer et al.,  Proc. Natl. Acad. Sci. USA  89:1827-1831 (1992). When sodium bisulfite is contacted to DNA, unmethylated cytosine is converted to uracil, while methylated cytosine is not modified. 
     In some embodiments, restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA is used to detect DNA methylation. See, e.g., Sadri &amp; Hornsby,  Nucl. Acids Res.  24:5058-5059 (1996); Xiong &amp; Laird,  Nucleic Acids Res.  25:2532-2534 (1997). 
     In some embodiments, a methylation-specific PCR (“MSP”) reaction is used alone or in combination with other methods to detect DNA methylation. An MSP assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated verses unmethylated DNA. See, Herman et al.  Proc. Natl. Acad. Sci. USA  93:9821-9826 (1996); U.S. Pat. No. 5,786,146. 
     In some embodiments, a MethyLight assay is used alone or in combination with other methods to detect DNA methylation (see, Eads et al.,  Cancer Res.  59:2302-2306 (1999)). Briefly, in the MethyLight process genomic DNA is converted in a sodium bisulfite reaction (the bisulfite process converts unmethylated cytosine residues to uracil). Amplification of a DNA sequence of interest is then performed using PCR primers that hybridize to CpG dinucleotides. By using primers that hybridize only to sequences resulting from bisulfite conversion of methylated DNA, (or alternatively to unmethylated sequences) amplification can indicate methylation status of sequences where the primers hybridize. Furthermore, the amplification product can be detected with a probe that specifically binds to a sequence resulting from bisulfite treatment of a unmethylated DNA. If desired, both primers and probes can be used to detect methylation status. Thus, kits for use with MethyLight can include sodium bisulfite as well as primers or detectably-labeled probes (including but not limited to Taqman or molecular beacon probes) that distinguish between methylated and unmethylated DNA that have been treated with bisulfite. Other kit components can include, e.g., reagents necessary for amplification of DNA including but not limited to, PCR buffers, deoxynucleotides; and a thermostable polymerase. 
     In some embodiments, a Ms-SNuPE (Methylation-sensitive Single Nucleotide Primer Extension) reaction is used alone or in combination with other methods to detect DNA methylation (see, Gonzalgo &amp; Jones,  Nucleic Acids Res.  25:2529-2531 (1997)). The Ms-SNuPE technique is a quantitative method for assessing methylation differences at specific CpG sites based on bisulfite treatment of DNA, followed by single-nucleotide primer extension (Gonzalgo &amp; Jones, supra). Briefly, genomic DNA is reacted with sodium bisulfite to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. Amplification of the desired target sequence is then performed using PCR primers specific for bisulfite-converted DNA, and the resulting product is isolated and used as a template for methylation analysis at the CpG site(s) of interest. 
     Typical reagents (e.g., as might be found in a typical Ms-SNuPE-based kit) for Ms-SNuPE analysis can include, but are not limited to: PCR primers for specific gene (or methylation-altered DNA sequence or CpG island); optimized PCR buffers and deoxynucleotides; gel extraction kit; positive control primers; Ms-SNuPE primers for a specific gene; reaction buffer (for the Ms-SNuPE reaction); and detectably-labeled nucleotides. Additionally, bisulfite conversion reagents may include: DNA denaturation buffer; sulfonation buffer; DNA recovery regents or kit (e.g., precipitation, ultrafiltration, affinity column); desulfonation buffer; and DNA recovery components. 
     Additional methylation detection methods include, but are not limited to, methylated CpG island amplification (see, Toyota et al.,  Cancer Res.  59:2307-12 (1999)) and those described in, e.g., U.S. Patent Publication 2005/0069879; Rein, et al.  Nucleic Acids Res.  26 (10): 2255-64 (1998); Olek, et al.  Nat. Genet.  17(3): 275-6 (1997); and PCT Publication No. WO 00/70090. 
     More detailed information in relation to several of these generally described methods is provided below: 
     (a) Probe or Primer Design and/or Production 
     Several methods described herein for the diagnosis of a neoplasia use one or more probes and/or primers. Methods for designing probes and/or primers for use in, for example, PCR or hybridization are known in the art and described, for example, in Dieffenbach and Dveksler (Eds) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995). Furthermore, several software packages are publicly available that design optimal probes and/or primers for a variety of assays, e.g. Primer 3 available from the Center for Genome Research, Cambridge, Mass., USA. 
     Clearly, the potential use of the probe or primer should be considered during its design. For example, should the probe or primer be produced for use in a methylation specific PCR or ligase chain reaction (LCR) assay the nucleotide at the 3′ end (or 5′ end in the case of LCR) should preferably correspond to a methylated nucleotide in a nucleic acid. 
     Probes and/or primers useful for detection of a sequence associated with a neoplasia are assessed, for example, to determine those that do not form hairpins, self-prime or form primer dimers (e.g. with another probe or primer used in a detection assay). Furthermore, a probe or primer (or the sequence thereof) is often assessed to determine the temperature at which it denatures from a target nucleic acid (i.e. the melting temperature of the probe or primer, or Tm). Methods for estimating Tm are known in the art and described, for example, in Santa Lucia,  Proc. Natl. Acad. Sci. USA,  95: 1460-1465, 1995 or Bresslauer et al.,  Proc. Natl. Acad. Sci. USA,  83: 3746-3750, 1986. 
     Methods for producing/synthesizing a probe or primer of the present invention are known in the art. For example, oligonucleotide synthesis is described, in Gait (Ed) (In: Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, 1984). For example, a probe or primer may be obtained by biological synthesis (e.g. by digestion of a nucleic acid with a restriction endonuclease) or by chemical synthesis. For short sequences (up to about 100 nucleotides) chemical synthesis is preferable. 
     For longer sequences standard replication methods employed in molecular biology are useful, such as, for example, the use of M13 for single stranded DNA as described by Messing,  Methods Enzymol,  101, 20-78, 1983. Other methods for oligonucleotide synthesis include, for example, phosphotriester and phosphodiester methods (Narang, et al.  Meth. Enzymol  68: 90, 1979) and synthesis on a support (Beaucage, et al.  Tetrahedron Letters  22:1859-1862, 1981) as well as phosphoramidate technique, Caruthers, M. H., et al.,  Methods in Enzymology , Vol. 154, pp. 287-314 (1988), and others described in “Synthesis and Applications of DNA and RNA,” S. A. Narang, editor, Academic Press, New York, 1987, and the references cited therein. Probes comprising locked nucleic acid (LNA) are synthesized as described, for example, in Nielsen et al.  J. Chem. Soc. Perkin Trans.,  1:3423, 1997; Singh and Wengel,  Chem. Commun.  1247, 1998. While, probes comprising peptide-nucleic acid (PNA) are synthesized as described, for example, in Egholm et al.,  Am. Chem. Soc.,  114:1895, 1992; Egholm et al.,  Nature,  365:566, 1993; and Orum et al.,  Nucl. Acids Res.,  21:5332, 1993. 
     (b) Methylation-Sensitive Endonuclease Digestion of DNA 
     In one example, the increased methylation in a sample is determined using a process comprising treating the nucleic acid with an amount of a methylation-sensitive restriction endonuclease enzyme under conditions sufficient for nucleic acid to be digested and then detecting the fragments produced. Exemplary methylation-sensitive endonucleases include, for example, HhaI or HpaII. Preferably, assays include internal controls that are digested with a methylation-insensitive enzyme having the same specificity as the methylation-sensitive enzyme employed. For example, the methylation-insensitive enzyme MspI is an isoschizomer of the methylation-sensitive enzyme HpaII. 
     Hybridization Assay Formats 
     In one example, the digestion of nucleic acid is detected by selective hybridization of a probe or primer to the undigested nucleic acid. Alternatively, the probe selectively hybridizes to both digested and undigested nucleic acid but facilitates differentiation between both forms, e.g., by electrophoresis. Suitable detection methods for achieving selective hybridization to a hybridization probe include, for example, Southern or other nucleic acid hybridization (Kawai et al.,  Mol. Cell. Biol.  14:7421-7427, 1994; Gonzalgo et al.,  Cancer Res.  57:594-599, 1997). 
     Suitable hybridization conditions are determined based on the melting temperature (Tm) of a nucleic acid duplex comprising the probe. The skilled artisan will be aware that optimum hybridization reaction conditions should be determined empirically for each probe, although some generalities can be applied. Preferably, hybridizations employing short oligonucleotide probes are performed at low to medium stringency. In the case of a GC rich probe or primer or a longer probe or primer a high stringency hybridization and/or wash is preferred. A high stringency is defined herein as being a hybridization and/or wash carried out in about 0.1×SSC buffer and/or about 0.1% (w/v) SDS, or lower salt concentration, and/or at a temperature of at least 65° C., or equivalent conditions. Reference herein to a particular level of stringency encompasses equivalent conditions using wash/hybridization solutions other than SSC known to those skilled in the art. 
     In accordance with the present example, a difference in the fragments produced for the test sample and a negative control sample is indicative of the subject having a neoplasia. Similarly, in cases where the control sample comprises data from a tumor, cancer tissue or a cancerous cell or pre-cancerous cell, similarity, albeit not necessarily absolute identity, between the test sample and the control sample is indicative of a positive diagnosis (i.e. cancer). 
     Amplification Assay Formats 
     In an alternative example, the fragments produced by the restriction enzyme are detected using an amplification system, such as, for example, polymerase chain reaction (PCR), rolling circle amplification (RCA), inverse polymerase chain reaction (iPCR), in situ PCR (Singer-Sam et al.,  Nucl. Acids Res.  18:687, 1990), strand displacement amplification (SDA) or cycling probe technology. 
     Methods of PCR are known in the art and described, for example, by McPherson et al., PCR: A Practical Approach. (series eds, D. Rickwood and B. D. Hames), IRL Press Limited, Oxford. pp 1-253, 1991 and by Dieffenbach (ed) and Dveksler (ed) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbour Laboratories, NY, 1995), the contents of which are each incorporated in their entirety by way of reference. Generally, for PCR two non-complementary nucleic acid primer molecules comprising at least about 18 nucleotides in length, and more preferably at least 20-30 nucleotides in length are hybridized to different strands of a nucleic acid template molecule at their respective annealing sites, and specific nucleic acid molecule copies of the template that intervene the annealing sites are amplified enzymatically. Amplification products may be detected, for example, using electrophoresis and detection with a detectable marker that binds nucleic acids. Alternatively, one or more of the oligonucleotides are labeled with a detectable marker (e.g. a fluorophore) and the amplification product detected using, for example, a lightcycler (Perkin Elmer, Wellesley, Mass., USA, Roche Applied Science, Indianapolis, Ind., USA). 
     Strand displacement amplification (SDA) utilizes oligonucleotide primers, a DNA polymerase and a restriction endonuclease to amplify a target sequence. The oligonucleotides are hybridized to a target nucleic acid and the polymerase is used to produce a copy of the region intervening the primer annealing sites. The duplexes of copied nucleic acid and target nucleic acid are then nicked with an endonuclease that specifically recognizes a sequence at the beginning of the copied nucleic acid. The DNA polymerase recognizes the nicked DNA and produces another copy of the target region at the same time displacing the previously generated nucleic acid. The advantage of SDA is that it occurs in an isothermal format, thereby facilitating high-throughput automated analysis. 
     Cycling Probe Technology uses a chimeric synthetic primer that comprises DNA-RNA-DNA that is capable of hybridizing to a target sequence. Upon hybridization to a target sequence the RNA-DNA duplex formed is a target for RNaseH thereby cleaving the primer. The cleaved primer is then detected, for example, using mass spectrometry or electrophoresis. 
     For primers that flank or are adjacent to a methylation-sensitive endonuclease recognition site, it is preferred that such primers flank only those sites that are hypermethylated in neoplasia to ensure that a diagnostic amplification product is produced. In this regard, an amplification product will only be produced when the restriction site is not cleaved, i.e., when it is methylated. Accordingly, detection of an amplification product indicates that the CpG dinucleotide/s of interest is/are methylated. 
     As will be known to the skilled artisan, the precise length of the amplified product will vary depending upon the distance between the primers. Clearly this form of analysis may be used to determine the methylation status of a plurality of CpG dinucleotides provided that each dinucleotide is within a methylation sensitive restriction endonuclease site. In these methods, one or more of the primers may be labeled with a detectable marker to facilitate rapid detection of amplified nucleic acid, for example, a fluorescent label (e.g. Cy5 or Cy3) or a radioisotope (e.g.  32 P). 
     The amplified nucleic acids are generally analyzed using, for example, non-denaturing agarose gel electrophoresis, non-denaturing polyacrylamide gel electrophoresis, mass spectrometry, liquid chromatography (e.g. HPLC or dHPLC), or capillary electrophoresis. (e.g. MALDI-TOF). High throughput detection methods, such as, for example, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), electrospray ionization (ESI), mass spectrometry (including tandem mass spectrometry, e.g. LC MS/MS), biosensor technology, evanescent fiber-optics technology or DNA chip technology (e.g., WO98/49557; WO 96/17958; Fodor et al.,  Science  767-773, 1991; U.S. Pat. Nos. 5,143,854; and 5,837,832, the contents of which are all incorporated herein by reference), are especially preferred for all assay formats described herein. Alternatively, amplification of a nucleic acid may be continuously monitored using a melting curve analysis method as described herein and/or in, for example, U.S. Pat. No. 6,174,670, which is incorporated herein by reference. 
     (c) Other Assay Formats 
     In an alternative example, the increased methylation in a sample is determined by performing a process comprising treating chromatin containing the nucleic acid with an amount of DNaseI under conditions sufficient for nucleic acid to be digested and then detecting the fragments produced. This assay format is predicated on the understanding that chromatin containing methylated DNA, e.g., hyper methylated DNA, has a more tightly-closed conformation than non-hyper methylated DNA and, as a consequence, is less susceptible to endonuclease digestion by DNase I. 
     In accordance with this method , DNA fragments of different lengths are produced by DNase I digestion of methylated compared to non-methylated DNA. Such different DNA fragments are detected, for example, using an assay described earlier. Alternatively, the DNA fragments are detected using PCR-SSCP essentially as described, for example, in Gregory and Feil,  Nucleic Acids Res.,  27, e32i-e32iv, 1999. In adapting PCR-SSCP to the present invention, amplification primers flanking or comprising one or more CpG dinucleotides in a nucleic acid that are resistant to DNase I digestion in a neoplasia sample but not resistant to DNase I digestion in a healthy/normal control or healthy/normal test sample are used to amplify the DNase I-generated fragments. In this case, the production of a specific nucleic acid fragment using DNase I is diagnostic of neoplasia, because the DNA is not efficiently degraded. In contrast, template DNA from a healthy/normal subject sample is degraded by the action of DNase I and, as a consequence, amplification fails to produce a discrete amplification product. Alternative methods to PCR-SSCP, such as for example, PCR-dHPLC are also known in the art and contemplated by the present invention. 
     (d) Selective Mutagenesis of Non Methylated DNA 
     In an alternative method the increased methylation in a sample is determined using a process comprising treating the nucleic acid with an amount of a compound that selectively mutates a non-methylated cytosine residue within a CpG dinucleotide under conditions sufficient to induce mutagenesis. 
     Preferred compounds mutate cytosine to uracil or thymidine, such as, for example, a salt of bisulfite, e.g., sodium bisulfite or potassium bisulfite (Frommer et al., 1992, supra). Bisulfite treatment of DNA is known to distinguish methylated from non-methylated cytosine residues, by mutating cytosine residues that are not protected by methylation, including cytosine residues that are not within a CpG dinucleotide or that are positioned within a CpG dinucleotide that is not subject to methylation. 
     Sequence Based Detection 
     In one example, the presence of one or more mutated nucleotides or the number of mutated sequences is determined by sequencing mutated DNA. One form of analysis comprises amplifying mutated nucleic acid using an amplification reaction described herein, for example, PCR. The amplified product is then directly sequenced or cloned and the cloned product sequenced. Methods for sequencing DNA are known in the art and include for example, the dideoxy chain termination method or the Maxam-Gilbert method (see Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989) or Zyskind et al., Recombinant DNA Laboratory Manual, (Acad. Press, 1988)). 
     As the treatment of nucleic acid with a compound, such as, for example, bisulfite results in non-methylated cytosines being mutated to uracil (and hence thymidine after an amplification process), analysis of the sequence determines the presence or absence of a methylated nucleotide. For example, by comparing the sequence obtained using a control sample or a sample that has not been treated with bisulfite, or the known nucleotide sequence of the region of interest with a treated sample facilitates the detection of differences in the nucleotide sequence. Any thymine residue detected at the site of a cytosine in the treated sample compared to a control or untreated sample may be considered to be caused by mutation as a result of bisulfite treatment. Suitable methods for the detection of methylation using sequencing of bisulfite treated nucleic acid are described, for example, in Frommer et al., 1992, supra or Clark et al.,  Nucl. Acids Res.  22:2990-2997, 1994. 
     In another method, the presence of a mutated or non-mutated nucleotide in a bisulfite treated sample is detected using pyrosequencing, such as, for example, as described in Uhlmann et al., Electrophoresis, 23: 4072-4079, 2002. Essentially this method is a form of real-time sequencing that uses a primer that hybridizes to a site adjacent or close to the site of a cytosine that is methylated. Following hybridization of the primer and template in the presence of a DNA polymerase each of four modified deoxynucleotide triphosphates are added separately according to a predetermined dispensation order. Only an added nucleotide that is complementary to the bisulfite treated sample is incorporated and inorganic pyrophosphate (PPi) is liberated. The PPi then drives a reaction resulting in production of detectable levels of light. Such a method allows determination of the identity of a specific nucleotide adjacent to the site of hybridization of the primer. 
     Methods of solid phase pyrosequencing are known in the art and reviewed in, for example, Landegren et al.,  Genome Res.,  8(8): 769-776, 1998. Such methods enable the high-throughput detection of methylation of a number of CpG dinucleotides. 
     A related method for determining the sequence of a bisulfite treated nucleotide is methylation-sensitive single nucleotide primer extension (Me-SnuPE) or SNaPmeth. Suitable methods are described, for example, in Gonzalgo and Jones, 1997, supra, or Uhlmann et al.,  Electrophoresis,  23:4072-4079, 2002. An oligonucleotide is used that hybridizes to the region of a nucleic acid adjacent to the site of a cytosine that is methylated. This oligonucleotide is then used in a primer extension protocol with a polymerase and a free nucleotide diphosphate or dideoxynucleotide triphosphate that corresponds to either or any of the possible bases that occur at this site following bisulfite treatment (i.e., thymine or cytosine). Preferably, the nucleotide-diphosphate is labeled with a detectable marker (e.g. a fluorophore). Following primer extension, unbound labeled nucleotide diphosphates are removed, e.g. using size exclusion chromatography or electrophoresis, or hydrolyzed, using for example, alkaline phosphatase, and the incorporation of the labeled nucleotide to the oligonucleotide is detected, indicating the base that is present at the site. 
     Clearly other high throughput sequencing methods are encompassed by the present invention. Such methods include, for example, solid phase minisequencing (as described, for example, in Southern et al.,  Genomics,  13:1008-1017, 1992), or minisequencing with FRET (as described, for example, in Chen and Kwok,  Nucleic Acids Res.  25:347-353, 1997). 
     Restriction Endonuclease-Based Assay Format 
     In one method, the presence of a non-mutated sequence is detected using combined bisulfite restriction analysis (COBRA) essentially as described in Xiong and Laird, 2001, supra. This method exploits the differences in restriction enzyme recognition sites between methylated and unmethylated nucleic acid after treatment with a compound that selectively mutates a non-methylated cytosine residue, e.g., bisulfite. 
     Following bisulfite treatment a region of interest comprising one or more CpG dinucleotides that are methylated and are included in a restriction endonuclease recognition sequence is amplified using an amplification reaction described herein, e.g., PCR. The amplified product is then contacted with the restriction enzyme that cleaves at the site of the CpG dinucleotide for a time and under conditions sufficient for cleavage to occur. A restriction site may be selected to indicate the presence or absence of methylation. For example, the restriction endonuclease TaqI cleaves the sequence TCGA, following bisulfite treatment of a non-methylated nucleic acid the sequence will be TTGA and, as a consequence, will not be cleaved. The digested and/or non-digested nucleic acid is then detected using a detection means known in the art, such as, for example, electrophoresis and/or mass spectrometry. The cleavage or non-cleavage of the nucleic acid is indicative of cancer in a subject. Clearly, this method may be employed in either a positive read-out or negative read-out system for the diagnosis of a cancer. 
     Positive Read-Out Assay Format 
     In one embodiment, the assay format of the invention comprises a positive read-out system in which DNA from a sample that has been treated, for example, with bisulfite is detected as a positive signal. Preferably, the non-hypermethylated DNA from a healthy or normal control subject is not detected or only weakly detected. 
     In a preferred embodiment, the increased methylation in a subject sample is determined using a process comprising: 
     (i) treating the nucleic acid with an amount of a compound that selectively mutates a non-methylated cytosine residue under conditions sufficient to induce mutagenesis thereby producing a mutated nucleic acid; 
     (ii) hybridizing a nucleic acid to a probe or primer comprising a nucleotide sequence that is complementary to a sequence comprising a methylated cytosine residue under conditions such that selective hybridization to the non-mutated nucleic acid occurs; and 
     (iii) detecting the selective hybridization. 
     In this context, the term “selective hybridization” means that hybridization of a probe or primer to the non-mutated nucleic acid occurs at a higher frequency or rate, or has a higher maximum reaction velocity, than hybridization of the same probe or primer to the corresponding mutated sequence. Preferably, the probe or primer does not hybridize to the non-methylated sequence carrying the mutation(s) under the reaction conditions used. 
     Hybridization-Based Assay Format 
     In one embodiment, the hybridization is detected using Southern, dot blot, slot blot or other nucleic acid hybridization means (Kawai et al., 1994, supra; Gonzalgo et al., 1997, supra). Subject to appropriate probe selection, such assay formats are generally described herein above and apply mutatis mutandis to the presently described selective mutagenesis approach. 
     Preferably, a ligase chain reaction format is employed to distinguish between a mutated and non-mutated nucleic acid. Ligase chain reaction (described in EP 320,308 and U.S. Pat. No. 4,883,750) uses at least two oligonucleotide probes that anneal to a target nucleic acid in such a way that they are juxtaposed on the target nucleic acid. In a ligase chain reaction assay, the target nucleic acid is hybridized to a first probe that is complementary to a diagnostic portion of the target sequence (the diagnostic probe) e.g., a nucleic acid comprising one or more methylated CpG dinucleotide(s), and with a second probe that is complementary to a nucleotide sequence contiguous with the diagnostic portion (the contiguous probe), under conditions wherein the diagnostic probe remains bound substantially only to the target nucleic acid. The diagnostic and contiguous probes can be of different lengths and/or have different melting temperatures such that the stringency of the hybridization can be adjusted to permit their selective hybridization to the target, wherein the probe having the higher melting temperature is hybridized at higher stringency and, following washing to remove unbound and/or non-selectively bound probe, the other probe having the lower melting temperature is hybridized at lower stringency. The diagnostic probe and contiguous probe are then covalently ligated such as, for example, using T4 DNA ligase, to thereby produce a larger target probe that is complementary to the target sequence, and the probes that are not ligated are removed by modifying the hybridization stringency. In this respect, probes that have not been ligated will selectively hybridize under lower stringency hybridization conditions than probes that have been ligated. Accordingly, the stringency of the hybridization can be increased to a stringency that is at least as high as the stringency used to hybridize the longer probe, and preferably at a higher stringency due to the increased length contributed by the shorter probe following ligation. 
     In another example, one or both of the probes is labeled such that the presence or absence of the target sequence can be tested by melting the target-probe duplex, eluting the dissociated probe, and testing for the label(s). Where both probes are labeled, different ligands are used to permit distinction between the ligated and unligated probes, in which case the presence of both labels in the same eluate fraction confirms the ligation event. If the target nucleic acid is bound to a solid matrix e.g., in a Southern hybridization, slot blot, dot blot, or microchip assay format, the presence of both the diagnostic and contiguous probes can be determined directly. 
     Methylation specific microarrays (MSO) are also useful for differentiating between a mutated and non-mutated sequence. A suitable method is described, for example, in Adorjan et al.  Nucl. Acids Res.,  30: e21, 2002. MSO uses nucleic acid that has been treated with a compound that selectively mutates a non-methylated cytosine residue (e.g., bisulfite) as template for an amplification reaction that amplifies both mutant and non-mutated nucleic acid. The amplification is performed with at least one primer that comprises a detectable label, such as, for example, a fluorophore, e.g., Cy3 or Cy5. 
     To produce a microarray for detection of mutated nucleic acid oligonucleotides are spotted onto, for example, a glass slide, preferably, with a degree of redundancy (for example, as described in Golub et al.,  Science,  286:531-537, 1999). Preferably, for each CpG dinucleotide analyzed two different oligonucleotides are used. Each oligonucleotide comprises a sequence N 2 -16CGN 2 -16 or N 2 -16TGN 2 -16 (wherein N is a number of nucleotides adjacent or juxtaposed to the CpG dinucleotide of interest) reflecting the methylated or non-methylated status of the CpG dinucleotides. 
     The labeled amplification products are then hybridized to the oligonucleotides on the microarray under conditions that enable detection of single nucleotide differences. Following washing to remove unbound amplification product, hybridization is detected using, for example, a microarray scanner. Not only does this method allow for determination of the methylation status of a large number of CpG dinucleotides, it is also semi-quantitative, enabling determination of the degree of methylation at each CpG dinucleotide analyzed. As there may be some degree of heterogeneity of methylation in a single sample, such quantification may assist in the diagnosis of cancer. 
     Amplification-Based Assay Format 
     In an alternative example, the hybridization is detected using an amplification system. In methylation-specific PCR formats (MSP; Herman et al.  Proc. Natl. Acad. Sci. USA  93:9821-9826, 1992), the hybridization is detected using a process comprising amplifying the bisulfite-treated DNA. Accordingly, by using one or more probe or primer that anneals specifically to the unmutated sequence under moderate and/or high stringency conditions an amplification product is only produced using a sample comprising a methylated nucleotide. Alternate assays that provide for selective amplification of either the methylated or the unmethylated component from a mixture of bisulfite-treated DNA are provided by Cottrell et al.,  Nucl. Acids Res.  32: e10, 2003 (HeavyMethyl PCR), Rand et al.  Nucl. Acids Res.  33:e127, 2005 (Headloop PCR), Rand et al.  Epigenetics  1:94-100, 2006 (Bisulfite Differential Denaturation PCR) and PCT/AU07/000389 (End-specific PCR). 
     Any amplification assay format described herein can be used, such as, for example, polymerase chain reaction (PCR), rolling circle amplification (RCA), inverse polymerase chain reaction (iPCR), in situ PCR (Singer-Sam et al., 1990, supra), strand displacement amplification, or cycling probe technology. PCR techniques have been developed for detection of gene mutations (Kuppuswamy et al.,  Proc. Natl. Acad. Sci. USA  88:1143-1147, 1991) and quantitation of allelic-specific expression (Szabo and Mann,  Genes Dev.  9: 3097-3108, 1995; and Singer-Sam et al.,  PCR Methods Appl.  1: 160-163, 1992). Such techniques use internal primers, which anneal to a PCR-generated template and terminate immediately 5′ of the single nucleotide to be assayed. Such as format is readily combined with ligase chain reaction as described herein above. The use of a real-time quantitative assay format is also useful. Subject to the selection of appropriate primers, such assay formats are generally described herein above and apply mutatis mutandis to the presently described selective mutagenesis approach. 
     Methylation-specific melting-curve analysis (essentially as described in Worm et al.,  Clin. Chem.,  47:1183-1189, 2001) is also contemplated by the present invention. This process exploits the difference in melting temperature in amplification products produced using bisulfite treated methylated or unmethylated nucleic acid. In essence, non-discriminatory amplification of a bisulfite treated sample is performed in the presence of a fluorescent dye that specifically binds to double stranded DNA (e.g., SYBR Green I). By increasing the temperature of the amplification product while monitoring fluorescence the melting properties and thus the sequence of the amplification product is determined. A decrease in the fluorescence reflects melting of at least a domain in the amplification product. The temperature at which the fluorescence decreases is indicative of the nucleotide sequence of the amplified nucleic acid, thereby permitting the nucleotide at the site of one or more CpG dinucleotides to be determined. As the sequence of the nucleic acids amplified using the present invention 
     The present invention also encompasses the use of real-time quantitative forms of PCR, such as, for example, TaqMan (Holland et al.,  Proc. Natl. Acad. Sci. USA,  88:7276-7280, 1991; Lee et al.,  Nucleic Acid Res.  21:3761-3766, 1993) to perform this embodiment. For example, the MethylLight method of Eads et al.,  Nucl. Acids Res.  28: E32, 2000 uses a modified TaqMan assay to detect methylation of a CpG dinucleotide. Essentially, this method comprises treating a nucleic acid sample with bisulfite and amplifying nucleic acid comprising one or more CpG dinucleotides that are methylated in a neoplastic cell and not in a control sample using an amplification reaction, e.g., PCR. The amplification reaction is performed in the presence of three oligonucleotides, a forward and reverse primer that flank the region of interest and a probe that hybridizes between the two primers to the site of the one or more methylated CpG dinucleotides. The probe is dual labeled with a 5′ fluorescent reporter and a 3′ quencher (or vice versa). When the probe is intact, the quencher dye absorbs the fluorescence of the reporter due to their proximity. Following annealing of to the PCR product the probe is cleaved by 5′ to 3′ exonuclease activity of, for example, Taq DNA polymerase. This cleavage releases the reporter from the quencher thereby resulting in an increased fluorescence signal that can be used to estimate the initial template methylation level. By using a probe or primer that selectively hybridizes to unmutated nucleic acid (i.e. methylated nucleic acid) the level of methylation is determined, e.g., using a standard curve. 
     Alternatively, rather than using a labeled probe that requires cleavage, a probe, such as, for example, a Molecular Beacon is used (see, for example, Mhlanga and Malmberg,  Methods  25:463-471, 2001). Molecular beacons are single stranded nucleic acid molecules with a stem-and-loop structure. The loop structure is complementary to the region surrounding the one or more CpG dinucleotides that are methylated in a neoplastic sample and not in a control sample. The stem structure is formed by annealing two “arms” complementary to each other, which are on either side of the probe (loop). A fluorescent moiety is bound to one arm and a quenching moiety that suppresses any detectable fluorescence when the molecular beacon is not bound to a target sequence is bound to the other arm. Upon binding of the loop region to its target nucleic acid the arms are separated and fluorescence is detectable. However, even a single base mismatch significantly alters the level of fluorescence detected in a sample. Accordingly, the presence or absence of a particular base is determined by the level of fluorescence detected. Such an assay facilitates detection of one or more unmutated sites (i.e. methylated nucleotides) in a nucleic acid. 
     Fluorescently labeled locked nucleic acid (LNA) molecules or fluorescently labeled protein-nucleic acid (PNA) molecules are useful for the detection of nucleotide differences (e.g., as described in Simeonov and Nikiforov,  Nucleic Acids Research,  30(17):1-5, 2002). LNA and PNA molecules bind, with high affinity, to nucleic acid, in particular, DNA. Fluorophores (in particular, rhodamine or hexachlorofluorescein) conjugated to the LNA or PNA probe fluoresce at a significantly greater level upon hybridization of the probe to target nucleic acid. However, the level of increase of fluorescence is not enhanced to the same level when even a single nucleotide mismatch occurs. Accordingly, the degree of fluorescence detected in a sample is indicative of the presence of a mismatch between the LNA or PNA probe and the target nucleic acid, such as, in the presence of a mutated cytosine in a methylated CpG dinucleotide. Preferably, fluorescently labeled LNA or PNA technology is used to detect at least a single base change in a nucleic acid that has been previously amplified using, for example, an amplification method known in the art and/or described herein. 
     As will be apparent to the skilled artisan, LNA or PNA detection technology is amenable to a high-throughput detection of one or more markers by immobilizing an LNA or PNA probe to a solid support, as described in Orum et al.,  Clin. Chem.  45:1898-1905, 1999. 
     Alternatively, a real-time assay, such as, for example, the so-called HeavyMethyl assay (Cottrell et al., 2003, supra) is used to determine the presence or level of methylation of nucleic acid in a test sample. Essentially, this method uses one or more non-extendible nucleic acid (e.g., oligonucleotide) blockers that bind to bisulfite-treated nucleic acid in a methylation specific manner (i.e., the blocker/s bind specifically to unmutated DNA under moderate to high stringency conditions). An amplification reaction is performed using one or more primers that may optionally be methylation specific but that flank the one or more blockers. In the presence of unmethylated nucleic acid (i.e., non-mutated DNA) the blocker/s bind and no PCR product is produced. Using a TaqMan assay essentially as described supra the level of methylation of nucleic acid in a sample is determined. 
     Other amplification-based methods for detecting methylated nucleic acid following treatment with a compound that selectively mutates a non-methylated cytosine residue include, for example, methylation-specific single stranded conformation analysis (MS-SSCA) (Bianco et al.,  Hum. Mutat.,  14:289-293, 1999), methylation-specific denaturing gradient gel electrophoresis (MS-DGGE) (Abrams and Stanton,  Methods Enzymol.,  212:71-74, 1992) and methylation-specific denaturing high-performance liquid chromatography (MS-DHPLC) (Deng et al.  Chin. J. Cancer Res.,  12:171-191, 2000). Each of these methods use different techniques for detecting nucleic acid differences in an amplification product based on differences in nucleotide sequence and/or secondary structure. Such methods are clearly contemplated by the present invention. 
     As with other amplification-based assay formats, the amplification product is analyzed using a range of procedures, including gel electrophoresis, gel filtration, mass spectrometry, and in the case of labeled primers, by identifying the label in the amplification product. In an alternative embodiment, restriction enzyme digestion of PCR products amplified from bisulfite-converted DNA is performed essentially as described by Sadri and Hornsby,  Nucl. Acids Res.  24:5058-5059, 1996; and Xiong and Laird,  Nucl. Acids Res.  25:2532-2534, 1997), to analyze the product formed. 
     High throughput detection methods, such as, for example, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), electrospray ionization (ESI), Mass spectrometry (including tandem mass spectrometry, e.g. LC MS/MS), biosensor technology, evanescent fiber-optics technology or DNA chip technology, can also be employed. 
     As with the other assay formats described herein that utilize hybridization and/or amplification detection systems, combinations of such processes as described herein above are particularly contemplated by the selective mutagenesis-based assay formats of the present invention. In one example, the increased methylation is detected by performing a process comprising: 
     (i) treating the nucleic acid with an amount of a compound that selectively mutates a non-methylated cytosine residue within a CpG dinucleotide under conditions sufficient to induce mutagenesis thereby producing a mutated nucleic acid; 
     (ii) hybridizing the nucleic acid to two non-overlapping and non-complementary primers each of which comprises a nucleotide sequence that is complementary to a sequence in the DNA comprising a methylated cytosine residue under conditions such that hybridization to the non-mutated nucleic acid occurs; 
     (iii) amplifying nucleic acid intervening the hybridized primers thereby producing a DNA fragment consisting of a sequence that comprises a primer sequence; 
     (iv) hybridizing the amplified DNA fragment to a probe comprising a nucleotide sequence that corresponds or is complementary to a sequence comprising a methylated cytosine residue under conditions such that hybridization to the non-mutated nucleic acid occurs; and detecting the hybridization. 
     Negative Read-Out Assays 
     In another example, the assay format comprises a negative read-out system in which reduced methylation of DNA from a healthy/normal control sample is detected as a positive signal and preferably, methylated DNA from a neoplastic sample is not detected or is only weakly detected. 
     In a preferred embodiment, the reduced methylation is determined using a process comprising: 
     (i) treating the nucleic acid with an amount of a compound that selectively mutates a non-methylated cytosine residue within a CpG island under conditions sufficient to induce mutagenesis thereby producing a mutated nucleic acid; 
     (ii) hybridizing the nucleic acid to a probe or primer comprising a nucleotide sequence that is complementary to a sequence comprising the mutated cytosine residue under conditions such that selective hybridization to the mutated nucleic acid occurs; and 
     (iii) detecting the selective hybridization. 
     In this context, the term “selective hybridization” means that hybridization of a probe or primer to the mutated nucleic acid occurs at a higher frequency or rate, or has a higher maximum reaction velocity, than hybridization of the same probe or primer to the corresponding non-mutated sequence. Preferably, the probe or primer does not hybridize to the methylated sequence (or non-mutated sequence) under the reaction conditions used. 
     Hybridization-Based Assay Format 
     In one embodiment the hybridization is detected using Southern, dot blot, slot blot or other nucleic acid hybridization means (Kawai et al., 1994, supra; Gonzalgo et al., 1997, supra). Subject to appropriate probe selection, such assay formats are generally described herein above and apply mutatis mutandis to the presently described selective mutagenesis approach. Preferably, a ligase chain reaction format is employed to distinguish between a non-mutated and mutated nucleic acid. In this respect, the assay requirements and conditions are as described herein above for positive read-out assays and apply mutatis mutandis to the present format. However the selection of probes will differ. For negative read-out assays, one or more probes are selected that selectively hybridize to the mutated sequence rather than the non-mutated sequence. 
     Preferably, the ligase chain reaction probe(s) have 3′-terminal and/or 5′-terminal sequences that comprise a CpG dinucleotide that is not methylated in a healthy control sample, but is hypermethylated in cancer, such that the diagnostic probe and contiguous probe are capable of being ligated only when the cytosine of the CpG dinucleotide is mutated to thymidine e.g., in the case of a non-methylated cytosine residue. 
     As will be apparent to the skilled artisan the MSO method described supra is amenable to either or both positive and/or negative readout assays. This is because the assay described detects both mutated and non-mutated sequences thereby facilitating determining the level of methylation. However, an assay detecting only methylated or non-methylated sequences is contemplated by the invention. 
     Amplification-Based Assay Format 
     In an alternative example, the hybridization is detected using an amplification system using any amplification assay format as described herein above for positive read-out assay albeit using primers (and probes where applicable) selectively hybridize to a mutated nucleic acid. 
     In adapting the HeavyMethyl assay described supra to a negative read-out format, the blockers that bind to bisulfite-treated nucleic acid in a methylation specific manner bind specifically to mutated DNA under moderate to high stringency conditions. An amplification reaction is performed using one or more primers that may optionally be methylation specific (i.e. only bind to mutated nucleic acid) but that flank the one or more blockers. In the presence of methylated nucleic acid (i.e., mutated DNA) the blocker/s bind and no PCR product is produced. 
     In one example, the reduced methylation in the normal/healthy control subject is detected by performing a process comprising: 
     (i) treating the nucleic acid with an amount of a compound that selectively mutates non-methylated cytosine residues under conditions sufficient to induce mutagenesis thereby producing a mutated nucleic acid; 
     (ii) hybridizing the nucleic acid to two non-overlapping and non-complementary primers each of which comprises a nucleotide sequence that is complementary to a sequence in the DNA comprising a mutated cytosine residue under conditions such that hybridization to the mutated nucleic acid occurs; 
     (iii) amplifying nucleic acid intervening the hybridized primers thereby producing a DNA fragment consisting of a sequence that comprises a primer sequence; 
     (iv) hybridizing the amplified DNA fragment to a probe comprising a nucleotide sequence that corresponds or is complementary to a sequence comprising a mutated cytosine residue under conditions such that hybridization to the mutated nucleic acid occurs; and 
     (v) detecting the hybridization. 
     As will be apparent to the skilled artisan a negative read-out assay preferably includes a suitable control sample to ensure that the negative result is caused by methylated nucleic acid rather than a reaction failing. 
     This invention also provides kits for the detection and/or quantification of the diagnostic sequences of the invention, or expression or methylation thereof using the methods described herein. 
     For kits for detection of methylation, the kits of the invention can comprise at least one polynucleotide that hybridizes to at least one of the diagnostic sequences of the invention and at least one reagent for detection of gene methylation. Reagents for detection of methylation include, e.g., sodium bisulfite, polynucleotides designed to hybridize to sequence that is the product of a biomarker sequence of the invention if the biomarker sequence is not methylated (e.g., containing at least one C→U conversion), and/or a methylation-sensitive or methylation-dependent restriction enzyme. The kits may also include control natural or synthetic DNA sequences representing methylated or unmethylated forms of the sequence. The kits can provide solid supports in the form of an assay apparatus that is adapted to use in the assay. The kits may further comprise detectable labels, optionally linked to a polynucleotide, e.g., a probe, in the kit. Other materials useful in the performance of the assays can also be included in the kits, including test tubes, transfer pipettes, and the like. The kits can also include written instructions for the use of one or more of these reagents in any of the assays described herein. 
     As detailed hereinbefore, hypermethylation is associated with transcriptional silencing. Accordingly, in addition to the increased level of methylation of these genes providing a basis upon which to screen for the predisposition to or onset of a large intestine neoplasm, the downregulation in the level of expression of these genes is also diagnostically valuable. In accordance with this aspect of the present invention, reference to a gene “expression product” or “expression of a gene” is a reference to either a transcription product (such as primary RNA or mRNA) or a translation product such as protein. In this regard, one can assess changes to the level of expression of a gene either by screening for changes to the level of expression product which is produced (i.e. RNA or protein), changes to the chromatin proteins with which the gene is associated, for example the presence of histone H3 methylated on lysine at amino acid position number 9 or 27 (repressive modifications) or changes to the DNA itself which acts to downregulate expression, such as changes to the methylation of the DNA. These genes and their gene expression products, whether they be RNA transcripts, changes to the DNA which act to downregulate expression or encoded proteins, are collectively referred to as “neoplastic markers”. 
     Accordingly, another aspect of the present invention is directed to a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the level of expression of a DNA region selected from:
     (i) the region, including 2 kb upstream of the transcription start site, defined by any two or more of Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798   (3) chr6:391739..411443; or   (4) chr12:52400748..52409671; and   (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of any two or more of:
       (1) BCAT1 (2) IKZF1 (3) IRF4 (4) GRASP and (5) CAHM
 
in a biological sample from said individual wherein a lower level of expression of at least one of the DNA regions of group (i) and/or (ii) relative to control levels is indicative of a large intestine neoplasm or a predisposition to the onset of a neoplastic state.
   
       

     In one embodiment, said method is directed to identifying biological samples in which any one of said DNA regions exhibits a higher level of methylation. 
     In another embodiment, said method is directed to identifying biological samples in which two or more of said DNA regions exhibits a higher level of methylation. 
     The method of this aspect of the present invention is predicated on the comparison of the level of the neoplastic markers of a biological sample with the control levels of these markers. The “control level” may be either a “normal level”, which is the level of marker expressed by a corresponding large intestine cell or cellular population which is not neoplastic. 
     As detailed hereinbefore, the normal (or “non-neoplastic”) level may be determined using tissues derived from the same individual who is the subject of testing. However, it would be appreciated that this may be quite invasive for the individual concerned and it is therefore likely to be more convenient to analyze the test results relative to a standard result which reflects individual or collective results obtained from individuals other than the patient in issue. 
     There is more particularly provided a method of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm in an individual, said method comprising assessing the level of expression of one or more genes or transcripts selected from:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393; and   (2) chr7:50344378...50472798;   and optionally one or more of (3) chr6:391739..411443, (4) chr12:52400748..52409671 and (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream or:
       (1) BCAT1; and   (2) IKZF1;   and optionally one or more of (3) IRF4, (4) GRASP and (5) CAHM
 
in a biological sample from said individual wherein a lower level of expression of at least one of the DNA regions of group (i) and/or group (ii) relative to control levels is indicative of a neoplastic large intestine neoplasm or a predisposition to the onset of a neoplastic state.
   
       

     Preferably, said control level is a non-neoplastic level. 
     In one embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798; and   (3) chr6:391739..411443; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCATI (2) IKZF1 and (3) IRF4   
       

     In another embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798; and   (4) chr12:52400748..52409671; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1 (2) IKZF1 and (4) GRASP.   
       

     In still another embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798; and   (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1 (2) IKZF1 and (5) CAHM   
       

     In still yet another embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798   (3) chr6:391739..411443; and   (4) chr12:52400748..52409671; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1 (2) IKZF1 (3) IRF4 and (4) GRASP   
       

     In yet still another embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798   (3) chr6:391739..411443; and   (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of:
       (1) BCAT1 (2) IKZF1 (3) IRF4 and (5) CAHM.   
       

     In a further embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798   (4) chr12:52400748..52409671; and   (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of any one or more of:
       (1) BCAT1 (2) IKZF1 (4) GRASP and (5) CAHM   
       

     In yet another further embodiment of this aspect, the gene marker panel which is screened for is:
     (i) the DNA regions, including 2 kb upstream of the transcription start site, defined by Hg19 coordinates:
       (1) chr12:24962958..25102393   (2) chr7:50344378...50472798   (3) chr6:391739..411443; or   (4) chr12:52400748..52409671; and   (5) chr6:163834097..163834982; or   
       (ii) the gene region, including 2 kb upstream of any one or more of:
       (1) BCAT1 (2) IKZF1 (3) IRF4 (4) GRASP and (5) CAHM.   
       

     In one embodiment, said method is directed to identifying biological samples in which any one of said DNA regions exhibits a higher level of methylation. 
     In another embodiment, said method is directed to identifying biological samples in which two or more of said DNA regions exhibits a higher level of methylation. 
     As detailed hereinbefore, the present invention is designed to screen for a neoplastic cell or cellular population, which is located in the large intestine. Accordingly, reference to “cell or cellular population” should be understood as a reference to an individual cell or a group of cells. Said group of cells may be a diffuse population of cells, a cell suspension, an encapsulated population of cells or a population of cells which take the form of tissue. 
     Reference to “expression” should be understood as a reference to the transcription and/or translation of a nucleic acid molecule. Reference to “RNA” should be understood to encompass reference to any form of RNA, such as primary RNA or mRNA or non-translated RNA (e.g. miRNAs etc.). Without limiting the present invention in any way, the modulation of gene transcription leading to increased or decreased RNA synthesis may also correlate with the translation of some of these RNA transcripts (such as mRNA) to produce a protein product. Accordingly, the present invention also extends to detection methodology which is directed to screening for modulated levels or patterns of the neoplastic marker protein products as an indicator of the neoplastic state of a cell or cellular population. Although one method is to screen for mRNA transcripts and/or the corresponding protein product, it should be understood that the present invention is not limited in this regard and extends to screening for any other form of neoplastic marker expression product such as, for example, a primary RNA transcript. 
     In terms of screening for the downregulation of expression of a marker it would also be well known to the person of skill in the art that changes which are detectable at the DNA level are indicative of changes to gene expression activity and therefore changes to expression product levels. Such changes include but are not limited to, changes to DNA methylation. Accordingly, reference herein to “screening the level of expression” and comparison of these “levels of expression” to control “levels of expression” should be understood as a reference to assessing DNA factors which are related to transcription, such as gene/DNA methylation patterns. These have, in part, been described in detail hereinbefore. 
     It would also be known to a person skilled in the art that changes in the structure of chromatin are indicative of changes in gene expression. Silencing of gene expression is often associated with modification of chromatin proteins, methylation of lysines at either or both positions 9 and 27 of histone H3 being well studied examples, while active chromatin is marked by acetylation of lysine 9 of histone H3. Thus association of gene sequences with chromatin carrying repressive or active modifications can be used to make an assessment of the expression level of a gene. 
     Reference to “nucleic acid molecule” should be understood as a reference to both deoxyribonucleic acid molecules and ribonucleic acid molecules and fragments thereof. The present invention therefore extends to both directly screening for mRNA levels in a biological sample or screening for the complementary cDNA which has been reverse-transcribed from an mRNA population of interest. It is well within the skill of the person of skill in the art to design methodology directed to screening for either DNA or RNA. As detailed above, the method of the present invention also extends to screening for the protein product translated from the subject mRNA or the genomic DNA itself. 
     In one preferred embodiment, the level of gene expression is measured by reference to genes which encode a protein product and, more particularly, said level of expression is measured at the protein level. 
     In another particularly preferred embodiment, said gene expression is assessed by the association of DNA with chromatin proteins carrying repressive modifications, for example, methylation of lysines 9 or 27 of histone H3. 
     The present invention should be understood to encompass methods of detection based on identifying both proteins and/or nucleic acid molecules in one or more biological samples. This may be of particular significance to the extent that some of the neoplastic markers of interest may correspond to genes or gene fragments which do not encode a protein product. Accordingly, to the extent that this occurs it would not be possible to test for a protein and the subject marker would have to be assessed on the basis of transcription expression profiles or changes to genomic DNA. 
     The term “protein” should be understood to encompass peptides, polypeptides and proteins (including protein fragments). The protein may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins. Reference herein to a “protein” includes a protein comprising a sequence of amino acids as well as a protein associated with other molecules such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins. 
     The proteins encoded by the neoplastic markers of the present invention may be in multimeric form meaning that two or more molecules are associated together. Where the same protein molecules are associated together, the complex is a homomultimer. An example of a homomultimer is a homodimer. Where at least one marker protein is associated with at least one non-marker protein, then the complex is a heteromultimer such as a heterodimer. 
     Reference to a “fragment” should be understood as a reference to a portion of the subject nucleic acid molecule or protein. This is particularly relevant with respect to screening for modulated RNA levels in stool samples since the subject RNA is likely to have been degraded or otherwise fragmented due to the environment of the gut. One may therefore actually be detecting fragments of the subject RNA molecule, which fragments are identified by virtue of the use of a suitably specific probe. 
     Although the preferred method is to detect the expression product or DNA changes of the neoplastic markers for the purpose of diagnosing neoplasia development or predisposition thereto, the detection of converse changes in the levels of said markers may be desired under certain circumstances, for example, to monitor the effectiveness of therapeutic or prophylactic treatment directed to modulating a neoplastic condition, such as adenoma or adenocarcinoma development. For example, where reduced expression of the subject markers indicates that an individual has developed a condition characterized by adenoma or adenocarcinoma development, for example, screening for an increase in the levels of these markers subsequently to the onset of a therapeutic regime may be utilized to indicate reversal or other form of improvement of the subject individual&#39;s condition. The method of the present invention is therefore useful as a one off test or as an on-going monitor of those individuals thought to be at risk of neoplasia development or as a monitor of the effectiveness of therapeutic or prophylactic treatment regimens directed to inhibiting or otherwise slowing neoplasia development. 
     Means of assessing the subject expressed neoplasm markers in a biological sample can be achieved by any suitable method, which would be well known to the person of skill in the art. To this end, it would be appreciated that to the extent that one is examining either a homogeneous cellular population (such as a tumor biopsy or a cellular population which has been enriched from a heterogeneous starting population) or a tissue section, one may utilize a wide range of techniques such as in situ hybridization, assessment of expression profiles by microassays, immunoassays and the like (hereinafter described in more detail) to detect the absence of or downregulation of the level of expression of one or more markers of interest. However, to the extent that one is screening a heterogenous cellular population or a bodily fluid in which heterogeneous populations of cells are found, such as a blood sample, the absence of or reduction in level of expression of a particular marker may be undetectable due to the inherent expression of the marker by non-neoplastic cells which are present in the sample. That is, a decrease in the level of expression of a subgroup of cells may not be detectable. In this situation, a more appropriate mechanism of detecting a reduction in a neoplastic subpopulation of the expression levels of one or more markers of the present invention is via indirect means, such as the detection of epigenetic changes. 
     Methods of detecting changes to gene expression levels (in addition to the methylation analyses hereinbefore described in detail), particularly where the subject biological sample is not contaminated with high numbers of non-neoplastic cells, include but are not limited to: 
     In vivo detection. 
     Molecular Imaging may be used following administration of imaging probes or reagents capable of disclosing altered expression of the markers in the intestinal tissues. 
     Molecular imaging (Moore et al.,  BBA,  1402:239-249, 1988; Weissleder et al.,  Nature Medicine  6:351-355, 2000) is the in vivo imaging of molecular expression that correlates with the macro-features currently visualized using “classical” diagnostic imaging techniques such as X-Ray, computed tomography (CT), MRI, Positron Emission Tomography (PET) or endoscopy. 
     Detection of downregulation of RNA expression in the cells by Fluorescent In Situ Hybridization (FISH), or in extracts from the cells by technologies such as Quantitative Reverse Transcriptase Polymerase Chain Reaction (QRTPCR) or Flow cytometric qualification of competitive RT-PCR products (Wedemeyer et al.,  Clinical Chemistry  48:9 1398-1405, 2002). 
     Assessment of expression profiles of RNA, for example by array technologies (Alon et al.,  Proc. Natl. Acad. Sci. USA:  96:6745-6750, June 1999). 
     A “microarray” is a linear or multi-dimensional array of preferably discrete regions, each having a defined area, formed on the surface of a solid support. The density of the discrete regions on a microarray is determined by the total numbers of target polynucleotides to be detected on the surface of a single solid phase support. As used herein, a DNA microarray is an array of oligonucleotide probes placed onto a chip or other surfaces used to amplify or clone target polynucleotides. Since the position of each particular group of probes in the array is known, the identities of the target polynucleotides can be determined based on their binding to a particular position in the microarray. 
     DNA microarray technology make it possible to conduct a large-scale assay of a plurality of target nucleic acid molecules on a single solid phase support. U.S. Pat. No. 5,837,832 (Chee et al.) and related patent applications describe immobilizing an array of oligonucleotide probes for hybridization and detection of specific nucleic acid sequences in a sample. Target polynucleotides of interest isolated from a tissue of interest are hybridized to the DNA chip and the specific sequences detected based on the target polynucleotides&#39; preference and degree of hybridization at discrete probe locations. One important use of arrays is in the analysis of differential gene expression, where the profile of expression of genes in different cells or tissues, often a tissue of interest and a control tissue, is compared and any differences in gene expression among the respective tissues are identified. Such information is useful for the identification of the types of genes expressed in a particular tissue type and diagnosis of conditions based on the expression profile. 
     Measurement of altered neoplastic marker protein levels in cell extracts, for example by immunoassay. 
     Testing for proteinaceous neoplastic marker expression product in a biological sample can be performed by any one of a number of suitable methods which are well known to those skilled in the art. Examples of suitable methods include, but are not limited to, antibody screening of tissue sections, biopsy specimens or bodily fluid samples. To the extent that antibody-based methods of diagnosis are used, the presence of the marker protein may be determined in a number of ways such as by Western blotting, ELISA or flow cytometry procedures. These, of course, include both single-site and two-site or “sandwich” assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labeled antibody to a target. 
     Determining altered expression of protein neoplastic markers on the cell surface, for example by immunohistochemistry. 
     Determining altered protein expression based on any suitable functional test, enzymatic test or immunological test in addition to those detailed in points (iv) and (v) above. 
     A person of ordinary skill in the art could determine, as a matter of routine procedure, the appropriateness of applying a given method to a particular type of biological sample. 
     A related aspect of the present invention provides a molecular array, which array comprises a plurality of: 
     (i) nucleic acid molecules comprising a nucleotide sequence corresponding to any two or more of the neoplastic marker DNA hereinbefore described or a sequence exhibiting at least 80% identity thereto or a functional derivative, fragment, variant or homologue of said nucleic acid molecule; or 
     (ii) nucleic acid molecules comprising a nucleotide sequence capable of hybridizing to any one or more of the sequences of (i) under medium stringency conditions or a functional derivative, fragment, variant or homologue of said nucleic acid molecules; or 
     (iii) nucleic acid probes or oligonucleotides comprising a nucleotide sequence capable of hybridizing to any two or more of the sequences of (i) under medium stringency conditions or a functional derivative, fragment, variant or homologue of said nucleic acid molecule; or 
     (iv) probes capable of binding to any two or more of the proteins encoded by the nucleic acid molecules of (i) or a derivative, fragment or, homologue thereof wherein the level of expression of said marker genes of (i)-(iii) or proteins of (iv) is indicative of the neoplastic state of a cell or cellular subpopulation derived from the large intestine. 
     Preferably, said percent identity is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. 
     “Hybridization” refers to the process by which a nucleic acid strand joins with a complementary strand through base pairing. Hybridization reactions can be sensitive and selective so that a particular sequence of interest can be identified even in samples in which it is present at low concentrations. Stringent conditions can be defined by, for example, the concentrations of salt or formamide in the prehybridization and hybridization solutions, or by the hybridization temperature, and are well known in the art. For example, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature, altering the time of hybridization, as described in detail, below. In alternative aspects, nucleic acids of the invention are defined by their ability to hybridize under various stringency conditions (e.g., high, medium, and low), as set forth herein. 
     Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30° C. to about 42° C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out T m =69.3+0.41 (G+C) % (Marmur and Doty,  J. Mol. Biol.  5:109, 1962). However, the T m  of a duplex DNA decreases by 1° C. with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey,  Eur. J. Biochem.  46:83, 1974). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6×SSC buffer, 0.1% w/v SDS at 25-42° C.; a moderate stringency is 2×SSC buffer, 0.1% w/v SDS at a temperature in the range 20° C. to 65° C.; high stringency is 0.1×SSC buffer, 0.1% w/v SDS at a temperature of at least 65° C. 
     Where nucleic acids of the invention are defined by their ability to hybridize under high stringency, these conditions comprise about 50% formamide at about 37° C. to 42° C. In one aspect, nucleic acids of the invention are defined by their ability to hybridize under reduced stringency comprising conditions in about 35% to 25% formamide at about 30° C. to 35° C. Alternatively, nucleic acids of the invention are defined by their ability to hybridize under high stringency comprising conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS, and a repetitive sequence blocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200 n/ml sheared and denatured salmon sperm DNA). In one aspect, nucleic acids of the invention are defined by their ability to hybridize under reduced stringency conditions comprising 35% formamide at a reduced temperature of 35° C. 
     Preferably, the subject probes are designed to bind to the nucleic acid or protein to which they are directed with a level of specificity which minimizes the incidence of non-specific reactivity. However, it would be appreciated that it may not be possible to eliminate all potential cross-reactivity or non-specific reactivity, this being an inherent limitation of any probe-based system. 
     In terms of the probes which are used to detect the subject proteins, they may take any suitable form including antibodies and aptamers. 
     A library or array of nucleic acid or protein probes provides rich and highly valuable information. Further, two or more arrays or profiles (information obtained from use of an array) of such sequences are useful tools for comparing a test set of results with a reference, such as another sample or stored calibrator. In using an array, individual probes typically are immobilized at separate locations and allowed to react for binding reactions. Primers associated with assembled sets of markers are useful for either preparing libraries of sequences or directly detecting markers from other biological samples. 
     A library (or array, when referring to physically separated nucleic acids corresponding to at least some sequences in a library) of gene markers exhibits highly desirable properties. These properties are associated with specific conditions, and may be characterized as regulatory profiles. A profile, as termed here refers to a set of members that provides diagnostic information of the tissue from which the markers were originally derived. A profile in many instances comprises a series of spots on an array made from deposited sequences. 
     A characteristic patient profile is generally prepared by use of an array. An array profile may be compared with one or more other array profiles or other reference profiles. The comparative results can provide rich information pertaining to disease states, developmental state, receptiveness to therapy and other information about the patient. 
     Another aspect of the present invention provides a diagnostic kit for assaying biological samples comprising one or more agents for detecting one or more neoplastic markers and reagents useful for facilitating the detection by said agents. Further means may also be included, for example, to receive a biological sample. The agent may be any suitable detecting molecule. 
     The present invention is further described by reference to the following non-limiting examples. 
     EXAMPLE 1 
     CAHM: Colorectal Adenocarcinoma HyperMethylated 
     Single-plex performance: 
     74 normals: 0.8 pg hypermethylated/mL plasma (95% CI: 0.2; 1.4) 
     73 adenomas: 9.1 pg hypermethylated/mL plasma (95% CI: −8; 27) 
     73 cancers: 1788 pg hypermethylated/mL plasma (95% CI: 231; 3344). 
     The CAHM assay (threshold cut of 3 pg/mL plasma) is 55% sensitive for colorectal cancer with a 93% specificity ( FIG. 1 ). 
     GRASP: General Receptor for Phosphoinositides 1-Associated Scaffold Protein. 
     Single plex performance: 
     34 normals: 1.3 pg hypermethylated/mL plasma (95% CI: 0.2; 2.5) 
     33 adenomas: 0.1 pg hypermethylated/mL plasma (95% CI: −0.1; 0.4) 
     33 cancers: 670.8 pg hypermethylated/mL plasma (95% CI: −470.7; 1812) 
     The GRASP assay is 58% sensitive for colorectal cancer with a 100% specificity using a threshold cut of 20 pg methylated/mL plasma ( FIG. 2 ). 
     IRF4: Interferon Regulatory Factor 4 
     Single plex performance: 
     24 normals: 6 pg hypermethylated/mL plasma (95% CI: 2.9; 9.0) 
     23 adenomas: 3.7 pg hypermethylated/mL plasma (95% CI: −0.4; 8.0) 
     33 cancers: 5340 pg hypermethylated/mL plasma (95% CI: −5369; 16049) 
     The IRF4 assay is 57% sensitive for colorectal cancer with a 96% specificity using a threshold cut of 20 pg methylated/mL plasma ( FIG. 3 ). 
     BCAT1: Branched Chain Amino Acid Transaminase 1, Cytosolic 
     Single plex performance summary 
     14 normals: 0 pg hypermethylated/mL plasma (95% CI: na) 
     13 adenomas: 0.4 pg hypermethylated/mL plasma (95% CI: −0.4; 1.3) 
     13 cancers: 4088 pg hypermethylated/mL plasma (95% CI: −4459; 10539) 
     The BCAT1 assay is 62% sensitive for colorectal cancer with a 100% specificity using a threshold cut of 3 pg methylated/mL plasma ( FIG. 4 ). 
     IKZF1: IKAROS Family Zinc Finger 1 
     Single plex performance summary 
     14 normals: 0 pg hypermethylated/mL plasma (95% CI: na) 
     13 adenomas: 0 pg hypermethylated/mL plasma (95% CI: na) 
     13 cancers: 113 pg hypermethylated/mL plasma (95% CI: −43; 270) 
     The IKZF1 assay is 54% sensitive for colorectal cancer with a 100% specificity using a threshold cut of 3 pg methylated/mL plasma ( FIG. 4 ). 
     The methylation levels of the five genes were measured in the same 14 normals, 13 adenomas and 13 cancers. The table below demonstrates the improvement in clinical utility when measuring the methylation in TWO of the genes: 
     
       
         
           
               
               
            
               
                   
               
               
                   
                 (% sens; % spec) 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 CAHM&gt; 6 pg/mL   
                 GRASP &gt;20 pg/mL   
                 IRF4 &gt;20 pg/mL    
                 BCAT &gt;3 pg/mL   
                 IKZF1 &gt;3 pg/mL   
               
               
                   
               
               
                 CAHM&gt; 6 pg/mL   
                 54%; 100% 
                 62%; 100% 
                 69%; 100% 
                 69%; 100% 
                 69%; 100% 
               
               
                 GRASP &gt;20   pg/mL   
                 — 
                 54%; 100% 
                 69%; 100% 
                 77%; 100% 
                 77%; 100% 
               
               
                 IRF4 &gt;20   pg/mL    
                 — 
                 — 
                 54%; 100% 
                 85%; 100% 
                 69%; 100% 
               
               
                 BCAT &gt;3   pg/mL   
                 — 
                 — 
                 — 
                 62%; 100% 
                 77%; 100% 
               
               
                 IKZF1 &gt;3   pg/mL   
                 — 
                 — 
                 — 
                 — 
                 54%; 100% 
               
               
                   
               
            
           
         
       
     
     Combining the methylation levels measured in any three of the five genes resulted in: 
     CAHM 6 pg/mL -GRASP 20 pg/mL -IRF4 20 pg/mL : 77% sensitivity and 100% specificity 
     CAHM 6 pg/mL -GRASP 20 pg/mL -BCAT 3 pg/mL : 77% sensitivity and 100% specificity 
     CAHM 6 pg/mL -GRASP 20 pg/mL -IKZF1 3 pg/mL : 77% sensitivity and 100% specificity 
     CAHM 6 pg/mL -IKZF1 3 pg/mL -BCAT 3 pg/mL : 77% sensitivity and 100% specificity 
     GRASP 20 pg/mL -IKZF1 3 pg/mL -BCAT 3 pg/mL : 85% sensitivity and 100% specificity 
     Combining the methylation levels from all five genes results in a 92% sensitivity and 100% specificity: 
     Targeted Amplicon Sequences for GRASP, CAHM, IRF4, IKZF1 and BCAT1 
     The wild-type DNA sequence of the CAHM MSP amplicon is located on Chromosome 6, plus strand; 163,834,393⇒163,834,455 (Hg19) GAAGGAAGCA TTTCGAGCAC GACTGACGCT CCCCTTATTA TTTGCTAAGC CGCTGCGCTC GGG 
     The wild-type DNA sequence of the GRASP MSP amplicon is located on Chromosome 12, plus strand; 52,400,886⇒52,400,973 (Hg19) cggaagtcgc gcccgccgct ccggtcccga ccccgggacc ccctgccgca gccgccaccc ctgggccccc agcggacgag ctgtacgc 
     The wild-type DNA sequence of the IKZF1 MSP amplicon is located on Chromosome 7, plus strand; 50,343,867⇒50,343,961 (Hg19) gacgacgcac cctctccgtg tcccgctctg cgcccttctg cgcgccccgc tccctgtacc ggagcagcga tccgggaggc ggccgagagg tg 
     The wild-type DNA sequence of the BCAT1 MSP amplicon is located on Chromosome 12, minus strand; 25,101,992⇒25,102,093 (Hg19) gtcttcctgc tgatgcaatc CGctaggtcg cgagtctccg ccgcgagagg gccggtctgc aatccagccc gccacgtgta ctcgccgccg cctcg 
     The wild-type DNA sequence of the IRF4 MSP amplicon is located on Chromosome 6, minus strand; 392,036⇒392,145 (Hg19) tgggtgcctt ggacggcccc gcctcagcca ctcctggggc cccgacagtc cggttagctc atcccgtcca gcttgtggcg accccgtcgc aggagcgcgg agggcaggcg 
     PCR Protocols Used to Measure the Methylation Levels Across GRASP, CAHM, IRF4, IKZF1 and BCAT1 in Plasma Specimens 
     Real-Time PCR Protocols GRASP, CAHM, IRF4, BCAT1, IKZF1 
       
     
       
         
           
               
            
               
                   
               
               
                 Real-time PCR protocols GRASP, CAHM, IRF4, BCAT1, IKZF1 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 CAHM 
                 GRASP 
                 IRF4 
                 BCAT1 
                 IKZF1 
               
               
                   
               
               
                 FWD 
                 5’GAAGGA 
                 5’CGGAAG 
                 5’TGGGTG 
                 5’GTTTTTTT 
                 5’GACGACGT 
               
               
                 Primer 
                 AGTATTT 
                 TCGCGTTC 
                 TTTTGGA 
                 GTTGATGT 
                 ATTTTTTTCG 
               
               
                   
                 CGAGTAC 
                 GTC 
                 CGGTTTC 
                 AATTCGTT 
                 TGTTTC 
               
               
                   
                 GATTGAC 
                 200 nM 
                 400 nM 
                 AGGTC 
                 200 nM 
               
               
                   
                 200 nM 
                 final 
                 final 
                 200 nM 
                 final 
               
               
                   
                 final 
                   
                   
                 final 
                   
               
               
                   
               
               
                 REV 
                 5’CCCGAA 
                 5’GCGTACA 
                 5’CGCCTA 
                 5’CAATACC 
                 5’GCGCACCT 
               
               
                 primer 
                 CGCAACG 
                 ACTCGTCC 
                 CCCTCCG 
                 CGAAACGA 
                 CTCGACCG 
               
               
                   
                 ACTTAA 
                 GCTAA 
                 CG 
                 CGACG 
                 200 nM 
               
               
                   
                 200 nM 
                 200 nM 
                 400 nM 
                 200 nM 
                 final 
               
               
                   
                 final 
                 final 
                 final 
                 final 
                   
               
               
                   
               
               
                 Probe 
                 na 
                 [HEX] 
                 [HEX] 
                 [HEX] 
                 na 
               
               
                   
                   
                 TTCGATTT 
                 TCGTTTA 
                 TTCGTCGC 
                   
               
               
                   
                   
                 CGGGATTT 
                 GTTTGTG 
                 GAGAGGGT 
                   
               
               
                   
                   
                 TTTGTCGT 
                 GCGATTT 
                 CGGTT 
                   
               
               
                   
                   
                 AGTC 
                 CGTCG 
                 [BHQ] 
                   
               
               
                   
                   
                 [BHQ1] 
                 [BHQ1] 
                 200 nM 
                   
               
               
                   
                   
                 100 nM 
                 200 nM 
                 final 
                   
               
               
                   
                   
                 final 
                 final 
                   
                   
               
               
                   
               
               
                 1x PCR 
                 Platinum 
                 Platinum 
                 Platinum 
                 Platinum 
                 GoTaq Hot 
               
               
                   
                 Taq DNA 
                 Taq 
                 Taq DNA 
                 Taq 
                 Start buffer. 
               
               
                   
                 polymerase 
                 DNA 
                 polymerase 
                 DNA 
                 1x buffer 
               
               
                   
                 (Invitrogen) 
                 polymerase 
                 (Invitrogen) 
                 polymerase 
                 contains 
               
               
                   
                   
                 (Invitrogen) 
                   
                 (Invitrogen) 
                 2 mM MgC12 
               
               
                   
                   
                   
                   
                   
                 (Promega) 
               
               
                   
               
               
                 Final 
                 4.0 
                 4.0 
                 3.0 
                 4.0 
                 +1.0 
               
               
                 MgCL 2   
                   
                   
                   
                   
                   
               
               
                 conc. 
                   
                   
                   
                   
                   
               
               
                 [Mm] 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 dNTPs 
                 0.2 mM 
                 0.2 mM 
                 0.2 mM 
                 0.2 mM 
                 — 
               
               
                 SYBR 
                 1/120,000 
                 na 
                 na 
                 na 
                 1/120,000 
               
               
                   
               
            
           
           
               
            
               
                 LC480 cycling conditions 
               
            
           
           
               
               
               
               
               
               
            
               
                 1x 
                 95° C.; 
                 95° C.; 
                 95° C.; 
                 95° C.; 
                 95° C.; 
               
               
                   
                 2 min 
                 2 min 
                 2 min 
                 2 min 
                 2 min 
               
               
                   
               
               
                 3x 
                 92° C.; 
                 92° C.; 
                 50 cycles: 
                 50 cycles: 
                 50 cycles: 
               
               
                   
                 15 sec 
                 15 sec 
                 95° C.; 
                 95° C.; 
                 95° C.; 
               
               
                   
                 62° C.; 
                 64° C.; 
                 10 
                 15 sec 
                 15 sec 
               
               
                   
                 15 sec 
                 15 sec 
                 sec 
                 62° C.; 
                 62°C; 
               
               
                   
                 72° C.; 
                 72° C.; 
                 61° C.; 
                 30 sec 
                 30 sec 
               
               
                   
                 20 sec 
                 20 sec 
                 20 
                 72° C.; 
                 72° C.; 
               
               
                   
                   
                   
                 sec 
                 30 sec 
                 30 sec 
               
               
                   
               
               
                 47x 
                 82° C.; 
                 85° C., 
                 (w/quant) 
                 (w/quant) 
                 (w/quant) 
               
               
                   
                 15 sec 
                 15 sec 
                   
                   
                   
               
               
                   
                 62° C.; 
                 64° C.; 
                   
                   
                   
               
               
                   
                 15 sec 
                 15 sec 
                   
                   
                   
               
               
                   
                 72° C.; 
                 72° C.; 
                   
                   
                   
               
               
                   
                 20 sec 
                 20 sec 
                   
                   
                   
               
               
                   
                 (w/quant) 
                 (w/quant) 
                   
                   
                   
               
               
                   
               
               
                 melt 
                 95° C.; 
                 na 
                 na 
                 na 
                 95° C.; 
               
               
                 curve 
                 5 sec 
                   
                   
                   
                 5 sec 
               
               
                 analysis 
                 65° C.; 
                   
                   
                   
                 65° C.; 
               
               
                   
                 1 min 
                   
                   
                   
                 1 min 
               
               
                   
                 97° C. 
                   
                   
                   
                 97° C. 
               
               
                   
                 continuous 
                   
                   
                   
                 continuous 
               
               
                   
                 0.11 deg/sec 
                   
                   
                   
                 0.11 deg/sec 
               
               
                   
               
               
                 Correct 
                 &lt;80° C. 
                 na 
                 na 
                 na 
                 82.9-83.2° C. 
               
               
                 Melt 
                   
                   
                   
                   
                   
               
               
                 Temp 
                   
                   
                   
                   
                   
               
               
                 (° C.) 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                   
                   
                 Current 
                   
                 Resulting 
                   
               
               
                   
                   
                 sub- 
                   
                 bisulphite 
                 Oligonucleotide 
               
               
                   
                   
                 region(s) 
                   
                 converted 
                 sequences for 
               
               
                   
                   
                 of 
                   
                 sequence 
                 measurement 
               
               
                   
                   
                 interest 
                 Coordinates 
                 (strands 
                 of 
               
               
                   
                 Genomic 
                 (genomic 
                 of sub- 
                 no longer 
                 methylation 
               
               
                 Gene 
                 Strand 
                 sequence) 
                 region(s) 
                 complementary) 
                 levels 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 BCAT1 
                 top 
                 5’- 
                 25,101,992- 
                 5’- 
                   
               
               
                   
                 strand 
                 cagtgccCGaggC 
                 25,102,093 
                 tagtgttCGaggCGgC 
                   
               
               
                   
                   
                 GgCGgCGagtac 
                   
                 GgCGagtataCGtgg 
                   
               
               
                   
                   
                 aCGtggC 
                   
                 CGggttgga 
                   
               
               
                   
                   
                 Gggctggattgcaga 
                   
                 ttgtagatCGgttttttCG 
                   
               
               
                   
                   
                 cCGgccctctCGC 
                   
                 CGgCGgagattCGC 
                   
               
               
                   
                   
                 GgCGgagactCG 
                   
                 GatttagC Ggattgtatta 
                   
               
               
                   
                   
                 CGacctagCGgatt 
                   
                 gtaggaagat 
                   
               
               
                   
                   
                 gcatcagcaggaaga 
                   
                   
                   
               
               
                   
                   
                 c 
                   
                   
                   
               
               
                   
                   
                 (SEQ ID NO. 1) 
                   
                   
                   
               
               
                   
               
               
                   
                 minus 
                 3’- 
                   
                 3’- 
                 5’- 
               
               
                   
                 strand 
                 gtcacggGCtccG 
                   
                 gttatggGCtttGCtGC 
                 gtttttttgttgatg 
               
               
                   
                   
                 CcGCcGCtcatgt 
                   
                 tGCttatgtGCattGCtt 
                 taattcgttaggtc 
               
               
                   
                   
                 GCaccGCc 
                   
                 gatttaatg 
                 5’- 
               
               
                   
                   
                 cgacctaacgtctgG 
                   
                 tttgGCtgggagaGCG 
                 caatacccgaaa 
               
               
                   
                   
                 CcgggagaGCGC 
                   
                 CtGCttttgaGCGCtg 
                 cgacgacg 
               
               
                   
                   
                 cGCctctgaGCG 
                   
                 gattGCttaatgtagttgtt 
                 5’- 
               
               
                   
                   
                 CtggatcGCctaac 
                   
                 tttttg 
                 ttcgtcgcgaga 
               
               
                   
                   
                 gtagtcgtccttctg 
                   
                   
                 gggtcggtt 
               
               
                   
                   
                   
                   
                   
                 5’- 
               
               
                   
                   
                   
                   
                   
                 tttttgttgatgtaa 
               
               
                   
                   
                   
                   
                   
                 ttcgttaggtc 
               
               
                   
                   
                   
                   
                   
                 5’- 
               
               
                   
                   
                   
                   
                   
                 attacaaaccga 
               
               
                   
                   
                   
                   
                   
                 ccctctcg 
               
               
                   
               
               
                   
                 top 
                 5’- 
                 25,101,909- 
                 This sequence 
                 5’- 
               
               
                   
                 strand 
                 agatcccaagggtC 
                 25,101,995 
                 is for 
                 agatcccaaggg 
               
               
                   
                   
                 GtagcccctggcCG 
                   
                 measuring CpG 
                 tcgtagc 
               
               
                   
                   
                 tgtgga cCGg   
                   
                 methylation 
                 5’- 
               
               
                   
                   
                 gtctgCGgctgcaga 
                   
                 levels 
                 actgccccaggt 
               
               
                   
                   
                   gCGC Gctcc CGg   
                   
                 using 
                 cttgct 
               
               
                   
                   
                 ctgcagcaagacctg 
                   
                 methylation 
                   
               
               
                   
                   
                 gggcagt (SEQ ID 
                   
                 sensitive restriction 
                   
               
               
                   
                   
                 NO. 2) 
                   
                 enzymes (e.g. 
                   
               
               
                   
                   
                   
                   
                 HbaII, HhaI 
                   
               
               
                   
                   
                   
                   
                 (underlined) 
                   
               
               
                   
               
               
                   
                 minus 
                 3’- 
                   
                   
                   
               
               
                   
                 strand 
                 tctagggttcccaGC 
                   
                   
                   
               
               
                   
                   
                 atcggggaccgGCa 
                   
                   
                   
               
               
                   
                   
                 cacctgGCc 
                   
                   
                   
               
               
                   
                   
                 cagacGCcgacgtc 
                   
                   
                   
               
               
                   
                   
                 tcGCGCcaggGC 
                   
                   
                   
               
               
                   
                   
                 cgacgtcgttctggac 
                   
                   
                   
               
               
                   
                   
                 cccgtca 
                   
                   
                   
               
               
                   
               
               
                 IKZF1 
                 top 
                 5’- 
                 50,343,867- 
                 5’- 
                 5’- 
               
               
                   
                 strand 
                 gaCGaCGcaccct 
                 50,343,961 
                 gaCGaCGtatttttttC 
                 gacgacgtattttt 
               
               
                   
                   
                 ctcCGtgtccCGct 
                   
                 GtgtttCGttttgCGtttt 
                 ttcgtgtttc 
               
               
                   
                   
                 ctgCGccctt 
                   
                 tttgCGCG 
                 5’- 
               
               
                   
                   
                 ctgC GCGcccCG 
                   
                 tttCGttttttgtatCGga 
                 gcgcacctctcg 
               
               
                   
                   
                 ctccctgtacCGgag 
                   
                 gtagCGattCGggagg 
                 accg 
               
               
                   
                   
                 cagCGatcCGgga 
                   
                 CGgtCGagaggtgC 
                 5’- 
               
               
                   
                   
                 ggCGgcCGagag 
                   
                 Gt 
                 tttgtatcggagta 
               
               
                   
                   
                 gtgCGc (SEQ ID 
                   
                   
                 gcgattcgggag 
               
               
                   
                   
                 NO. 3) 
                   
                   
                   
               
               
                   
               
               
                   
                 minus 
                 3’- 
                   
                 3’- 
                   
               
               
                   
                 strand 
                 ctGCtGCgtggga 
                   
                 ttGCtGCgtgggagag 
                   
               
               
                   
                   
                 gagGCacaggGC 
                   
                 GCataggGCgagatG 
                   
               
               
                   
                   
                 gagacGCgg 
                   
                 Cgggaa 
                   
               
               
                   
                   
                 gaagacGCGCgg 
                   
                 gatGCGCgggGCga 
                   
               
               
                   
                   
                 gGCgagggacatg 
                   
                 gggatatgGCtttgttG 
                   
               
               
                   
                   
                 GCctcgtcGCtag 
                   
                 CtagGCtttttGCtgG 
                   
               
               
                   
                   
                 GCcctccGCcgG 
                   
                 CtttttatGCg 
                   
               
               
                   
                   
                 CtctccacGCg 
                   
                   
                   
               
               
                   
               
               
                   
                 top 
                 5’- 
                 50,343,804- 
                 This sequence 
                 5’- 
               
               
                   
                 strand 
                   cCGg agttgCGgct 
                 50,343,895 
                 is for 
                 ggagttgcggct 
               
               
                   
                   
                 gagaC GCGCGc   
                   
                 measuring CpG 
                 gagac 
               
               
                   
                   
                 C GCGC G 
                   
                 methylation levels 
                 5’- 
               
               
                   
                   
                 a gcCGg ggaactC 
                   
                 using methylation 
                 agagcgggaca 
               
               
                   
                   
                 GgCGaCGgggC 
                   
                 sensitive 
                 cggaga 
               
               
                   
                   
                 GgggaCGggaCG 
                   
                 restriction 
                   
               
               
                   
                   
                 aCGcaccctctcCG 
                   
                 enzymes (e.g. 
                   
               
               
                   
                   
                 tgtccCGctct 
                   
                 HbaII, HhaI 
                   
               
               
                   
                   
                 (SEQ ID NO. 4) 
                   
                 (underlined) 
                   
               
               
                   
               
               
                   
                 minus 
                 3’- 
                   
                   
                   
               
               
                   
                 strand 
                 gGCctcaacGCcg 
                   
                   
                   
               
               
                   
                   
                 actctGCGCGCg 
                   
                   
                   
               
               
                   
                   
                 GCGCGC 
                   
                   
                   
               
               
                   
                   
                 tcgGCcccctgaG 
                   
                   
                   
               
               
                   
                   
                 CcGCtGCcccGC 
                   
                   
                   
               
               
                   
                   
                 ccctGCcctGCtG 
                   
                   
                   
               
               
                   
                   
                 CgtgggagagGCa 
                   
                   
                   
               
               
                   
                   
                 caggGCgaga 
                   
                   
                   
               
               
                   
               
               
                 IRF4 
                 top 
                 5’- 
                 392,036- 
                 5’- 
                 5’- 
               
               
                   
                 strand 
                 CGcctgccctcCG 
                 392,145 
                 CGtttgtttttCGCGtttt 
                 gtttttgcgacgg 
               
               
                   
                   
                 CGctcctgCGaC 
                   
                 tgCGaCGgggtCGtt 
                 ggtc 
               
               
                   
                   
                 GgggtCGcc 
                   
                 ataagttg 
                 5’- 
               
               
                   
                   
                 acaagctggaCGgg 
                   
                 gaCGggatgagttaatC 
                 taaaaccccgac 
               
               
                   
                   
                 atgagctaacCGga 
                   
                 GgattgtCGgggttttag 
                 aatccg 
               
               
                   
                   
                 ctgtCGgggcccca 
                   
                 gagtggttgaggCGgg 
                   
               
               
                   
                   
                 ggagtggctgaggC 
                   
                 gtCGtttaaggtattta 
                   
               
               
                   
                   
                 GgggcCGtccaag 
                   
                   
                   
               
               
                   
                   
                 gcaccca (SEQ ID 
                   
                   
                   
               
               
                   
                   
                 NO. 5) 
                   
                   
                   
               
               
                   
               
               
                   
                 minus 
                 3’- 
                   
                 3’- 
                 5’- 
               
               
                   
                 strand 
                 GCggacgggagG 
                   
                 GCggatgggagGCG 
                 tgggtgttttgga 
               
               
                   
                   
                 CGCgaggacGCt 
                   
                 CgaggatGCtGCttta 
                 cggtttc 
               
               
                   
                   
                 GCcccaGC 
                   
                 GCggtgtttg 
                 5’- 
               
               
                   
                   
                 ggtgttcgacctGCc 
                   
                 atttGCtttatttgattgG 
                 tagttatttttggg 
               
               
                   
                   
                 ctactcgattgGCctg 
                   
                 CttgataGCtttggggttt 
                 gtttcgatagttc 
               
               
                   
                   
                 acaGCcccggggtc 
                   
                 ttattgattttGCtttgGC 
                 5’- 
               
               
                   
                   
                 ctcaccgactccGCc 
                   
                 aggttttgtgggt 
                 cgcctaccctcc 
               
               
                   
                   
                 ccgGCaggttccgtg 
                   
                   
                 gcg 
               
               
                   
                   
                 ggt 
                   
                   
                 5’- 
               
               
                   
                   
                   
                   
                   
                 tcgtttagtttgtg 
               
               
                   
                   
                   
                   
                   
                 gcgatttcgtcg 
               
               
                   
               
               
                 GRASP 
                 top 
                 5’- 
                 52,400,821- 
                 5’- 
                 5’- 
               
               
                   
                 strand 
                 caggaagctgcagca 
                 52,401,051 
                 taggaagttgtagtagaag 
                 cggaagtcgcgt 
               
               
                   
                   
                 gaaggaggaggCG 
                   
                 gaggaggCGgCGgtt 
                 tcgtc 
               
               
                   
                   
                 gCGgcca 
                   
                 atttCGga 
                 5’- 
               
               
                   
                   
                 cccCGgacccCGc 
                   
                 tttCGtCGttCGgatttt 
                 gcgtacaactcgt 
               
               
                   
                   
                 CGccCGgactccC 
                   
                 CGattCGgaagtCGC 
                 ccgctaa 
               
               
                   
                   
                 GactCGgaagtCG 
                   
                 GttCGtCGtttCGgttt 
                 5’- 
               
               
                   
                   
                 CGccCGcCGctc 
                   
                 CGatttCGggattttttgt 
                 ttcgatttcgggat 
               
               
                   
                   
                 CGgtccCGacccC 
                   
                 CGtagtCGttatttttgg 
                 tttttgtcgtagtc 
               
               
                   
                   
                 GggaccccctgcC 
                   
                 gtttttagCGgaCGagtt 
                 5’- 
               
               
                   
                   
                 GcagcCGccaccc 
                   
                 gtaCGCGgCGttgga 
                 cggattttcgattc 
               
               
                   
                   
                 ctgggcccccagCG 
                   
                 ggattattattttgtC Gagt 
                 ggaagt 
               
               
                   
                   
                 gaCGagctgtaCG 
                   
                 tgtatCGCGCGttCG 
                   
               
               
                   
                   
                 CGgCGctggagga 
                   
                 tCGtgttCGggggtattt 
                   
               
               
                   
                   
                 ctatcaccctgcCGa 
                   
                 tgtttCGtCGaaaggtg 
                   
               
               
                   
                   
                 gctgtacCGCGC 
                   
                 CGtttttCGttCGttttta 
                   
               
               
                   
                   
                 GctCGcCGtgtcC 
                   
                 ggatttgtttagttttttttC 
                   
               
               
                   
                   
                 Gggggcaccctgcc 
                   
                 Gattttttatagggtttgttg 
                   
               
               
                   
                   
                 cCGcCGaaaggtg 
                   
                 atttCG 
                   
               
               
                   
                   
                 CGtccccCGccC 
                   
                   
                   
               
               
                   
                   
                 Gccttcaggatctgct 
                   
                   
                   
               
               
                   
                   
                 cagcccctctcCGac 
                   
                   
                   
               
               
                   
                   
                 tccctacagggcctgc 
                   
                   
                   
               
               
                   
                   
                 tgactcCG (SEQ 
                   
                   
                   
               
               
                   
                   
                 ID NO. 6) 
                   
                   
                   
               
               
                   
                   
                 3’- 
                   
                   
                   
               
               
                   
               
               
                   
                 minus 
                 gtccttcgacgtcgtct 
                   
                   
                   
               
               
                   
                 strand 
                 tcctcctccGCcGC 
                   
                 3’- 
                   
               
               
                   
                   
                 cggtgggG 
                   
                 gttttttgatgttgtttttt 
                   
               
               
                   
                   
                 CctgggGCgGCg 
                   
                 ttttttGCtGCtggtgggGC 
                   
               
               
                   
                   
                 gGCctgaggGCtg 
                   
                 ttgggGC 
                   
               
               
                   
                   
                 aGCctcaGCGCg 
                   
                 gGCggGCttgaggG 
                   
               
               
                   
                   
                 gGCgGCgagGC 
                   
                 CtgaGCtttaGCGCg 
                   
               
               
                   
                   
                 caggGCtgggGCc 
                   
                 gGCgGCgagGCtag 
                   
               
               
                   
                   
                 ctgggggacgGCgt 
                   
                 gGCtgggGCtttgggg 
                 5’- 
               
               
                   
                   
                 cgGCggtggggacc 
                   
                 gatgGCgttgGCggtg 
                 ggtagggtgtttt 
               
               
                   
                   
                 cgggggtcGCctG 
                   
                 gggatttgggggttGCtt 
                 cggatac 
               
               
                   
                   
                 CtcgacatGCGCc 
                   
                 GCttgatatGCGCtG 
                 5’- 
               
               
                   
                   
                 GCgacctcctgatag 
                   
                 Cgattttttgatagtgggat 
                 aacgaacgaact 
               
               
                   
                   
                 tgggacgGCtcgac 
                   
                 gGCttgatatgGCGC 
                 atacgcgac 
               
               
                   
                   
                 atgGCGCGCga 
                   
                 GCgaGCgGCatagG 
                   
               
               
                   
                   
                 GCgGCacagGCc 
                   
                 CttttgtgggatgggGC 
                   
               
               
                   
                   
                 cccgtgggacgggG 
                   
                 gGCtttttatGCagggg 
                   
               
               
                   
                   
                 CgGCtttccacGC 
                   
                 GCggGCggaagtttta 
                   
               
               
                   
                   
                 aggggGCggGCg 
                   
                 gatgagttggggagagG 
                   
               
               
                   
                   
                 gaagtcctagacgagt 
                   
                 Ctgagggatgttttggatg 
                   
               
               
                   
                   
                 cggggagagGCtga 
                   
                 attgagGC 
                   
               
               
                   
                   
                 gggatgtcccggacg 
                   
                   
                   
               
               
                   
                   
                 actgagGC 
                   
                   
                   
               
               
                   
               
               
                   
                 top 
                 5’- 
                 52,401,407- 
                 5’- 
                   
               
               
                   
                 strand 
                 gacagagacagcccc 
                 52,401,664 
                 gatagagatagttttaggta 
                   
               
               
                   
                   
                 aggcaagttgaaggtc 
                   
                 agttgaaggttCGagagt 
                   
               
               
                   
                   
                 CGagagc 
                   
                 tttCGgtgg 
                   
               
               
                   
                   
                 cccCGgtgggagaa 
                   
                 gagaagCGggtCGgt 
                   
               
               
                   
                   
                 gCGggcCGgtggc 
                   
                 ggttgCGtCGCGtgC 
                   
               
               
                   
                   
                 tgCGcCGCGtgC 
                   
                 Gtttttattttgaggaagtg 
                   
               
               
                   
                   
                 Gttctcactctgagga 
                   
                 CGtggggagtC Gttgat 
                   
               
               
                   
                   
                 agtgCGtggggagc 
                   
                 ttCGgatagtatattttttC 
                   
               
               
                   
                   
                 CGctgactcCGgat 
                   
                 GaggggatttttCGatttt 
                   
               
               
                   
                   
                 agcacacccttcCGa 
                   
                 tgggttgggggtttgtCG 
                   
               
               
                   
                   
                 ggggactcccCGatt 
                   
                 tttggttttaCGtttgaCG 
                   
               
               
                   
                   
                 cctgggctgggggcc 
                   
                 taCGgggCGCGagg 
                   
               
               
                   
                   
                 tgcCGcctggcccc 
                   
                 gttattgttttttg 
                   
               
               
                   
                   
                 aCGtctgaCGtaC 
                   
                 gatttttgt 
                   
               
               
                   
                   
                 GgggCGCGaggg 
                   
                 CGgaatCGgaCGtag 
                   
               
               
                   
                   
                 ccactgctccctggac 
                   
                 tgggaggggtCGtagg 
                   
               
               
                   
                   
                 ttctgtCGgaacCG 
                   
                   
                   
               
               
                   
                   
                 gaCGcagtgggagg 
                   
                   
                   
               
               
                   
                   
                 ggtCGcagg 
                   
                   
                   
               
               
                   
                   
                 (SEQ ID NO. 7) 
                   
                   
                   
               
               
                   
               
               
                   
                 minus 
                 3’- 
                   
                 3’- 
                 5’- 
               
               
                   
                 strand 
                 ctgtctctgtcggggtc 
                   
                 ttgtttttgttggggtttgttt 
                 cggagttagcgg 
               
               
                   
                   
                 cgttcaacttccagG 
                   
                 aatttttagGCttttgggg 
                 ttttttacg 
               
               
                   
                   
                 Ctctcgggg 
                   
                 GCtattttttt 
                 5’- 
               
               
                   
                   
                 GCcaccctcttcGC 
                   
                 tGCttgGCtattgatGC 
                 cgataaaaaaaa 
               
               
                   
                   
                 ccgGCcaccgacG 
                   
                 gGCGCatGCaagagt 
                 cgaaccga 
               
               
                   
                   
                 CgGCGCacGCa 
                   
                 gagatttttttatGCatttttt 
                 5’- 
               
               
                   
                   
                 agagtgagactccttc 
                   
                 gGCgattgagGCttatt 
                 agagtgagaacg 
               
               
                   
                   
                 acGCacccctcgG 
                   
                 gtgtgggaagGCttttttg 
                 tacgcggc 
               
               
                   
                   
                 CgactgagGCctat 
                   
                 agggGCtaaggatttgat 
                   
               
               
                   
                   
                 cgtgtgggaagGCt 
                   
                 ttttggatgGCggattgg 
                   
               
               
                   
                   
                 cccctgagggGCta 
                   
                 ggtGCagattGCatG 
                   
               
               
                   
                   
                 aggacccgacccccg 
                   
                 CtttGCGCttttggtgat 
                   
               
               
                   
                   
                 gacgGCggaccgg 
                   
                 gagggatttgaagataG 
                   
               
               
                   
                   
                 ggtGC agactGC at 
                   
                 CtttgGCttGCgttatttt 
                   
               
               
                   
                   
                 GCcccGCGCtcc 
                   
                 ttttaGCgttt 
                   
               
               
                   
                   
                 cggtgacgagggacc 
                   
                   
                   
               
               
                   
                   
                 tgaagacaGCcttg 
                   
                   
                   
               
               
                   
                   
                 GCctGCgtcaccct 
                   
                   
                   
               
               
                   
                   
                 ccccaGCgtcc 
                   
                   
                   
               
               
                   
               
               
                   
                 top 
                 5’- 
                   
                 This sequence 
                 5’- 
               
               
                   
                 strand 
                 gacagagacagcccc 
                   
                 is for 
                 caagttgaaggt 
               
               
                   
                   
                 aggcaagttgaaggtc 
                   
                 measuring CpG 
                 ccgagagc 
               
               
                   
                   
                 CGagag 
                   
                 methylation 
                 5’- 
               
               
                   
                   
                 cccc CGg tgggag 
                   
                 levels 
                 cgcacttcctcag 
               
               
                   
                   
                 agCGgg cCGg tgg 
                   
                 using 
                 agtgaga 
               
               
                   
                   
                 ct gCGc CGCGta 
                   
                 methylation 
                   
               
               
                   
                   
                 CGttctcactctgagg 
                   
                 sensitive 
                   
               
               
                   
                   
                 aagtgCGtggggag 
                   
                 restriction 
                   
               
               
                   
                   
                 cCGctaactc CGg   
                   
                 enzymes (e.g. 
                   
               
               
                   
                   
                 atagcacacccttcC 
                   
                 HbaII, HhaI 
                   
               
               
                   
                   
                 GaggggactcccC 
                   
                 (underlined) 
                   
               
               
                   
                   
                 Gattcctgggctggg 
                   
                   
                   
               
               
                   
                   
                 ggcctgcCGcctgg 
                   
                   
                   
               
               
                   
                   
                 ccccaCGtctgaCG 
                   
                   
                   
               
               
                   
                   
                 taCGgg gCGC Ga 
                   
                   
                   
               
               
                   
                   
                 gggccactgctccctg 
                   
                   
                   
               
               
                   
                   
                 gacttctgtCGgaa c   
                   
                   
                   
               
               
                   
                   
                   CGg aCGcagtggg 
                   
                   
                   
               
               
                   
                   
                 aggggtCGcagg 
                   
                   
                   
               
               
                   
               
               
                   
                 minus 
                 3’- 
                   
                   
                   
               
               
                   
                 strand 
                 ctgtctctgtcggggtc 
                   
                   
                   
               
               
                   
                   
                 cgttcaacttccagG 
                   
                   
                   
               
               
                   
                   
                 Ctctcgggg 
                   
                   
                   
               
               
                   
                   
                   GCc accctcttcGC 
                   
                   
                   
               
               
                   
                   
                 ccgGCcaccga cG   
                   
                   
                   
               
               
                   
                   
                   Cg GCGCacGCa 
                   
                   
                   
               
               
                   
                   
                 agagtgagactccttc 
                   
                   
                   
               
               
                   
                   
                 acGCacccctcgG 
                   
                   
                   
               
               
                   
                   
                 Cgactga gGCc tat 
                   
                   
                   
               
               
                   
                   
                 cgtgtgggaagGCt 
                   
                   
                   
               
               
                   
                   
                 cccctgagggGCta 
                   
                   
                   
               
               
                   
                   
                 aggacccgacccccg 
                   
                   
                   
               
               
                   
                   
                 gacgGCggaccgg 
                   
                   
                   
               
               
                   
                   
                 ggtGCagactGCat 
                   
                   
                   
               
               
                   
                   
                 GCcc cGCG Ctcc 
                   
                   
                   
               
               
                   
                   
                 cggtgacgagggacc 
                   
                   
                   
               
               
                   
                   
                 tgaagacaGCcttg 
                   
                   
                   
               
               
                   
                   
                   GCc tGCgtcaccct 
                   
                   
                   
               
               
                   
                   
                 ccccaGCgtcc 
                   
                   
                   
               
               
                   
               
               
                 CAHM 
                 top 
                 5’- 
                 163,834,295 
                 5’- 
                 5’- 
               
               
                   
                 strand 
                 atctgtaaaaatgttga 
                 163,834,500 
                 atttgtaaaaatgttgattttt 
                 gaaggaagtattt 
               
               
                   
                   
                 cttctgcttttcagacta 
                   
                 gttttttagattaCGCGta 
                 cgagtacgattg 
               
               
                   
                   
                 CGCGcac 
                   
                 tagtttttttatt 
                 acc 5’- 
               
               
                   
                   
                 agcctctttatttcctac 
                   
                 ttttattgCGgttttattttttt 
                 cccgaacgcaac 
               
               
                   
                   
                 tgCGgcttcattccct 
                   
                 aCGgaatattgaCGtta 
                 gacttaa 
               
               
                   
                   
                 caCGgaacactgaC 
                   
                 tCGCGaaggaagtattt 
                 5’- 
               
               
                   
                   
                 GccatCGCGaag 
                   
                 CGagtaCGattgaCGt 
                 gcctctaaaaaa 
               
               
                   
                   
                 gaagcatttCGagca 
                   
                 tttttttattatttgttaagtC 
                 acgatcttattaca 
               
               
                   
                   
                 CGactgaCGctccc 
                   
                 GttgCGttCGggtttgg 
                 cc 
               
               
                   
                   
                 cttattatttgctaagc 
                   
                 ttaCGatttgtttttagaat 
                   
               
               
                   
                   
                 CGctgCGctCGg 
                   
                 aaCGggaaggtgtaata 
                   
               
               
                   
                   
                 gtctggctaCGatttg 
                   
                 aga 
                   
               
               
                   
                   
                 ctttcagaataaCGg 
                   
                   
                   
               
               
                   
                   
                 gaaggtgcaacaaga 
                   
                   
                   
               
               
                   
                   
                 (SEQ ID NO. 8) 
                   
                   
                   
               
               
                   
               
               
                   
                 minus 
                 3’- 
                   
                   
                   
               
               
                   
                 strand 
                 ctgtctctgtcggggtc 
                   
                   
                   
               
               
                   
                   
                 cgttcaacttccagG 
                   
                   
                   
               
               
                   
                   
                 Ctctcgggg 
                   
                   
                   
               
               
                   
                   
                 GCcaccctcttcGC 
                   
                   
                   
               
               
                   
                   
                 ccgGCcaccgacG 
                   
                   
                   
               
               
                   
                   
                 CgGCGCacGCa 
                   
                   
                   
               
               
                   
                   
                 agagtgagactccttc 
                   
                   
                   
               
               
                   
                   
                 acGCacccctcgG 
                   
                   
                   
               
               
                   
                   
                 CgactgagGCctat 
                   
                   
                   
               
               
                   
                   
                 cgtgtgggaagGCt 
                   
                   
                   
               
               
                   
                   
                 cccctgagggGCta 
                   
                   
                   
               
               
                   
                   
                 aggacccgacccccg 
                   
                   
                   
               
               
                   
                   
                 gacgGCggaccgg 
                   
                   
                   
               
               
                   
                   
                 ggtGCagactGCat 
                   
                   
                   
               
               
                   
                   
                 GCcccGCGCtcc 
                   
                   
                   
               
               
                   
                   
                 cggtgacgagggacc 
                   
                   
                   
               
               
                   
                   
                 tgaagacaGCcttg 
                   
                   
                   
               
               
                   
                   
                 GCctGCgtcaccct 
                   
                   
                   
               
               
                   
                   
                 ccccaGCgtcc 
                   
                   
                   
               
               
                   
               
               
                 CAHM 
                 top 
                 5’- 
                 163,834,295 
                 5’- 
                 5’- 
               
               
                   
                 strand 
                 atctgtaaaaatgttga 
                 163,834,500 
                 atttgtaaaaatgttgattttt 
                 gaaggaagtattt 
               
               
                   
                   
                 cttctgcttttcagacta 
                   
                 gttttttagattaCGCGta 
                 cgagtacgattg 
               
               
                   
                   
                 CGCGcac 
                   
                 tagtttttttatt 
                 acc 5’- 
               
               
                   
                   
                 agcctctttatttcctac 
                   
                 ttttattgCGgttttattttttt 
                 cccgaacgcaac 
               
               
                   
                   
                 tgCGgcttcattccct 
                   
                 aCGgaatattgaCGtta 
                 gacttaa 
               
               
                   
                   
                 caCGgaacactgaC 
                   
                 tCGCGaaggaagtattt 
                 5’- 
               
               
                   
                   
                 GccatCGCGaag 
                   
                 CGagtaCGattgaCGt 
                 gcctctaaaaaa 
               
               
                   
                   
                 gaagcatttCGagca 
                   
                 tttttttattatttgttaagtC 
                 acgatcttattaca 
               
               
                   
                   
                 CGactgaCGctccc 
                   
                 GttgCGttCGggtttgg 
                 cc 
               
               
                   
                   
                 cttattatttgctaagc 
                   
                 ttaCGatttgtttttagaat 
                   
               
               
                   
                   
                 CGctgCGctCGg 
                   
                 aaCGggaaggtgtaata 
                   
               
               
                   
                   
                 gtctggctaCGatttg 
                   
                 aga 
                   
               
               
                   
                   
                 ctttcagaataaCGg 
                   
                   
                   
               
               
                   
                   
                 gaaggtgcaacaaga 
                   
                   
                   
               
               
                   
                   
                 (SEQ ID NO. 8) 
                   
                   
                   
               
               
                   
               
               
                   
                 minus 
                 3’- 
                   
                 3’- 
                 5’- 
               
               
                   
                 strand 
                 tagacatttttacaactg 
                   
                 tagatatttttataattgaag 
                 gaaacactaacg 
               
               
                   
                   
                 aagacgaaaagtctg 
                   
                 atgaaaagtttgatGCG 
                 ccatcg 
               
               
                   
                   
                 atGCGCg 
                   
                 Cgtgttggag 
                 5’- 
               
               
                   
                   
                 tgtcggagaaataaag 
                   
                 aaataaaggatgatGCtg 
                 cgtagttagattc 
               
               
                   
                   
                 gatgacGCcgaagt 
                   
                 aagtaagggagtGCtttg 
                 gagcgtag 
               
               
                   
                   
                 aagggagtGCcttgt 
                   
                 tgattGCggtaGCGCt 
                 5’- 
               
               
                   
                   
                 gactGCggtaGCG 
                   
                 ttttttgtaaaGCttgtGC 
                 aggggagcgtta 
               
               
                   
                   
                 CttccttcgtaaaGCt 
                   
                 tgattGCgaggggaata 
                 gtcgtgttcgaaa 
               
               
                   
                   
                 cgtGCtgactGCga 
                   
                 ataaatgatttgGCgatG 
                   
               
               
                   
                   
                 ggggaataataaacg 
                   
                 CgaGCttagattgatG 
                   
               
               
                   
                   
                 attcgGCgacGCg 
                   
                 CtaaatgaaagttttattG 
                   
               
               
                   
                   
                 aGCccagaccgatG 
                   
                 Cttttttatgttgtttt 
                   
               
               
                   
                   
                 Ctaaacgaaagtctta 
                   
                   
                   
               
               
                   
                   
                 ttGCccttccacgttg 
                   
                   
                   
               
               
                   
                   
                 ttct 
                   
                   
                   
               
               
                   
               
               
                   
                 minus 
                 3’- 
                 163,834,621 
                 tgGCatgatgaaaggttg 
                 5’- 
               
               
                   
                 strand 
                 cgGCacgacgaaag 
                 163,834,906 
                 gagagttgtttagtGCttg 
                 gtttttttcggcga 
               
               
                   
                   
                 gtcggagagtcgttta 
                   
                 tgGCtttttttggtGCtG 
                 taaagc 
               
               
                   
                   
                 gtGCttgt 
                   
                 CtGCtGCtttttttGCa 
                 5’- 
               
               
                   
                   
                 gGCtttcttcggtGC 
                   
                 GCGCGCatgaaggg 
                 cgcctctacgaa 
               
               
                   
                   
                 cGCcGCtGCcctc 
                   
                 aGCtGCtgttttGCtttt 
                 actctacg 
               
               
                   
                   
                 cccGCaGCGCG 
                   
                 gGCttGCGCgGCtg 
                 5’- 
               
               
                   
                   
                 CacgaagggaGCc 
                   
                 GCttttGCggGCtGC 
                 cgtcggtcgagg 
               
               
                   
                   
                 GCtgtttcGCcctcg 
                   
                 gttttaggGCgtttttGCt 
                 gcgttc 
               
               
                   
                   
                 GCccGCGCgGC 
                   
                 tGCgGC GCtgtGC 
                   
               
               
                   
                   
                 cgGCtcccGCgg 
                   
                 GCggaGCtttttggagt 
                   
               
               
                   
                   
                 GCcGCgtctcagg 
                   
                 ttgagaataggaGCtga 
                   
               
               
                   
                   
                 GCgtctccGCctG 
                   
                 gaggGCggggtggag 
                   
               
               
                   
                   
                 CgGCGCcgtGC 
                   
                 GCgggGCgttggttttg 
                   
               
               
                   
                   
                 GCggaGCttttcgg 
                   
                 gGCGCgGCattGCt 
                   
               
               
                   
                   
                 agtttgagaatagga 
                   
                 tggGCtGCtggtttttttt 
                   
               
               
                   
                   
                 GCcgagaggGCg 
                   
                 gggtggttgggagGC 
                   
               
               
                   
                   
                 gggtggagGCggg 
                   
                 tgGCat 
                   
               
               
                   
                   
                 GCgtcggttctggG 
                   
                   
                   
               
               
                   
                   
                 CGCgGCaccGC 
                   
                   
                   
               
               
                   
                   
                 ccggGCtGCcggtt 
                   
                   
                   
               
               
                   
                   
                 cctttcgggtggtcgg 
                   
                   
                   
               
               
                   
                   
                 gagGCtgGCac 
               
               
                   
               
            
           
         
       
     
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