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AU2015258259B2 - Epigenetic markers of colorectal cancers and diagnostic methods using the same - Google Patents

Epigenetic markers of colorectal cancers and diagnostic methods using the same Download PDF

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AU2015258259B2
AU2015258259B2 AU2015258259A AU2015258259A AU2015258259B2 AU 2015258259 B2 AU2015258259 B2 AU 2015258259B2 AU 2015258259 A AU2015258259 A AU 2015258259A AU 2015258259 A AU2015258259 A AU 2015258259A AU 2015258259 B2 AU2015258259 B2 AU 2015258259B2
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chrl
chrl2
chr5
dna
chr7
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AU2015258259A1 (en
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Michael Buckley
Horace Drew
Konsta Rainer Duesing
Susan Margaret Mitchell
Peter Laurence Molloy
Jason Peter Ross
Zheng-zhou XU
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Commonwealth Scientific and Industrial Research Organization CSIRO
Clinical Genomics Pty Ltd
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Commonwealth Scientific and Industrial Research Organization CSIRO
Clinical Genomics Pty Ltd
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Priority claimed from AU2010904116A external-priority patent/AU2010904116A0/en
Priority claimed from PCT/AU2011/001176 external-priority patent/WO2012034170A1/en
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Priority to AU2015258259A priority Critical patent/AU2015258259B2/en
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Priority to AU2018200932A priority patent/AU2018200932B2/en
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Abstract

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

Description

Epigenetic markers of colorectal cancers and diagnostic methods using the same
FIELD OF THE INVENTION
The present invention relates generally to nucleic acid molecules in respect of which changes to DNA methylation levels are indicative of the onset or predisposition to the onset of a neoplasm. More particularly, the present invention is directed to nucleic acid molecules in respect of which changes to DNA methylation levels are indicative of the onset and/or progression of a large intestine neoplasm, such as an adenoma or adenocarcinoma. The DNA methylation status of the present invention is useful in a range of applications including, but not limited to, those relating to the diagnosis and/or monitoring of colorectal neoplasms, such as colorectal adenocarcinomas. Accordingly, in a related aspect the present invention is directed to a method of screening for the onset, predisposition to the onset and/or progression of a neoplasm by screening for modulation in DNA methylation of one or more nucleic acid molecules.
BACKGROUND OF THE INVENTION
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 (TNM 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 tumours, or neoplasms, of epithelial origin which are derived from glandular tissue or exhibit clearly defined glandular structures. Some adenomas show recognisable 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 tumours arising from glandular structures, which are constituent parts of many organs of the body. The term adenocarcinoma is also applied to tumours showing a glandular growth pattern. These tumours 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 tumours 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 centimetre. Such a development is estimated at 40 to 50% in adenomas which are greater than 4 centimetres 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 is has metastasised. 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 anaemia 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. • Faecal 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 JN (December 1999). “Genetic cancer risk assessment. Putting it all together”. Cancer 86 (11 Suppl): 2483-92). • Endoscopy: o Sigmoidoscopy: A lit probe (sigmoidoscope) is inserted into the rectum and lower colon to check for polyps and other abnormalities, o 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'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 hese 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.
In work leading up to the present invention it has been determined that changes to the methylation of certain genes is indicative of the development of neoplasms of the large intestine, such as adenomas and adenocarcinomas. Still further, the identification of specific genomic DNA cytosine nucleotides which become hypermethylated has enabled the development of very simple and specific amplification reactions for routine use in the context of diagnosis. Diagnosis can therefore be made based on screening for one or more of a panel of these differentially methylated genes. Accordingly, the inventors have identified a panel of genes which facilitate the diagnosis of adenocarcinoma and adenoma development and/or the monitoring of conditions characterised by the development of these types of neoplasms.
SUMMARY OF THE INVENTION
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 programme Patentln Version 3.5, presented herein after the bibliography. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (eg. <2101, <2102, etc). The length, type of sequence (DNA, etc) and source organism for each sequence is indicated by information provided in the numeric indicator fields <211 >, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are identified by the indicator SEQ ID NO: followed by the sequence identifier (eg. SEQ ID N0:1, SEQ ID N0:2, etc.). The sequence identifier referred to in the specification correlates to the information provided in numeric indicator field <400 in the sequence listing, which is followed by the sequence identifier (eg. <400>1, <400>2, etc). That is SEQ ID N0:1 as detailed in the specification correlates to the sequence indicated as <400>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 defined by any one or more of Hg 19 coordinates and 2kb upstream of the transcription start site: (1) chrl 2:52400748..52409671 (34) chr 10:101292690.. 101296281 (2) chr5:3596168..3601517 (35) chr4:4190530..4228621 (3) chr!3:95361876..95364389 (36) chrl 2:54943404..54973023 (4) chr4:81187742..81212171 (37) chr5:176047210..176057557 (5) chr!9:57019212..57040270 (38) chrl2:22346325..22487648 (6) chr3:33191537..33260707 (39) chrl 9:56894648..56904889 (7) chrl5:60296421..60298142 (40) chr20:21491648..21494664 (8) chrl 3:28494168..28500451 (41) chrl :50883225..50889141 (9) chr7:96649702..96654143, (42) chr7:27180996..27183287 (10) chr8:140,811,770-141,537,860 (43) chrl 1:2016406..2019065 (11) chr5:2746279..2751769 (44) chrl4:57267425.,57277184 (12) chrl8:55102917..55158530 (45)chr4:126237567..126414087 (13) chr20:37353101..37358016 (46) chr8:23559964..23563922 (14) chr8:2792875..4852328 (47) chrlO: 131633547..131762091 (15) chrl6:66613351..66622178 (48) chr4:62362839..62938168 (16) chr5:37815753..37839782 (49) chrl :47901689..47906363 (17) chrl :63788730..63790797 (50) chrl7:77768176..77770890 (18) chrl 5:37156644..37178734 (51) chrl 7:93598762..93604831 (19) chr7:27139973..27142394 (52) chrl :33789224..33841194 (20) chr20:21686297..21696620 (53) chr9:124,004,679-124,030,840 (21) chrI6:51169886..51185183 (54) chr4:158141736.. 158287227 (22) chrl 2:85253267..85306606 (55) chrl2:9445136..9462559 (23) chr8:6357172. .6420784 (56) chrl2:24964278..25102308 (24) chrl 4:85996488..86094270 (57) chrX:21542357..21690352 (25) chr2:182541194.. 1825453 81 (58) chr20:52769988..52790516 (26) chr7:30951468..30965131 (59) chr3:172162951.. 172166203 (27) chr8:131792547..132052835 (60) chrl 3:28366780..28368089 (28) chr3:128749292,.128759583 (61) chr7:50344378...50472799 (29) chrl0:101088856..101154087 (62) chr7:149412148..149431664 (30) chr7:27282164. .27286192 (63) chr7:24323809..24331477 (31) chrl0:129535538.. 129539450 (64) chr4:30722037..31148421 (32) chrl9:49316274..49339934 (65) chrl0:47083534.,47088320 (33) chr6:391752..411443; or (ii) the gene region, including 2kb upstream of any one or more of: (1) GRASP (18) ANGPT2 (35)NKX2-6 (52) HOXA5
(2) 1RXI (19) LHX6 (36)PAX1 (53) GDNF (3) SOX21 (20) NEUROD1 (37)FOXD2 (54) FAT4 (4) FGF5 (21) AC149644.1 (38)SLC6A15 (55) HOXA2 (5) ZNF471 (22) CCDC48 (39) PHC2 (56) LPHN3 (6) SUSD5 (23) EVX1 (40) FLRT2 (57)ADCYAP1 (7) FOXB1 (24) GHSR (41)GATA2 (58) GR1A2 (8) PDX1 (25) HSD17B14 (42)ADCY8 (59)AQP1 (9) DLX5 (26) KRBA1 (43)CNNM1 (60)BCAT1 (10) ONECUT2 (27) OTOP1 (44)IKZF1 (61)CYP24A1 (11) DMRTA2 (28) PPYR1 (45)NKX2-3 (62)FOX!2 (12) CMTM2 (29) SRMS (46)PCDH7 (63)GSX1 (13) OTX2 (30) ZNF582 (47) SNCB (64)IRF4
(14) LOCI45845 (31)IRX2 (48)ST8S1AI (65)NPY
(15) EBF3 (32) CSMD1 (49)TRAPPC9 (66)PDE1B (16) SALL1 (33) MIR675.H19 (50)NKX2-2 (17) CBX8 (34) FOXD3 (51) SLC32A1 in a biological sample from said individual wherein a higher level of methylation of the DMA 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 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 a DNA region selected from: (i) the region defined by any one or more of Hgl 9 coordinates and 2kb upstream of the transcription start site: (1) chrl 2:52400748..52409671 (6) chr3:33191537..33260707 (2) chr5:3596168..3601517 (7) chrl 5:60296421 ..60298142 (3) chrl 3:95361876..95364389 (8) chrl3:28494168..28500451 (4) chr4:81187742..81212171 (9) chr7:96649702..96654143 (5) chrl 9:57019212..57040270 (ii) the gene region, including 2kb upstream of any one or more of: (1) GRASP (4) FGF5 (6) SUSD5 (8)PDX1 (2) 1RX1 (5) ZNF471 (7)FOXBl (9)DLX5 (3) SOX21 in a biological sample from said individual wherein a higher level of methylation 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) Chrl2:52400748-52409671 (GRASP) subregions Chrl2:52399672-52399922 (SEQ ID NO: 15), Chrl2:52400821 -52401119 (SEQ IDNO:16) and Chrl2:52401407-52401664 (SEQ ID NO: 17). (2) Chr8:97505882-97624037 (SDC2) subregions Chr8:97506813-97507045 (SEQ ID NO: 18) and Chr8:97507037-97507257 (SEQ ID NO: 19). (3) Chrl3:l 10959094-110959330 (SEQ ID NO:20) (COL4A1). (4) Chr5:3596168-3601517 (IRX1) subregions Chr5:3597227-3597480 (SEQ ID NO:21), Chr5:3599990-3600175 (SEQ 1DN0:22), Chr5:3600160-3600352 (SEQ ID NO:23) and Chr5:3594657-3594847 (SEQ ID NO:24). (5) Chrl3:95361879-95364389 (SOX21) subregions Chrl3:95364013-95364178 (SEQ ID NO:25) and Chrl3:95364515-95364784 (SEQ ID NO:26). (6) Chr4:81187742-81212171 (FGF5) subregions Chr4:81186918-81187228 (SEQ ID NO:27), Chr4:81187326-81187578 (SEQ ID NO:28) and Chr4:81187571-81187792 (SEQ ID NO:29). (7) Chrl9:57019212-57040269 (ZNF471) subregions Chr19:57018955-57019135 (SEQ IDNO:30) and Chrl9:57019294-57019573 (SEQ ID NO:31). (8) Chr3:33191537-33260707 (SUSD5) subregion Chr3:33260566-33260818 (SEQ ID NO:32). (9) Chr2:56093097-56151298 (EFEMP1) subregion Chr2:56150356-56150606 (SEQ ID NO:33). (10) Chr6:134210259-134216675 (TCF21) subregions Chr6:134210545-134210749 (SEQ ID NO:34), Chr6:134210712-134210951 (SEQ ID NO:35) and Chr6:134210994-134211274 (SEQ ID NO:36). (11) Chr7:94023873-94060544 (COL1A2) subregions Chr7:94023751-94023975 (SEQ ID NO:37) and Chr7:94024141-94024345 (SEQ ID NO:38). (12) Chrl 5:60296421-60298142 (FOXB1) subregions Chrl5:60296522-60296719 (SEQ ID NO:39) and Chrl 5:60297024-60297305 (SEQ IDNO:40). (13) Chrl 6:86544133-86548070 (FOXF1) subregions Chrl6:86544560-86544770 (SEQ ID NO:41), Chrl6:86544265-86544584 (SEQ IDNO:42) and Chr 16:86544795-86545110 (SEQ ID NO:43). (14) Chr 13:28494168-28500451 (PDX1) subregions Chr 13:28502100-28502311 (SEQ ID NO:44), Chrl3:28502417-28502603 (SEQ ID NO:45) and Chrl 3:28503006-28503210 (SEQ ID NO:46). (15) Chr7:96649702-96654143 (DLX5) subregions Chr7:96650026-96650127 (SEQ ID NO:47), Chr7:96651454-96651618 (SEQ ID NO:48) and Chr7:96653553-96653732 (SEQ ID NO :49). (16) Chr21:28208606-28217728 (ADAMTS1) subregions Chr21:28217946-28218109 (SEQ ID NO:50), Chr21:28218494-28218777 (SEQ ID NO:51)and Chr21:28218859-28219017 (SEQ ID NO:52). (17) Chr 13:110959631-111165373 (COL4A2) subregions Chr 13:110960787-110961141 (SEQ ID NO:53); Chrl 3:110961331-110961659 (SEQ ID NO:54) and
Chrl3:l 10959932-110960181 (SEQ ID NO:82);
(18) Chr5:83238126-83680611 (EDIL3) subregions Chr5:83679544-83679807 (SEQ ID NO:55), Chr5:83679784-83679988 (SEQ ID NO:56), Chr5:83679960-83680263 (SEQ ID NO:57), Chr5:83680075-83680383 (SEQ ID NO:58) and Chr5:83680356-83680630 (SEQ ID NO:59). (19) Chrl5:48700503-48937985 (FBN1) subregion Chrl5:48938136-48938384 (SEQ ID NO:60). (20) Chrl :47901689-47906363 (FOXD2) subregions Chrl :47899091-47899337 (SEQ ID NO:61) and Chrl :47909944-47910172 (SEQ IDNO:62). (21) Chr2:66662532-66799891 (MEIS1) subregions Chr2:66662009-66662219 (SEQ ID NO:63) and Chr2:66662177-66662430 (SEQ ID NO:64). (22) Chrl6:55513081-55540586 (MMP2) subregions Chrl6:55512662-55512856 (SEQ ID NO:65) and Chrl6:55513916-55514215 (SEQ ID NO:66). (23) Chr7:24323807-24331484 (NPY) subregions Chr7:24323765-24323936 (SEQ ID NO:67), Chr7:24324150-24324342 (SEQ IDNO:68) and Chr7:24324513-24324717 (SEQ ID NO:69). (24) Chrl9:38741877-38747172 (PPP1R14A) subregions Chrl9:38747251-38747424 (SEQ ID NO:70) and Chrl 9:38746653-38746912 (SEQ ID NO:71).
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 of one or more cytosine residues selected from: Chrl2:52400748-52409671 (GRASP)
Chrl 2:52399713 Chrl2:52399731 Chrl 2:52399749 Chrl 2:52399783
Chrl2:52399796 Chrl2:52399808 Chrl 2:52399823 Chrl2:52399835
Chrl2:52399891
Chrl 2:52400847 Chrl2:52400850 Chrl 2:52400859 Chrl 2:52400866
Chrl 2:52400869 Chrl2:52400873 Chrl 2:52400881 Chrl 2:52400886
Chrl 2:52400893 Chrl2:52400895 Chrl2:52400899 Chrl 2:52400902
Chrl 2:52400907 Chrl2:52400913 Chrl 2:52400919 Chrl 2:52400932
Chrl2:52400938 Chrl 2:52400958 Chrl 2:52400962 Chrl 2:52400971
Chrl 2:52400973 Chrl 2:52400976 Chrl2:52400998 Chrl2:52401008
Chrl2:52401010 Chrl2:52401012 Chrl 2:52401016 Chrl 2:52401019
Chrl 2:52401025 Chrl2:52401041 Chrl2:52401044 Chrl2:52401053
Chrl 2:52401060 Chrl2:52401064 Chrl 2:52401092 Chrl2:52401118
Chrl2:52401438 Chrl2:52401448 Chrl 2:52401460 Chrl 2:52401465
Chrl2:52401474 Chrl2:52401477 Chrl 2:52401479 Chrl 2:52401483
Chrl 2:52401504 Chrl2:52401514 Chrl 2:52401523 Chrl 2:52401540
Chrl 2:52401553 Chrl 2:52401576 Chrl 2:52401588 Chrl2:52401595
Chrl 2:52401599 Chrl 2:52401604 Chrl2:52401606 Chrl2:52401634
Chrl 2:52401640 Chrl2:52401644 Chrl2:52401659
Chr8:97505882-97624037 (SDC2)
Chr8:97506843 Chr8:97506845 Chr8:97506861 Chr8:97506876
Chr8:97506902 Chr8:97506909 Chr8:97506911 Chr8:97506913
Chr8:97506924 Chr8:97506932 Chr8:97506934 Chr8:97506940
Chr8:97506977 Chr8:97506993 Chr8:97507005 Chr8:97507008
Chr8:97507014
Chr8:97507038 Chr8:97507061 Chr8:97507064 Chr8:97507078
Chr8:97507082 Chr8:97507084 Chr8:97507089 Chr8:97507119
Chr8:97507128 Chr8:97507136 Chr8:97507144 Chr8:97507153
Chr8:97507162 Chr8:97507214 Chr8:97507216
Chrl3:110959094-110959330 (COL4A1).
Chrl3:97507038 Chr 13:97507061 Chrl3:97507064 Chrl 3:97507078
Chrl3:97507082 Chrl 3:97507084 Chrl 3:97507089 Chrl 3:97507119
Chrl3:97507128 Chrl 3:97507136 Chrl3:97507144 Chrl 3:97507153
Chrl3:97507162 Chrl3:97507214 Chrl 3:97507216
Chr5:3596168-3601517 (1RX1)
Chr5:3597229 Chr5:3597299 Chr5:3597311 Chr5:3597321
Chr5:3597330 Chr5:3597350 Chr5:3597352 Chr5:3597373
Chr5:3597413 Chr5:3597427 Chr5:3597431 Chr5:3597447
Chr5:3597450 Chr5:3597454 Chr5:3597456
Chr5:3599998 Chr5:3600019 Chr5:3600037 Chr5:3600050
Chr5:360005 8 Chr5:3600061 Chr5:3600070 Chr5:3600074
Chr5:3600076 Chr5:3600079 Chr5:3600089 Chr5:3600091
Chr5:3600104 Chr5:3600116 Chr5:3600121 Chr5:3600124
Chr5:3600131 Chr5:3600143 Chr5:3600149 Chr5:3600169
Chr5:3600173
Chr5:3600163 Chr5:3600190 Chr5:3600201 Chr5:3600205
Chr5:3600208 Chr5:3600214 Chr5:3600250 Chr5:3600252
Chr5:3600255 Chr5:3600264 Chr5:3600270 Chr5:3600284
Chr5:3600309 Chr5:3600318 Chr5:3600345
Chr5:3 594663 Chr5:3594679 Chr5:3594684 Chr5:3594697
Chr5:3 594703 Chr5:3594711 Chr5:3594725 Chr5:3594738
Chr5:3594745 Chr5:3594758 Chr5:3594768 Chr5:3594774
Chr5:3594778 Chr5:3594794 Chr5:3594799 Chr5:3594818
Chr5:3594840
Chrl3:95361879-95364389 ($0X21)
Chrl 3:95364016 Chrl 3:95364019 Chrl3:95364042 Chrl 3:95364050
Chrl 3:95364054 Chrl 3:95364061 Chrl 3:95364064 Chrl 3:95364074
Chrl 3:95364080 Chrl3:95364082 Chrl 3:95364106 Chrl 3:95364119
Chrl3:95364123 Chrl3:95364125 Chrl3:95364128 Chr 13:95364141
Chrl 3:95364163 Chrl3:95364171
Chrl 3:95364543 Chrl 3:95364545 Chrl 3:95364548 Chrl 3:95364551
Chrl 3:95364560 Chrl 3:95364571 Chrl 3:953645 82 Chrl 3:95364584
Chrl 3:95364587 Chrl 3:95364602 Chrl 3:95364623 Chrl 3:95364627
Chrl 3:95364640 Chrl 3:95364649 Chrl 3:95364653 Chrl 3:95364656
Chrl3:95364665 Chrl3:95364684 Chrl 3:95364696 Chrl 3:95364712
Chrl3:95364729 Chrl 3:95364732 Chrl 3:95364734 Chrl 3:95364748
Chrl 3:95364751
Chr4:81187742-81212171 (FGF5)
Chr4:81186919 Chr4:81186947 Chr4:81186964 Chr4:81186968
Chr4:81186973 Chr4:81186975 Chr4:81186983 Chr4:81187001
Chr4:81187011 Chr4:81187041 Chr4:81187056 Chr4:81187062
Chr4:81187093 Chr4:81187116 Chr4:81187125 Chr4:8l 187161
Chr4:81187191 Chr4:81187196 Chr4:81187198
Chr4:81187367 Chr4:81187389 Chr4:81187458 Chr4:81187467
Chr4:81187495 Chr4:81187498 Chr4:81187504 Chr4:81187512
Chr4:81187514 Chr4:81187530 Chr4:81187539 Chr4:81187547
Chr4:81187549 Chr4:81187551 Chr4:81187554 Chr4:81187556
Chr4:81187575
Chr4:81187575 Chr4:81187592 Chr4:81187601 Chr4:81187605
Chr4:81187610 Chr4:81187648 Chr4:81187652 Chr4:81187679
Chr4:81187685 Chr4:81187691 Chr4:81187693 Chr4:81187707
Chr4:81187712 Chr4:81187733 Chr4:81187735 Chr4:81187752
Chr4:81187758 Chr4:81187764 Chr4:81187784
Chr19:57019212-57040269 (ZNF471)
Chrl 9:57018990 Chrl9:57018994 Chrl9:57019003 Chrl9:57019010
Chrl9:57019018 Chrl9:57019020 Chrl9:57019025 Chrl 9:57019029
Chrl9:57019044 Chrl 9:57019047 Chrl 9:57019067 Chrl9:57019073
Chrl 9:57019084 Chrl9:57019101 Chrl 9:57019118
Chr 19:57019315 Chrl 9:57019321 Chrl 9:57019346 Chrl9:57019351
Chr 19:57019355 Chrl9:57019361 Chrl 9:57019364 Chrl 9:57019366
Chrl 9:57019371 Chrl9:57019373 Chrl9:57019385 Chrl 9:57019387
Chrl9:57019405 Chrl 9:57019428 Chr 19:57019433 Chrl 9:57019435
Chrl9:57019437 Chrl9:57019443 Chrl9:57019451 Chrl 9:57019456
Chrl9:57019463 Chrl 9:57019465 Chrl9:57019470 Chrl 9:57019483
Chrl9:57019487 Chrl9:57019492 Chrl 9:57019502 Chrl 9:57019505
Chr3:33191537-33260707 (SUSD5)
Chr3:33260601 Chr3:33260621 Chr3:3326063I Chr3:33260640
Chr3:33260651 Chr3:33260665 Chr3:33260676 Chr3:33260694
Chr3:33260698 Chr3:33260711 Chr3:33260715 Chr3:33260732
Chr3:33260742 Chr3:33260748 Chr3:33260755 Chr3:33260760
Chr3:33260769 Chr3:33260776 Chr3:33260778 Chr3:33260780
Chr3:33260788 Chr3:33260806
Chr2:56093097-56151298 (EFEMP1)
Chr2:56150376 Chr2:56150389 Chr2:56150394 Chr2:56150415
Chr2:56150419 Chr2:56150423 Chr2:56150433 Chr2:56150473
Chr2:56150475 Chr2:56150478 Chr2:56150499 Chr2:56l 50537
Chr2:56150549 Chr2:56150580 Chr2:56150601
Chr6:134210259-134216675 (TCF21)
Chr6:134210556 Chr6:134210598 Chr6:134210615 Chr6:134210640
Chr6:134210649 Chr6:134210667 Chr6:134210692 Chr6:134210694
Chr6:134210697 Chr6:134210720 Chr6:134210745
Chr6:134210720 Chr6:134210745 Chr6:134210776 Chr6:134210781
Chr6:134210784 Chr6:134210790 Chr6:134210792 Chr6:134210794
Chr6:134210800 Chr6:134210806 Chr6:134210812 Chr6:134210868
Chr6:134210894 Chr6:134210906 Chr6:134210919 Chr6:134210946
Chr6:134211050 Chr6:134211061 Chr6:134211076 Chr6:134211081
Chr6:134211103 Chr6:134211110 Chr6:134211121 Chr6:134211125
Chr6:134211131 Chr6:134211153 Chr6:134211155 Chr6:134211162
Chr6:134211179 Chr6:134211182 Chr6:134211184 Chr6:134211208
Chr6:134211210 Chr6:134211212 Chr6:134211218 Chr6:134211220
Chr6:134211227 Chr6:1342ll233 Chr6:134211241 Chr6:134211245
Chr6:134211247 Chr6:134211270
Chr7:94023873-94060544 (COL1A2)
Chr7:94024172 Chr7:94024191 Chr7:94024214 Chr7:94024230
Chr7:94024254 Chr7:94024266 Chr7:94024268 Chr7:94024272
Chr7:94024288 Chr7:94024291 Chr7:94024310
Chrl5:60296421-60298142 (FOXB1)
Chrl 5:60296555 Chrl 5:60296561 Chrl 5:60296563 Chrl 5:60296578
Chrl5:60296585 Chrl 5:60296598 Chrl 5:60296601 Chrl 5:60296614
Chrl 5:60296616 Chrl 5:60296619 Chrl 5:60296627 Chrl 5:60296633
Chrl 5:60296639 Chrl 5:60296643 Chrl 5:60296647 Chrl 5:60296654
Chrl 5:60296665 Chrl 5:60296668 Chrl 5:60296670 Chrl 5:60296675
Chrl 5:60296679 Chrl 5:60296684 Chrl 5:60296689 Chrl 5:60296694
Chrl 5:60297035 Chrl 5:60297050 Chrl 5:60297053 Chrl 5:60297109
Chrl 5:60297118 Chrl 5:60297121 Chrl 5:60297126 Chrl 5:60297128
Chrl 5:60297130 Chrl 5:60297152 Chrl 5:60297169 Chrl 5:60297174
Chrl5:60297178 Chrl 5:60297185 Chrl 5:60297192 Chrl 5:60297203
Chrl 5:60297212 Chrl 5:60297221 Chrl 5:60297228 Chrl 5:60297252
Chrl 5:60297266 Chrl 5:60297273 Chrl 5:60297298
Chrl6:86544133-86548070 (FOXF1)
Chrl6:86544571 Chrl 6:86544587 Chrl 6:86544590 Chrl6:86544593
Chrl 6:86544597 Chrl 6:86544599 Chrl 6:86544601 Chrl6:86544608
Chrl6:86544624 Chrl 6:86544652 Chrl 6:86544658 Chrl 6:86544675
Chrl 6:86544685 Chrl 6:86544699 Chrl 6:86544703 Chrl 6:86544706
Chrl6:86544714 Chrl 6:86544720 Chrl 6:86544735 Chrl 6:86544745
Chrl6:86544763
Chrl 6:86544268 Chrl 6:86544273 ' Chrl 6:86544295 Chrl 6:86544298
Chrl6:86544305 Chrl 6:86544308 Chrl 6:86544312 Chrl 6:86544321
Chrl 6:86544337 Chrl 6:86544339 Chrl 6:86544346 Chrl 6:86544377
Chrl6:86544384 Chrl 6:86544391 Chrl 6:86544416 Chrl 6:86544431
Chrl 6:86544460 Chrl 6:86544464 Chrl 6:86544477 Chrl 6:86544484
Chrl6:86544518 Chrl 6:86544523 Chrl 6:86544547 Chrl 6:86544552
Chrl 6:86544559 Chrl 6:86544571
Chrl 6:86544810 Chrl 6:86544832 Chrl 6:86544835 Chr16:86544843
Chrl 6:86544853 Chrl 6:86544859 Chrl 6:86544862 Chrl 6:86544865
Chrl 6:86544867 Chrl 6:86544870 Chrl 6:86544874 Chrl 6:86544877
Chrl6:86544885 Chrl 6:86544892 Chrl 6:86544900 Chr 16:86544907
Chrl 6:86544915 Chrl 6:86544928 Chrl 6:86544931 Chrl 6:86544934
Chr 16:86544951 Chrl 6:86544955 Chrl 6:86544958 Chrl 6:86544966
Chrl6:86544972 Chrl 6:86544975 Chrl 6:86544978 Chrl6:86544987
Chrl6:86544993 Chrl 6:86544996 Chrl 6:86545000 Chrl6:86545002
Chr 16:86545005 Chrl 6:86545015 Chrl 6:86545018 Chrl6:86545060
Chr 16:86545062 Chrl6:86545078 Chrl 6:86545092
Chrl3:28494168-28500451 (PDX1)
Chr 13:28502109 Chrl 3:28502153 Chrl3:28502161 Chrl 3:28502177
Chr 13:28502179 Chrl 3:28502191 Chrl3:28502198 Chrl3:28502205
Chr 13:28502207 Chrl 3:28502210 Chrl 3:28502245 Chrl 3:28502309
Chrl 3:28502442 Chrl 3:28502449 Chrl 3:28502461 Chrl 3:28502464
Chrl 3:28502475 Chrl 3:28502507 Chrl 3:28502512 Chrl 3:28502538
Chrl 3:28502544 Chrl 3:28502549 Chrl 3:28502559 Chrl 3:28502564
Chrl 3:28502585
Chrl 3:28503045 Chrl 3:28503049 Chrl 3:28503081 Chrl 3:28503099
Chrl 3:28503114 Chrl 3:28503127 Chrl3:28503138 Chrl 3:28503147
Chrl 3:28503155 Chrl 3:28503157 Chrl3:28503179
Chr7:96649702-96654143 (DLX5)
Chr7:96650062 Chr7:96650072 Chr7:96650078 Chr7:96650096
Chr7:96650099 Chr7:96650102
Chr7:96651485 Chr7:96651488 Chr7:96651518 Chr7:96651523
Chr7:96651532 Chr7:96651535 Chr7:96651537 Chr7:96651542
Chr7:96651550 Chr7:96651586
Chr7:96653596 Chr7:96653605 Chr7:96653607 Chr7:96653617
Chr7:96653620 Chr7:96653623 Chr7:96653644 Chr7:96653656
Chr7:96653683 Chr7:96653686 Chr7:96653692 Chr7:96653698
Chr7:96653701
Chr21:28208606-28217728 (ADAMTS1)
Chr21:28217973 Chr21:28218002 Chr21:28218015 Chr21:28218018
Chr21:28218047 Chr21:28218051 Chr21:28218057 Chr21:28218072
Chr21:28218074 Chr21:28218084 Chr21:28218105
Chr21:28218514 Chr21:28218516 Chr21:28218550 Chr21:28218568
Chr21:28218579 Chr21:28218586 Chr21:28218596 Chr21:28218635
Chr21:28218638 Chr21:28218646 Chr21:28218671 Chr21:28218684
Chr21:28218688 Chr21:28218704 Chr21:28218729 Chr21:28218741
Chr21:28218893 Chr21:28218906 Chr21:28218914 Chr21:28218916
Chr21:28218928 Chr21:28218934 Chr21:28218938 Chr21:28218949
Chr21:28218953 Chr21:28218959 Chr21:28218974 Chr21:28218976
Chr21:28218978 Chr21:28218984 Chr21:28218986 Chr21:28218996
Chr21:28219008 Chr21:28219016
Chrl3:110959631-1111653 73 (COL4A2)
Chrl 3:110960813 Chrl 3:110960827 Chrl 3:110960849 Chrl 3:110960875
Chrl 3:110960925 Chrl3:l 10960930 Chrl 3:110960938 Chrl 3:110960978
Chrl3:l 10961001 Chrl 3:110961003 Chrl3:l 10961025 Chrl 3:110961045
Chrl3:l 10961049 Chrl 3:110961069 Chrl3:l 10961072 Chrl 3:110961088
Chrl3:l 10961091 Chrl 3:110961095 Chrl3:l 10961110 Chrl3:l 10961116
Chrl3:l 10961131 Chrl3:110960786
Chrl 3:110961358 Chrl3:l 10961386 Chrl 3:110961404 Chrl 3:110961406
Chrl3:l 10961445 Chrl3:l 10961579 Chrl3:l 10961594 Chrl 3:110961606
Chrl3:l 10961631
Chrl3:110959935 Chrl 3:110959965 Chrl 3:110959968 Chrl3:l 10959974
Chrl 3:110959979 Chrl 3:110959981 Chrl 3:110959986 Chrl 3:110959988
Chr 13:110959997 Chrl 3:110959999 Chrl 3:110960004 Chrl 3:110960006
Chr13:110960011 Chrl 3:110960027 Chrl 3:110960035 Chrl 3:110960046
Chrl3:l 10960051 Chrl 3:110960071 Chrl 3:110960099 Chrl3:l 10960108
Chrl3:l 10960115 Chrl 3:110960156 Chrl 3:110960180
Chr5:83238126-83680611 (EDIL3)
Chr5:83679825 Chr5:83679827 Chr5:83679836 Chr5:83679843
Chr5:83679846 Chr5:83679860 Chr5:83679864 Chr5:83679883
Chr5:83679894 Chr5:83679900 Chr5:83679906 Chr5:83679910
Chr5:83679937 Chr5:83679953 Chr5:83679957 Chr5:83679961
Chr5:83679976 Chr5:83679982 Chr5:83679987 Chr5:83680003
Chr5:83680007 Chr5:83680011 Chr5:83680026 Chr5:83680032
Chr5:83680086 Chr5:83680090 Chr5:83680103 Chr5:83680106
Chr5:83680122 Chr5:83680126
Chr5:83679546 Chr5:83679568 Chr5:83679571 Chr5:83679585
Chr5:83679602 Chr5:83679617 Chr5:83679630 Chr5:83679640
Chr5:83679667 Chr5:83679675 Chr5:83679689 Chr5:83679693
Chr5:83679699 Chr5:83679701 Chr5:83679733
Chr5:83679973 Chr5:83679989 Chr5:83679992 Chr5:83680001
Chr5.83680003 Chr5:83680006 Chr5:83680012 Chr5:83680015
Chr5:83680019 Chr5:83680033 Chr5:83680036 Chr5:83680045
Chr5:83680051 Chr5:83680061 Chr5:83680063 Chr5:83680066
Chr5:83680108 Chr5:83680115 Chr5:83680118 Chr5:83680132
Chr5:83680147 Chr5:83680159 Chr5:83680174 Chr5:83680181
Chr5:83680185 Chr5:83680198 Chr5:83680205 Chr5:83680209
Chr5:83680217 Chr5:83680232 Chr5:83680235 Chr5:83680237
Chr5:83680253
Chr5:83680108 Chr5:83680115 Chr5:83680118 Chr5:83680132
Chr5:83680147 Chr5:83680159 Chr5:83680174 Chr5:83680181
Chr5:83680185 Chr5:83680198 Chr5:83680205 Chr5:83680209
Chr5:83680217 Chr5:83680232 Chr5:83680235 Chr5:83680237
Chr5:83680253 Chr5:83680264 Chr5:83680284 Chr5:83680310
Chr5:83680326 Chr5:83680355
Chr5:83680401 Chr5:83680406 Chr5:83680409 Chr5:836804! 1
Chr5:83680413 Chr5:83680430 Chr5:83680438 Chr5:83680441
Chr5:83680444 Chr5:83680458 Chr5:83680463 Chr5:83680474
Chr5:83680483 Chr5:83680485 Chr5:83680488 Chr5:83680492
Chr5:83680497 Chr5:83680499 Chr5:83680507 Chr5:83680509
Chr5:83680513 Chr5:83680518 Chr5:83680529 Chr5:83680531
Chr5:83680548 Chr5:83680559 Chr5:83680564 Chr5:83680568
Chr5:83680572 Chr5:83680579 Chr5:83680589 Chr5:83680591
Chr5:83680593 Chr5:83680596 Chr5:83680602
Chrl5:48700503-48937985 (FBN1)
Chrl 5:48938149 Chrl 5:48938163 Chrl 5:48938172 Chrl 5:48938181
Chrl 5:48938183 Chrl 5:48938218 Chrl 5:48938236 Chrl5:48938238
Chrl5:48938254 Chrl 5:48938267 Chrl 5:48938273 Chrl 5:48938280
Chrl 5:48938291 Chrl 5:48938349
Chrl:47901689-47906363 (FOXD2)
Chrl :47899102 Chrl :47899126 Chrl:47899129 Chrl :47899143
Chrl :47899152 Chrl :47899168 Chrl :47899184 Chrl :47899205
Chrl :47899212 Chrl :47899230 Chrl :47899232 Chrl :47899253
Chrl :47899286 Chrl :47899327
Chrl :47909988 Chrl :47909995 Chrl :47910005 Chrl :47910036
Chrl :47910051 Chrl :47910056 Chrl :47910062 Chrl :47910074
Chrl :47910103 Chrl:47910122 Chrl:47910137 Chrl :47910142
Chrl :47910144 Chrl :47910146
Chr2:66662532-66799891 (ME1S1)
Chr2:66662043 Chr2:66662047 Chr2:66662049 Chr2:66662063
Chr2:66662073 Chr2:66662l07 Chr2:66662111 Chr2:66662124
Chr2:66662145 Chr2:66662156 Chr2:66662161 Chr2:66662163
Chr2:66662178 Chr2:66662218
Chr2:66662178 Chr2:66662218 Chr2:66662220 Chr2:66662246
Chr2:66662265 Chr2:66662280 Chr2:66662306 Chr2:66662310
Chr2:66662323 Chr2:66662333 Chr2:66662337
Chrl6:55513081-55540586 (MMP2)
Chrl6:55512695 Chrl6:55512711 Chrl6:55512732 Chrl 6:55512750
Chrl 6:55512753 Chrl6:55512793
Chrl6:55513961 Chrl 6:55513997 Chrl 6:55514025 Chrl 6:55514059
Chrl 6:55514061 Chrl 6:55514092 Chrl 6:55514120 Chrl 6:55514131
Chrl6:55514158 Chrl6:55514205
Chr7:24323807-24331484 (NPY)
Chr7:24323767 Chr7:24323792 Chr7:24323794 Chr7:24323799
Chr7:24323817 Chr7:24323834 Chr7:24323840 Chr7:24323844
Chr7:24323848 Chr7:24323866 Chr7:24323876 Chr7:24323880
Chr7:24323882 Chr7:24323884 Chr7:24323894 Chr7:24323905
Chr7:24323910 Chr7:24323930 Chr7:24323934
Chr7:24324180 Chr7:24324186 Chr7:24324206 Chr7:24324217
Chr7:24324222 Chr7:24324225 Chr7:24324244 Chr7:24324249
Chr7:24324251 Chr7:24324268 Chr7:24324274 Chr7:24324280
Chr7:24324311 Chr7:24324313
Chr7:24324549 Chr7:24324570 Chr7:24324592 Chr7:24324595
Chr7:24324597 Chr7:24324599 Chr7:24324621 Chr7:24324625
Chr7:24324633 Chr7:24324635 Chr7:24324668 Chr7:24324672
Chr7:24324676 Chr7:24324679 Chr7:24324683 Chr7:24324690
Chr19:38741877-38747172 (PPP1R14A)
Chrl 9:38747280 Chrl 9:38747296 Chrl 9:38747300 Chrl 9:38747313
Chrl 9:38747319 Chrl 9:38747322 Chrl 9:38747328 Chrl 9:38747334
Chrl 9:38747355 Chrl 9:38747361 Chrl9:38747371 Chrl 9:38747382
Chrl 9:38747389 Chrl9:38747410
Chr 19:38746654 Chrl9:38746680 Chrl 9:38746690 Chrl 9:38746701
Chr 19:38746715 Chrl 9:38746726 Chrl 9:38746728 Chrl 9:38746732
Chrl 9:38746749 Chrl 9:38746759 Chrl 9:38746770 Chr 19:38746784
Chrl 9:38746786 Chrl 9:38746804 Chrl 9:38746808 Chrl 9:38746811
Chr 19:38746826 Chrl 9:38746834 Chrl 9:38746837 Chrl 9:38746849
Chrl 9:38746852 Chrl 9:38746865 Chrl 9:38746871 Chrl 9:38746873
Chrl 9:38746875 Chrl 9:38746877 Chrl 9:38746882 Chrl 9:38746898 or a corresponding cytosine at position n+1 on the opposite DNA strand, in a biological sample from said individual wherein a higher level of methylation of one or more of said residues relative to the methylation level of a corresponding residue in a control sample is indicative of a large intestine neoplasm or a predisposition to the onset of a neoplastic state.
In yet another aspect 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 defined by any one or more of Hg 19 coordinates and 2kb upstream of the transcription start site: (1) chrl2:52400748..52409671 (34) chrlO: 101292690.. 101296281 (2) chr5:3596168..3601517 (35) chr4:4190530..4228621 (3) chrl3:95361876..95364389 (36) chr 12:54943404..54973023 (4) chr4:81187742..81212171 (37) chr5:176047210..176057557 (5) chrl 9:57019212..57040270 (38) chrl 2:22346325..22487648 (6) chr3:33191537..33260707 (39) chrl 9:56894648..56904889 (7) chr 15:60296421..60298142 (40) chr20:21491648..21494664 (8) chrl 3:28494168..28500451 (41) chrl :50883225..50889141 (9) chr7:96649702..96654143, (42) chr7:27180996..27183287 (10) chr8:140,811,770-141,537,860 (43) chrl 1:2016406..2019065 (11) chr5:2746279..2751769 (44) chrl4:57267425.,57277184 (12) chrl 8:55102917..55158530 (45) chr4:126237567..126414087 (13) chr20:37353101..37358016 (46) chr8:23559964,.23563922 (14) chr8:2792875..4852328 (47) chrl0:131633547..131762091 (15) chrl 6:66613351 ..66622178 (48) chr4:62362839. .6293 8168 (16) chr5:37815753..37839782 (49) chrl :47901689..47906363 (17) chrl :63788730..63790797 (50) chrl 7:77768176..77770890 (18) chrl5:37156644..37178734 (51) chrl 7:93598762..93604831 (19) chr7:27139973 ..27142394 (52) chrl :33789224..33841194 (20) chr20:21686297. .21696620 (53) chr9:124,004,679-124,030,840 (21) chrl 6:51169886. .51185183 (54) chr4:158141736.. 158287227 (22) chrl2:85253267..85306606 (55) chrl2:9445136..9462559 (23) chr8:6357172..6420784 (56) chrl2:24964278..25102308 (24) chrl4:85996488..86094270 (57) chrX:21542357..21690352 (25) chr2:182541194..182545381 (58) chr20:52769988..527905l6 (26) chr7:30951468..30965131 (59) chr3:l 72162951.. 172166203 (27) chr8:131792547.. 132052835 (60) chrl 3:28366780..28368089 (28) chr3:128749292.. 128759583 (61) chr7:50344378...50472799 (29) chrl 0:101088856.. 101154087 (62) chr7:149412148.. 149431664 (30) chr7:27282164..27286192 (63) chr7:24323809..2433!477 (31) chr10:129535538.. 129539450 (64) chr4:30722037..31148421 (32) chrl9:49316274..49339934 (65) chrl 0:47083534..47088320 (33) chr6:391752..411443 (ii) the gene region, including 2kb upstream of any one or more of: (1) GRASP (18) ANGPT2 (35)NKX2-6 (52) HOXA5
(2) 1RX1 (19) LHX6 (36)PAX1 (53) GDNF (3) SOX21 (20) NEUROD1 (37) FOXD2 (54) FAT4 (4) FGF5 (21) AC 149644.1 (38)SLC6A15 (55) HOXA2 (5) ZNF471 (22) CCDC48 (39) PHC2 (56) LPHN3 (6) SUSD5 (23) EVX1 (40) FLRT2 (57)ADCYAP1 (7) FOXB1 (24) GHSR (41)GATA2 (58) GRIA2 (8) PDX1 (25) HSD17B14 (42)ADCY8 (59)AQP1 (9) DLX5 (26) KRBA1 (43)CNNM1 (60)BCAT1 (10) ONECUT2 (27) OTOP1 (44)IKZF1 (61)CYP24A1 (11) DMRTA2 (28) PPYR1 (45)NKX2-3 (62) FOXI2 (12) CMTM2 (29) SRMS (46) PCDH7 (63)GSX1 (13) OTX2 (30) ZNF582 (47) SNCB (64) IRF4
(14) LOCI 45845 (31)IRX2 (48)ST8SIA1 (65)NPY
(15) EBF3 (32) CSMD1 (49)TRAPPC9 (66)PDE1B (16) SALL1 (33) MIR675.H19 (50)NKX2-2 (17) CBX8 (34) FOXD3 (51) SLC32A1 in a biological sample from said individual wherein a lower level of expression 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. 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 one 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 hybridising 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 molecule; or (iii) nucleic acid probes or oligonucleotides comprising a nucleotide sequence capable of hybridising 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 molecule; or (iv) probes capable of binding to any one 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) or proteins of (iv) is indicative of the neoplastic state of a cell or cellular subpopulation derived from the large intestine.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1 and 2 depict the differential methylation plots for the genes shown in Table 3 and 4, respectively. The chromosome co-ordinates of the tiled regions are shown on the X-axis. The positions of TaqI and MspI restriction sites are indicated by dashed vertical lines. On the Y-axis is shown (log2 scale) the hybridisation intensities. In a column above each probe position are represented the signals of the methylated fractions of normal and cancer DNAs (GN and GT respectively) and their unmethylated fractions (AN and AT respectively). The “double-differences” (GT-GN) - (AT-AN), that represents the differential methylation between cancer and normal is shown as black DD for probes with the highest 17% quality score, as grey DD for those in the top 43% and as grey dots for those probes with lower quality scores or that lie beyond 300 bp from a TaqI or MspI restriction site. All DD probe points are circled for clarity.
Figures 3-27 show the profiles of DNA methylation obtained from bisulphite sequence analysis of one or more amplicons from 24 genes. In each figure the upper box shows the chromosomal co-ordinates of the gene (Hgl9 sequence) and of the individual amplicons. The DNA methylation plots show the fraction of methylated cytosine (Y-axis) at each CpG site as determined by sequence analysis. The positions of each CpG site within the amplicons are shown on the X-axis. Individual grey dots represent methylation fractions at each CpG site for the 10 cancer DNAs; the average methylation of the ten samples is shown by the continuous grey line and the interquartile range by the shaded area. Similarly, the methylation fraction of normal DNA samples is shown by black dots, the average methylation and interquartile range by the black line and shaded black area respectively. The methylation fraction in blood DNA is shown by a light grey dashed line (this is often obscured by the methylation data for normal tissue DNA). The figures also include the sequence region for each PCR amplicon, prior to bisulphite modification. In some cases have been designed to the bottom strand relative to the direction of gene transcription and these are labelled BS. The pictograms at the bottom of the figures show the chromosomal positions of the genes and maps that show the location and exon structure of the gene transcripts, the position of Affymetrix U133 Plus probesets and location of CpG islands, as well as the location of the different amplified regions.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is predicated, in part, on the elucidation of DNA methylation status which characterises large intestine neoplasms. This finding has now facilitated the development of routine means of screening for the onset or predisposition to the onset of or monitoring a large intestine neoplasm based on increased methylation of certain genes relative to control levels.
In accordance with the present invention, it has been determined that certain 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 of the tiling regions of Nimblegen promoter tiling arrays are consistent with the human genome database version Hgl8 (referred to herein as “Hgl8 coordinates” and are detailed in Tables 3 and 4). To the extent that chromosomal coordinates corresponding to gene names are listed, these are consistent with the human genome database version Hgl9 which was released in February 2009 (herein referred to as “Hgl9 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 defined by any one or more of Hgl 9 coordinates and 2kb upstream of the transcription start site: (1) chrl2:52400748..52409671 (34) chrl 0:101292690.. 101296281 (2) chr5:3596168..3601517 (35) chr4:4190530..4228621 (3) chrl 3:95361876..95364389 (36) chrl2:54943404..54973023 (4) chr4:81187742..81212171 (37) chr5:176047210..176057557 (5) chrl 9:57019212..57040270 (38) chrl2:22346325..22487648 (6) chr3:33191537..33260707 (39) chrl9:56894648.,56904889 (7) chrl 5:60296421..60298142 (40) chr20:21491648..21494664 (8) chrl 3:28494168..28500451 (41) chrl :50883225..50889141 (9) chr7:96649702..96654143, (42) chr7:27180996..27183287 (10) chr8:140,811,770-141,537,860 (43) chrl 1:2016406..2019065 (11) chr5:2746279..2751769 (44) chrl4:57267425..57277184 (12) chrl 8:55102917..55158530 (45) chr4:l26237567..126414087 (13) chr20:37353101..37358016 (46) chr8:23559964..23563922 (14) chr8:2792875..4852328 (47) chrl 0:131633547.. 131762091 (15) chrl 6:66613351..66622178 (48) chr4:62362839..62938168 (16) chr5:37815753..37839782 (49) chrl :47901689..47906363 (17) chrl :63788730..63790797 (50) chrl 7:77768176..77770890 (18) chrl 5:37156644..37178734 (51) chrl 7:93598762..93604831 (19) chr7:27139973..27142394 (52) chrl :33789224..33841194 (20) chr20:21686297..21696620 (53) chr9:124,004,679-124,030,840 (21) chrl 6:51169886..51185183 (54) chr4:158141736.. 158287227 (22) chrl 2:85253267..85306606 (55) chrl2:9445136..9462559 (23) chr8:6357172..6420784 (56) chrl 2:24964278..25102308 (24) chrl 4:85996488..86094270 (57) chrX:21542357..21690352 (25) chr2:182541194..182545381 (58) chr20:52769988..527905l6 (26) chr7:30951468..30965131 (59) chr3:172162951.. 172166203 (27) chr8:131792547.. 132052835 (60) chrl 3:28366780..28368089 (28) chr3:128749292.. 128759583 (61) chr7:50344378...50472799 (29) chrl 0:101088856.. 101154087 (62) chr7:149412148..149431664 (30) chr7:27282164. .27286192 (63) chr7:24323809..24331477 (31) chrlO: 129535538..129539450 (64) chr4:30722037..31148421 (32) chrl 9:49316274..49339934 (65) chrl 0:47083534..47088320 (33) chr6:391752..411443; or (ii) the gene region, including 2kb upstream of any one or more of: (1) GRASP (18) ANGPT2 (35)NKX2-6 (52) HOXA5
(2) 1RX1 (19) LHX6 (36)PAX1 (53) GDNF (3) SOX21 (20) NEUROD1 (37) FOXD2 (54)FAT4 (4) FGF5 (21) AC 149644.1 (38)SLC6A15 (55) HOXA2 (5) ZNF47I (22) CCDC48 (39)PHC2 (56) LPHN3 (6) SUSD5 (23) EVX1 (40)FLRT2 (57)ADCYAP1 (7) F0XB1 (24) GHSR (41)GATA2 (58) GRIA2 (8) PDX1 (25) HSD17B14 (42)ADCY8 (59)AQP1 (9) DLX5 (26) KRBA1 (43)CNNM1 (60) BCAT1 (10) ONECUT2 (27) OTOP1 (44)1KZF1 (61)CYP24A1 (11) DMRTA2 (28) PPYR1 (45)NKX2-3 (62)FOXI2 (12) CMTM2 (29) SRMS (46)PCDH7 (63)GSX1 (13) OTX2 (30) ZNF582 (47)SNCB (64)IRF4
(14) LOCI 45845 (31)IRX2 (48)ST8SIA1 (65)NPY
(15) EBF3 (32) CSMD1 (49)TRAPPC9 (66)PDE1B (16) SALL1 (33) MIR675.H19 (50)NKX2-2 (17) CBX 8 (34) FOXD3 (51) SLC32A1 in a biological sample from said individual wherein a higher level of methylation 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.
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, tumour 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 tumour 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 for regions assayed on Nimblegen promoter tiling arrays correspond to the Hgl 8 version of the genome, while those describing the associated gene symbol correspond to the Hgl 9 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, localised 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, several of these genes provide particularly good sensitivity and specificity, either or both because elevated methylation is observed in a higher proportion of neoplasms or because their greater difference in level of DNA methylation between neoplastic tissue and either normal colon tissue or peripheral blood leukocytes.
According to this 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 a DNA region selected from: (i) the region defined by any one or more of Hg 19 coordinates and 2kb upstream of the transcription start site: (1) chrl2:52400748..52409671 (6) chr3:33191537..33260707 (2) chr5:3596168..3601517 (7) chr 15:60296421..60298142 (3) chrl3:95361876..95364389 (8) chrl3:28494168..28500451 (4) chr4:81187742..81212171 (9) chr7:96649702..96654143 (5) chrl9:57019212..57040270 (ii) the gene region, including 2kb upstream of,any one or more of: (1) GRASP (4) FGF5 (6)SUSD5 (8)PDX1 (2) IRX1 (5) ZNF471 (7)FOXBl (9) DLX5 (3) SOX21 in a biological sample from said individual wherein a higher level of methylation 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, in yet another embodiment said control level is a non-neoplastic level.
According to these aspects of the present invention, 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.
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. To this end, it has also been determined that in the context of a further 15 DNA regions which were previously known to be downregulated in terms of their level of expression in the context of large intestine neoplasms, there in fact occur discrete subregions which exhibit a particularly high density of hypermethylated CpG dinucleotides. 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) Chrl2:52400748-52409671 (GRASP) subregions Chr12:52399672-52399922 (SEQ ID NO: 15), Chr 12:52400821 -52401119 (SEQ ID NO: 16) and Chr 12:52401407-52401664 (SEQ ID NO: 17). (2) Chr8:97505882-97624037 (SDC2) subregions Chr8:97506813-97507045 (SEQ ID NO: 18) and Chr8:97507037-97507257 (SEQ ID NO: 19). (3) Chrl3:l 10959094-110959330 (SEQ ID NO:20) (COL4A1). (4) Chr5:3596168-3601517 (1RX1) subregions Chr5:3597227-3597480 (SEQ ID NO:21), Chr5:3599990-3600175 (SEQ IDNO:22), Chr5:3600160-3600352 (SEQ IDNO:23) and Chr5:3594657-3594847 (SEQ ID NO:24). (5) Chrl3:95361879-95364389 (SOX21) subregions Chrl3:95364013-95364178 (SEQ ID NO:25) and Chrl 3:95364515-95364784 (SEQ ID NO:26). (6) Chr4:81187742-81212171 (FGF5) subregions Chr4:81186918-81187228 (SEQ ID NO:27), Chr4:81187326-81187578 (SEQ ID NO:28) and Chr4:81187571-81187792 (SEQ ID NO:29). (7) Chrl9:57019212-57040269 (ZNF471) subregions Chrl9:57018955-57019135 (SEQ ID NO:30) and Chrl9:57019294-57019573 (SEQ ID NO:31). (8) Chr3:33191537-33260707 (SUSD5) subregion Chr3:33260566-33260818 (SEQ ID NO:32). (9) Chr2:56093097-56151298 (EFEMP1) subregion Chr2:56150356-56150606 (SEQ ID NO:33). (10) Chr6:134210259-134216675 (TCF21) subregions Chr6:134210545-134210749 (SEQ ID NO:34), Chr6:134210712-134210951 (SEQ ID NO:35) and Chr6:134210994-134211274 (SEQ ID NO:36). (11) Chr7:94023873-94060544 (COL1A2) subregions Chr7:94023751-94023975 (SEQ ID NO:37) and Chr7:94024141-94024345 (SEQ ID NO:38). (12) Chr 15:60296421-60298142 (FOXB1) subregions Chrl 5:60296522-60296719 (SEQ ID NO:39) and Chrl 5:60297024-60297305 (SEQ ID NO:40). (13) Chrl6:86544133-86548070 (FOXF1) subregions Chrl6:86544560-86544770 (SEQ ID NO:41), Chrl6:86544265-86544584 (SEQ IDNO:42) and Chrl 6:86544795-86545110 (SEQ IDNO:43). (14) Chrl3:28494168-28500451 (PDX1) subregions Chrl3:28502100-28502311 (SEQ ID NO:44), Chrl 3:28502417-28502603 (SEQ IDNO:45) and Chrl 3:28503006-28503210 (SEQ ID NO:46). (15) Chr7:96649702-96654143 (DLX5) subregions Chr7:96650026-96650127 (SEQ ID NO:47), Chr7:96651454-96651618 (SEQ ID NO:48) and Chr7:96653553-96653732 (SEQ ID NO:49). (16) Chr21:28208606-28217728 (ADAMTS1) subregions Chr21:28217946-28218109 (SEQ ID NO:50), Chr21:28218494-28218777 (SEQ IDNO:51)and Chr21:28218859-28219017 (SEQ ID NO:52). (17) Chr 13:110959631 -111165373 (COL4A2) subregions Chr 13:110960787-110961141 (SEQ IDNO:53); Chrl3:110961331-110961659 (SEQ ID NO:54) and
Chrl 3:110959932-110960181 (SEQ IDNO:82); (18) Chr5:83238126-83680611 (EDIL3) subregions Chr5:83679544-83679807 (SEQ ID NO:55), Chr5:83679784-83679988 (SEQ IDNO:56), Chr5:83679960-83680263 (SEQ ID NO:57), Chr5:83680075-83680383 (SEQ ID NO:58) and Chr5:83680356-83680630 (SEQ ID NO:59). (19) Chrl5:48700503-48937985 (FBN1) subregion Chrl5:48938136-48938384 (SEQ ID NO :60). (20) Chrl:47901689-47906363 (FOXD2) subregions Chrl:47899091-47899337 (SEQ ID NO:61) and Chrl :47909944-47910172 (SEQ IDNO:62). (21) Chr2:66662532-66799891 (MEIS1) subregions Chr2:66662009-66662219 (SEQ ID NO:63) and Chr2:66662177-66662430 (SEQ ID NO:64). (22) Chrl6:55513081-55540586 (MMP2) subregions Chrl6:555l2662-555128S6 (SEQ ID NO:65) and Chrl 6:55513916-55514215 (SEQ IDNO:66). (23) Chr7:24323807-24331484 (NPY) subregions Chr7:24323765-24323936 (SEQ ID NO:67), Chr7:24324150-24324342 (SEQ ID NO:68) and Chr7:24324513-24324717 (SEQ ID NO :69). (24) Chrl9:38741877-38747172 (PPP1R14A) subregions Chrl9:38747251-38747424 (SEQ ID 140:70) and Chrl 9:38746653-38746912 (SEQ ID NO:71).
According to this embodiment there is therefore 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 one or more DNA regions selected from regions defined by Hgl9 coordinates: (1) Chrl 2:52399672-52399922 (30) Chrl 3:28502100-28502311 (2) Chrl 2:52400821-52401119 (31) Chrl3:28502417-28502603 (3) Chr12:52401407-52401664 (32) Chrl3:28503006-28503210 (4) Chr8:97506813-97507045 (33) Chr7:96650026-96650127 (5) Chr8:97507037-97507257 (34) Chr7:96651454-96651618 (6) Chrl 3:110959094-110959330 (35) Chr7:96653553-96653732 (7) Chr5:3597227-3597480 (36) Chr21:28217946-28218109 (8) Chr5:3599990-3600175 (37) Chr21:28218494-28218777 (9) Chr5:3600160-3600352 (38) Chr21:28218859-28219017 (10) Chr5:3594657-3594847 (39) Chrl 3:110960787-110961141 (11) Chrl 3:95364013-95364178 (40) Chrl3:l 10961331-110961659 (12) Chrl 3:95364515-95364784 (41) Chr5:83679544-83679807 (13) Chr4:81186918-81187228 (42) Chr5:83679784-83679988 (14) Chr4:81187326-81187578 (43) Chr5:83679960-83680263 (15) Chr4:81187571-81187792 (44) Chr5:83680075-83680383 (16) Chrl9:57018955-57019135 (45) Chr5:83680356-83680630 (17) Chrl 9:57019294-57019573 (46) Chrl 5:48938136-48938384 (18) Chr3:33260566-33260818 (47) Chrl :47899091-47899337 (19) Chr2:56150356-56150606 (48) Chrl :47909944-47910172 (20) Chr6:134210545-134210749 (49) Chr2:66662009-66662219 (21) Chr6:134210712-134210951 (50) Chr2:66662177-66662430 (22) Chr6:134210994-134211274 (51) Chrl6:55512662-55512856 (23) Chr7:94023751-94023975 (52) Chrl6:55513916-55514215 (24) Chr7:94024141-94024345 (53) Chr7:24323765-24323936 (25) Chrl 5:60296522-60296719 (54) Chr7:24324150-24324342 (26) Chrl 5:60297024-60297305 (55) Chr7:24324513-24324717 (27) Chrl 6:86544560-86544770 (56) Chrl 9:38747251-38747424 (28) Chrl 6:86544265-86544584 (57) Chrl9:38746653-38746912 (29) Chrl6:86544795-86545110 (58) Chrl3:110959932-110960181 in a biological sample from said individual when a higher level of methylation of said DNA region relative to control levels is indicative of a large intestine neoplasia or a predisposition to the onset of a large intestine neoplastic state.
In another embodiment, said neoplastic cell is an adenoma or adenocarcinoma and even more preferably a colorectal adenoma or adenocarcinoma.
In one particular embodiment there is therefore provided a method of screening for the onset or predisposition to the onset of or monitoring or monitoring a large intestine neoplasm in an individual, said method comprising assessing the methylation status of one or more DNA regions selected from regions defined by Hgl9 coordinates: (1) Chrl 2:52399672-52399922 (19) Chr2:56150356-56150606 (2) Chrl2:52400821-52401119 (20) Chr6:134210545-134210749 (3) Chrl 2:52401407-52401664 (21) Chr6:134210712-134210951 (4) Chr8:97506813-97507045 (22) Chr6:134210994-134211274 (5) Chr8:97507037-97507257 (23) Chr7:94023751-94023975 (6) Chrl3:l 10959094-110959330 (24) Chr7:94024141-94024345 (7) Chr5:3597227-3597480 (25) Chrl 5:60296522-60296719 (8) Chr5:3599990-3600175 (26) Chrl 5:60297024-60297305 (9) Chr5:3600160-3600352 (27) Chrl 6:86544560-86544770 (10) Chr5:3594657-3594847 (28) Chrl 6:86544265-86544584 (11) Chrl 3:95364013-95364178 (29) Chrl 6:86544795-86545110 (12) Chrl 3:95364515-95364784 (30) Chrl 3:28502100-28502311 (13) Chr4:81186918-81187228 (31) Chrl 3:28502417-28502603 (14) Chr4:81187326-81187578 (32) Chrl 3:28503006-28503210 (15) Chr4:81187571-81187792 (33) Chr7:96650026-96650127 (16) Chrl9:57018955-57019135 (34) Chr7:96651454-96651618 (17) Chrl 9:57019294-57019573 (35) Chr7:96653 5 53-96653732 (18) Chr3:33260566-33260818 in a biological sample from said individual when a higher level of methylation of said DNA region relative to control levels is indicative of a large intestine neoplasia or a predisposition to the onset of a large intestine neoplastic state.
In yet another embodiment said DNA regions are selected from regions defined by Hg 19 coordinates: (1) Chrl 2:52399672-52399922 (18) Chr3:33260566-33260818 (2) Chrl 2:52400821-52401119 (19) Chr2:56150356-56150606 (4) Chr8:97506813-97507045 (20) Chr6:134210545-134210749 (5) Chr8:97507037-97507257 (21) Chr6:134210712-134210951 (6) Chrl3:110959094-110959330 (24) Chr7:94024141-94024345 (10) Chr5:3594657-3594847 (25) Chrl 5:60296522-60296719 (11) Chrl 3:95364013-95364178 (26) Chrl 5:60297024-60297305 (12) Chrl 3:95364515-95364784 (28) Chrl 6:86544265-86544584 (13) Chr4:81186918-81187228 (30) Chr 13:28502100-28502311 (16) Chrl 9:57018955-57019135 (31) Chrl 3:28502417-28502603 (17) Chrl 9:57019294-57019573 (35) Chr7:96653553-96653732
In another embodiment, said neoplastic cell is an adenoma or adenocarcinoma and even more preferably a colorectal adenoma or adenocarcinoma.
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 remodelling 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 methylstransferase 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 analyse 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 analysed. 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”. 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, listed below 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 Hgl9, 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, here 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 Hgl9 chromosomal coordinates and the gene region name.
According to this embodiment there is therefore 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 of one or more cytosine residues selected from:
Chr12:52400748-52409671 (GRASP)
Chrl 2:52399713 Chr12:52399731 Chrl 2:52399749 Chrl 2:52399783
Chr 12:52399796 Chrl 2:52399808 Chrl 2:52399823 Chrl 2:52399835
Chrl 2:52399891
Chrl2:52400847 Chrl 2:52400850 Chrl 2:52400859 Chrl 2:52400866
Chrl 2:52400869 Chrl 2:52400873 Chrl 2:52400881 Chrl 2:52400886
Chrl 2:52400893 Chrl 2:52400895 Chrl 2:52400899 Chrl2:52400902
Chrl 2:52400907 Chrl2:52400913 Chrl 2:52400919 Chrl2:52400932
Chrl 2:52400938 Chrl2:52400958 Chrl 2:52400962 Chrl 2:52400971
Chrl 2:52400973 Chrl 2:52400976 Chrl 2:52400998 Chrl2:52401008
Chrl2:52401010 Chrl2:52401012 Chrl2:52401016 Chrl2:52401019
Chr 12:52401025 Chrl 2:52401041 Chrl 2:52401044 Chrl2:52401053
Chr 12:52401060 Chrl 2:52401064 Chrl2:52401092 Chrl2:52401118
Chrl2:52401438 Chrl2:52401448 Chr 12:52401460 Chrl2:52401465
Chrl 2:52401474 Chrl2:52401477 Chrl2:52401479 Chrl2:52401483
Chrl2:52401504 Chrl 2:52401514 Chrl2:52401523 Chrl2:52401540
Chrl2:52401553 Chrl 2:52401576 Chrl2:52401588 Chrl2:52401595
Chrl 2:52401599 Chr 12:52401604 Chrl 2:52401606 Chrl 2:52401634
Chrl 2:52401640 Chrl2:52401644 Chrl 2:52401659
Chr8:97505882-97624037 (SDC2)
Chr8:97506843 Chr8:97506845 Chr8:97506861 Chr8:97506876
Chr8:97506902 Chr8:97506909 Chr8:97506911 Chr8:97506913
Chr8:97506924 Chr8:97506932 Chr8:97506934 Chr8:97506940
Chr8:97506977 Chr8:97506993 Chr8:97507005 Chr8:97507008
Chr8:97507014
Chr8:97507038 Chr8:97507061 Chr8:97507064 Chr8:97507078
Chr8:97507082 Chr8:97507084 Chr8:97507089 Chr8:97507119
Chr8:97507128 Chr8:97507136 Chr8:97507144 Chr8:97507153
Chr8:97507162 Chr8:97507214 Chr8:97507216
Chrl3:110959094-110959330 (COL4A1).
Chrl 3:97507038 Chrl3:97507061 Chrl 3:97507064 Chrl 3:97507078
Chrl 3:97507082 Chrl 3:97507084 Chrl 3:97507089 Chrl 3:97507119
Chrl3:97507128 Chrl 3:97507136 Chrl3:97507144 Chrl3:97507153
Chr 13:97507162 Chrl3:97507214 Chr 13:97507216
Chr5:3596168-3601517 (1RX1)
Chr5:3 597229 Chr5:3597299 Chr5:3597311 Chr5:3597321
Chr5:3597330 Chr5:3597350 Chr5:3597352 Chr5:3597373
Chr5:3597413 Chr5:3597427 Chr5:3597431 Chr5:3597447
Chr5:3597450 Chr5:3597454 Chr5:3597456
Chr5:3599998 Chr5:3600019 Chr5:3600037 Chr5:3600050
Chr5:3600058 Chr5:3600061 Chr5:3600070 Chr5:3600074
Chr5:3600076 Chr5:3600079 Chr5:3600089 Chr5:3600091
Chr5:3600104 Chr5:3600116 Chr5:3600121 Chr5:3600l24
Chr5:3600131 Chr5:3600143 Chr5:3600149 Chr5:3600169
Chr5:3600173
Chr5:3600163 Chr5:3600190 Chr5:3600201 Chr5:3600205
Chr5:3600208 Chr5:3600214 Chr5:3600250 Chr5:3600252
Chr5:3600255 Chr5:3600264 Chr5:3600270 Chr5:3600284
Chr5:3600309 Chr5:3600318 Chr5:3600345
Chr5:3594663 Chr5:3594679 Chr5:3594684 Chr5:3594697
Chr5:3594703 Chr5:3594711 Chr5:3594725 Chr5:3594738
Chr5:3 594745 Chr5:3594758 Chr5:3594768 Chr5:3594774
Chr5:3594778 Chr5:3594794 Chr5:3594799 Chr5:3594818
Chr5:3594840
Chr13:95361879-95364389 (SOX21)
Chrl 3:95364016 Chrl3:95364019 Chrl 3:95364042 Chrl 3:95364050
Chrl 3:95364054 Chrl 3:95364061 Chrl 3:95364064 Chrl 3:95364074
Chrl 3:95364080 Chrl 3:95364082 Chrl 3:95364106 Chrl3:95364119
Chr 13:95364123 Chrl3:95364125 Chrl 3:95364128 Chrl3:95364141
Chrl3:95364163 Chrt3:95364171
Chrl 3:95364543 Chr 13:95364545 Chrl 3:95364548 Chrl 3:95364551
Chrl 3:95364560 Chrl 3:95364571 Chrl 3:95364582 Chrl 3:95364584
Chrl 3:95364587 Chrl 3:95364602 Chrl 3:95364623 Chrl 3:95364627
Chrl 3:95364640 Chrl 3:95364649 Chrl 3:95364653 Chrl 3:95364656
Chrl 3:95364665 Chrl 3:95364684 Chrl3:95364696 Chrl3:95364712
Chrl 3:95364729 Chrl3:95364732 Chrl3:95364734 Chrl3:95364748
Chrl 3:95364751
Chr4:81187742-81212171 (FGF5)
Chr4:81186919 Chr4:81186947 Chr4:8l 186964 Chr4:81186968
Chr4:81186973 Chr4:81186975 Chr4:81186983 Chr4:81187001
Chr4:81187011 Chr4:81187041 Chr4:8!187056 Chr4:81187062
Chr4:81187093 Chr4:81187116 Chr4:81187125 Chr4:81187161
Chr4:81187191 Chr4:81187196 Chr4:81187198
Chr4:81187367 Chr4:81187389 Chr4:81187458 Chr4:81187467
Chr4:81187495 Chr4:81187498 Chr4:81187504 Chr4:81187512
Chr4:81187514 Chr4:81187530 Chr4:81187539 Chr4:81187547
Chr4:81187549 Chr4:81187551 Chr4:81187554 Chr4:81187556
Chr4:81187575
Chr4:81187575 Chr4:81187592 Chr4:81187601 Chr4:81187605
Chr4:81187610 Chr4:81187648 Chr4:81187652 Chr4:81187679
Chr4:81187685 Chr4:81187691 Chr4:81187693 Chr4:81187707
Chr4:81187712 Chr4:81187733 Chr4:81187735 Chr4:81187752
Chr4:81187758 Chr4:81187764 Chr4:81187784
Chr19:57019212-57040269 (ZNF471)
Chrl9:57018990 Chrl9:57018994 Chrl9:57019003 Chrl9:570l9010
Chrl9:57019018 Chrl 9:57019020 Chrl9:57019025 Chrl9:57019029
Chrl9:57019044 Chrl9:57019047 Chrl9:57019067 Chrl9:57019073
Chr 19:57019084 Chrl 9:57019101 Chrl9:57019118
Chrl9:57019315 Chrl9:57019321 Chrl 9:57019346 Chrl9:57019351
Chrl9:57019355 Chrl9:57019361 Chrl9:57019364 Chrl 9:57019366
Chrl9:57019371 Chrl9:57019373 Chr19:57019385 Chrl9:57019387
Chrl9:57019405 Chrl 9:57019428 Chrl 9:57019433 Chrl9:57019435
Chrl9:57019437 Chrl9:57019443 Chr 19:57019451 Chrl 9:57019456
Chrl9:57019463 Chrl9:57019465 Chrl9:57019470 Chrl 9:57019483
Chrl9:57019487 Chrl9:57019492 Chrl 9:57019502 Chrl9:570I9505
Chr3:33191537-33260707 (SUSD5)
Chr3:33260601 Chr3:33260621 Chr3:33260631 Chr3:33260640
Chr3:33260651 Chr3:33260665 Chr3:33260676 Chr3:33260694
Chr3:33260698 Chr3:33260711 Chr3:33260715 Chr3:33260732
Chr3:33260742 Chr3:33260748 Chr3:33260755 Chr3:33260760
Chr3:33260769 Chr3:33260776 Chr3:33260778 Chr3:33260780
Chr3:33260788 Chr3:33260806
Chr2:56093097-56151298 (EFEMP1)
Chr2:56150376 Chr2:56150389 Chr2:56150394 Chr2:56150415
Chr2:56150419 Chr2:56150423 Chr2:56150433 Chr2:56150473
Chr2:56150475 Chr2:56150478 Chr2:56150499 Chr2:56150537
Chr2:56150549 Chr2:56150580 Chr2:56150601
Chr6:134210259-134216675 (TCF21)
Chr6:134210556 Chr6:134210598 Chr6:134210615 Chr6:134210640
Chr6:134210649 Chr6:134210667 Chr6:134210692 Chr6:134210694
Chr6:134210697 Chr6:134210720 Chr6:134210745
Chr6:134210720 Chr6:134210745 Chr6:134210776 Chr6:134210781
Chr6:134210784 Chr6:134210790 Chr6:134210792 Chr6:134210794
Chr6:134210800 Chr6:134210806 Chr6:134210812 Chr6:134210868
Chr6:134210894 Chr6:134210906 Chr6:134210919 Chr6:134210946
Chr6:134211050 Chr6:134211061 Chr6:134211076 Chr6:l 34211081
Chr6:134211103 Chr6:134211110 Chr6:134211121 Chr6:134211125
Chr6:134211131 Chr6:134211153 Chr6:134211155 Chr6:134211162
Chr6:134211179 Chr6:134211182 Chr6:134211184 Chr6:134211208
Chr6:134211210 Chr6:134211212 Chr6:134211218 Chr6:134211220
Chr6:134211227 Chr6:134211233 Chr6:134211241 Chr6:134211245
Chr6:134211247 Chr6:134211270 Chr7:94023873-94060544 (COL1A2)
Chr7:94024172 Chr7:94024191 Chr7:94024214 Chr7:94024230
Chr7:94024254 Chr7:94024266 Chr7:94024268 Chr7:94024272
Chr7:94024288 Chr7:94024291 Chr7:94024310
Chr15:60296421-60298142 (FOXB1)
Chrl 5:60296555 Chrl 5:60296561 Chrl 5:60296563 Chrl 5:60296578
Chrl 5:60296585 Chrl 5:60296598 Chrl 5:60296601 Chrl 5:60296614
Chrl5:60296616 Chrl 5:60296619 Chrl 5:60296627 Chr 15:60296633
Chrl 5:60296639 Chrl 5:60296643 Chrl 5:60296647 Chrl 5:60296654
Chrl 5:60296665 Chrl 5:60296668 Chrl 5:60296670 Chrl 5:60296675
Chrl 5:60296679 Chrl 5:60296684 Chrl 5:60296689 Chrl 5:60296694
Chrl 5:60297035 Chrl 5:60297050 Chrl 5:60297053 Chrl 5:60297109
Chrl5:60297118 Chrl 5:60297121 Chrl5:60297126 Chrl 5:60297128
Chrl 5:60297130 Chrl 5:60297152 Chrl5:60297169 Chrl 5:60297174
Chrl 5:60297178 Chr15:60297185 Chrl 5:60297192 Chrl 5:60297203
Chrl 5:60297212 Chrl 5:60297221 Chrl 5:60297228 Chrl 5:60297252
Chrl5:60297266 Chrl 5:60297273 Chrl 5:60297298
Chr16:86544133-86548070 (FOXF1)
Chrl6:86544571 Chrl 6:86544587 Chrl 6:86544590 Chrl 6:86544593
Chrl 6:86544597 Chrl 6:86544599 Chrl 6:86544601 Chrl 6:86544608
Chrl6:86544624 Chrl 6:86544652 Chrl 6:86544658 Chrl6:86544675
Chrl6:86544685 Chrl 6:86544699 Chrl 6:86544703 Chrl6:86544706
Chrl6:86544714 Chrl 6:86544720 Chrl6:86544735 Chrl 6:86544745
Chrl 6:86544763
Chrl6:86544268 Chrl 6:86544273 Chr 16:86544295 Chrl 6:86544298
Chrl 6:86544305 Chrl 6:86544308 Chr 16:86544312 Chrl 6:86544321
Chrl 6:86544337 Chrl 6:86544339 Chrl 6:86544346 Chrl 6:86544377
Chrl 6:86544384 Chrl 6:86544391 Chrl 6:86544416 Chrl 6:86544431
Chrl 6:86544460 Chrl 6:86544464 Chrl 6:86544477 Chrl 6:86544484
Chrl 6:86544518 Chrl 6:86544523 Chrl 6:86544547 Chrl 6:86544552
Chrl 6:86544559 Chrl 6:86544571
Chrl 6:86544810 Chrl 6:86544832 Chrl 6:86544835 Chrl 6:86544843
Chrl 6:86544853 Chrl 6:86544859 Chrl 6:86544862 Chrl 6:86544865
Chrl 6:86544867 Chrl 6:86544870 Chrl 6:86544874 Chrl6:86544877
Chrl 6:86544885 Chrl 6:86544892 Chrl6:86544900 Chrl6:86544907
Chrl 6:86544915 Chrl 6:86544928 Chrl 6:86544931 Chrl 6:86544934
Chrl 6:86544951 Chrl 6:86544955 Chrl 6:86544958 Chrl 6:86544966
Chrl 6:86544972 Chrl 6:86544975 Chrl 6:86544978 Chrl 6:86544987
Chrl 6:86544993 Chrl 6:86544996 Chrl 6:86545000 Chrl 6:86545002
Chrl 6:86545005 Chrl 6:86545015 Chrl 6:86545018 Chrl 6:86545060
Chrl 6:86545062 Chrl 6:86545078 Chrl 6:86545092
Chrl 3:28494168-28500451 (PDX1)
Chrl3:28502109 Chrl 3:28502153 Chrl3:28502161 Chrl 3:28502177
Chrl 3:28502179 Chrl 3:28502191 Chrl3:28502198 Chrl3:28502205
Chrl 3:28502207 Chrl3:28502210 Chrl 3:28502245 Chrl 3:28502309
Chrl 3:28502442 Chrl 3:28502449 Chr!3:2850246l Chrl 3:28502464
Chrl 3:28502475 Chrl 3:28502507 Chrl3:28502512 Chrl 3:28502538
Chrl 3:28502544 Chrl 3:28502549 Chrl 3:28502559 Chrl 3:28502564
Chrl 3:28502585
Chrl 3:28503045 Chrl 3:28503049 Chrl3:28503081 Chrl 3:28503099
Chrl 3:28503114 Chrl 3:28503127 Chrl 3:28503138 Chrl 3:28503147
Chrl 3:28503155 Chrl 3:28503157 Chrl 3:28503179 CAr7:96649702-96654143 (DLX5)
Chr7:96650062 Chr7:96650072 Chr7:96650078 Chr7:96650096
Chr7:96650099 Chr7:96650102
Chr7:96651485 Chr7:96651488 Chr7:96651518 Chr7:96651523
Chr7:96651532 Chr7:96651535 Chr7:96651537 Chr7:96651542
Chr7:96651550 Chr7:96651586
Chr7:96653596 Chr7:96653605 Chr7:96653607 Chr7:96653617
Chr7:96653620 Chr7:96653623 Chr7:96653644 Chr7:96653656
Chr7:96653683 Chr7:96653686 Chr7:96653692 Chr7:96653698
Chr7:96653701
Chr21:28208606-28217728 (ADAMTS1)
Chr21:28217973 Chr21:28218002 Chr21:28218015 Chr2l :28218018
Chr21:28218047 Chr21:28218051 Chr21:28218057 Chr21:28218072
Chr21:28218074 Chr21:28218084 Chr21:28218105
Chr21:28218514 Chr21:28218516 Chr21:28218550 Chr21:28218568
Chr21:28218579 Chr21:28218586 Chr21:28218596 Chr21:28218635
Chr21:28218638 Chr21:28218646 Chr21:28218671 Chr21:28218684
Chr21:28218688 Chr21:28218704 Chr21:28218729 Chr21:28218741
Chr21:28218893 Chr21:28218906 Chr21:28218914 Chr21:28218916
Chr21:28218928 Chr21:28218934 Chr21:28218938 Chr21:28218949
Chr21:28218953 Chr21:28218959 Chr21:28218974 Chr21:28218976
Chr21:28218978 Chr21:28218984 Chr21:28218986 Chr21:28218996
Chr21:28219008 Chr21:28219016
Chr13:110959631-111165373 (COL4A2)
Chrl3:l 10960813 Chrl 3:110960827 Chrl 3:110960849 Chrl 3:110960875
Chrl3:l 10960925 Chrl 3:110960930 Chrl 3:110960938 Chrl 3:110960978
Chrl3:l 10961001 Chrl3:l 10961003 Chrl3:l 10961025 Chrl3:l 10961045
Chrl3:l 10961049 Chrl 3:110961069 Chrl3:110961072 Chrl 3:110961088
Chrl3:l 10961091 Chrl3:l 10961095 Chrl3:l 10961110 Chrl3:l 10961116
Chrl3:l 10961131 Chr 13:110960786
Chrl3:l 10961358 Chrl3:l 10961386 Chrl3:l 10961404 Chrl3:l 10961406
Chrl3:l 10961445 Chrl 3:110961579 Chrl3:110961594 Chrl 3:110961606
Chrl3:l 10961631
Chrl3:l 10959935 Chrl 3:110959965 Chrl 3:110959968 Chrl 3:110959974
Chrl 3:110959979 Chrl 3:110959981 Chrl3:l 10959986 Chrl 3:110959988
Chrl 3:110959997 Chrl 3:110959999 Chrl 3:110960004 Chrl 3:110960006
Chrl3:l 10960011 Chrl 3:110960027 Chrl3:l 10960035 Chrl 3:110960046
Chrl 3:110960051 Chrl 3:110960071 Chrl 3:110960099 Chrl 3:110960108
Chrl3:l 10960115 Chrl3:l 10960156 Chrl3:110960180
Chr5:83238126-83680611 (ED1L3)
Chr5:83679825 Chr5:83679827 Chr5:83679836 Chr5:83679843
Chr5:83679846 Chr5:83679860 Chr5:83679864 Chr5:83679883
Chr5:83679894 Chr5:83679900 Chr5:83679906 Chr5:83679910
Chr5:83679937 Chr5:83679953 Chr5:83679957 Chr5:83679961
Chr5:83679976 Chr5:83679982 Chr5:83679987 Chr5:83680003
Chr5:83680007 Chr5:83680011 Chr5:83680026 Chr5:83680032
Chr5:83680086 Chr5:83680090 Chr5:83680103 ChrS:83680106
Chr5:83680122 Chr5:83680126
Chr5:83679546 Chr5:83679568 Chr5:83679571 Chr5:83679585
Chr5:83679602 Chr5:83679617 Chr5:83679630 Chr5:83679640
Chr5:83679667 Chr5:83679675 Chr5:83679689 Chr5:83679693
Chr5:83679699 Chr5:83679701 Chr5:83679733
Chr5:83679973 Chr5:83679989 Chr5:83679992 Chr5:83680001
Chr5:83680003 Chr5:83680006 Chr5:83680012 Chr5:83680015
Chr5:83680019 Chr5:83680033 Chr5:83680036 Chr5:83680045
Chr5:83680051 Chr5:83680061 Chr5:83680063 Chr5:83680066
Chr5:83680108 Chr5:83680115 Chr5:83680118 Chr5:83680132
Chr5:83680147 Chr5:83680159 Chr5:83680174 Chr5:83680181
Chr5:83680185 Chr5:83680198 Chr5:83680205 Chr5:83680209
Chr5:83680217 Chr5:83680232 Chr5:83680235 Chr5:83680237
Chr5:83680253
ChrS :83680108 Chr5:83680115 Chr5:83680118 Chr5:83680132
Chr5:83680147 Chr5:83680159 Chr5:83680174 Chr5:83680181
Chr5:83680185 Chr5:83680198 Chr5:83680205 Chr5:83680209
Chr5:83680217 Chr5:83680232 Chr5:83680235 Chr5:83680237
Chr5:83680253 Chr5:83680264 Chr5:83680284 Chr5:83680310
Chr5:83680326 Chr5:83680355
Chr5:83680401 Chr5:83680406 Chr5:83680409 Chr5:83680411
Chr5:83680413 Chr5:83680430 Chr5:83680438 Chr5:83680441
Chr5:83680444 Chr5:83680458 Chr5:83680463 Chr5:83680474
Chr5:83680483 Chr5:83680485 Chr5:83680488 Chr5:83680492
Chr5:83680497 Chr5:83680499 Chr5:83680507 Chr5:83680509
Chr5:83680513 Chr5:83680518 Chr5:83680529 Chr5:83680531
Chr5:83680548 Chr5:83680559 Chr5:83680564 Chr5:83680568
Chr5:83680572 Chr5:83680579 Chr5:83680589 Chr5:83680591
Chr5:83680593 Chr5:83680596 Chr5:83680602
ChrlS:48700503-48937985 (FBN1)
Chrl 5:48938149 Chrl5:48938163 ChrlS :48938172 Chrl5:48938181
Chrl 5:48938183 Chrl5:48938218 Chrl 5:48938236 Chrl5:48938238
Chrl 5:48938254 Chrl 5:48938267 Chrl 5:48938273 Chrl5:48938280
Chrl 5:48938291 Chrl 5:48938349
Chrl:47901689-47906363 (FOXD2)
Chrl :47899102 Chrl :47899126 Chrl :47899129 Chrl :47899143
Chrl :47899152 Chrl :47899168 Chrl :47899184 Chrl :47899205
Chrl :47899212 Chrl:47899230 Chrl :47899232 Chrl:47899253
Chrl :47899286 Chrl :47899327
Chrl :47909988 Chrl :47909995 Chrl :47910005 Chrl :47910036
Chrl :47910051 Chrl:47910056 Chrl:47910062 Chrl:47910074
Chrl :47910103 Chrl :47910122 Chrl :47910137 Chrl :47910142
Chrl :47910144 Chrl :47910146
Chr2:66662532-66799891 (MEIS1)
Chr2:66662043 Chr2:66662047 Chr2:66662049 Chr2:66662063
Chr2:66662073 Chr2:66662107 Chr2:66662111 Chr2:66662124
Chr2:66662145 Chr2:66662156 Chr2:66662161 Chr2:66662163
Chr2:66662178 Chr2:66662218
Chr2:66662178 Chr2:66662218 Chr2:66662220 Chr2:66662246
Chr2:66662265 Chr2:66662280 Chr2:66662306 Chr2:66662310
Chr2:66662323 Chr2:66662333 Chr2:66662337
Chr16:55513081-55540586 (MMP2)
Chrl6:55512695 Chrl 6:55512711 Chrl6:55512732 Chrl 6:55512750
Chrl 6:55512753 Chrl6:55512793
Chrl 6:55513961 Chrl6:55513997 Chrl 6:55514025 Chrl 6:55514059
Chrl6:55514061 Chrl6:55514092 Chrl6:55514120 Chrl 6:55514131
Chrl6:55514158 Chrl 6:55514205
Chr7:24323807-24331484 (NPY)
Chr7:24323767 Chr7:24323792 Chr7:24323794 Chr7:24323799
Chr7:24323817 Chr7:24323834 Chr7:24323840 Chr7:24323844
Chr7:24323848 Chr7:24323866 Chr7:24323876 Chr7:24323880
Chr7:24323882 Chr7:24323884 Chr7:24323894 Chr7:24323905
Chr7:24323910 Chr7:24323930 Chr7:24323934
Chr7:24324180 Chr7:24324186 Chr7:24324206 Chr7:24324217
Chr7:24324222 Chr7:24324225 Chr7:24324244 Chr7:24324249
Chr7:24324251 Chr7:24324268 Chr7:24324274 Chr7:24324280
Chr7:24324311 Chr7:24324313
Chr7:24324549 Chr7:24324570 Chr7:24324592 Chr7:24324595
Chr7:24324597 Chr7:24324599 Chr7:24324621 Chr7:24324625
Chr7:24324633 Chr7:24324635 Chr7:24324668 Chr7:24324672
Chr7:24324676 Chr7:24324679 Chr7:24324683 Chr7:24324690
Chr19:38741877-38747172 (PPPIR14A)
Chrl9:38747280 Chrl9:38747296 Chrl9:38747300 Chrl 9:38747313
Chrl9:38747319 Chrl 9:38747322 Chrl 9:38747328 Chrl 9:38747334
Chrl9:38747355 Chrl9:38747361 Chrl 9:38747371 Chrl 9:38747382
Chrl9:38747389 Chrl9:38747410
Chrl9:38746654 Chrl9:38746680 Chrl 9:38746690 Chrl 9:38746701
Chrl9:38746715 Chrl9:38746726 Chrl9:38746728 Chrl 9:38746732
Chrl9:38746749 Chrl9:38746759 Chrl 9:38746770 Chrl9:38746784
Chrl9:38746786 Chrl 9:38746804 Chrl 9:38746808 Chrl 9:38746811
Chrl9:38746826 Chrl9:38746834 Chrl 9:38746837 Chrl 9:38746849
Chrl9:38746852 Chrl 9:38746865 Chrl9:38746871 Chrl 9:38746873
Chrl9:38746875 Chrl9:38746877 Chrl9:38746882 Chrl 9:38746898 or a corresponding cytosine at position n+1 on the opposite DNA strand, in a biological sample from said individual wherein a higher level of methylation of one or more of said residues relative to the methylation level of a corresponding residue in a control sample is indicative of a large intestine neoplasm or a predisposition to the onset of a neoplastic state.
In another embodiment said one or more cytosine residues are selected from GRASP. SDC2, COL4A1, IRX1, SOX21, FGF5, ZNF471, SUSD5, EFEMP1, TCF21, COL1A2, FOXB1, FOXF1, PDX1 and DLX5.
In one particular embodiment said one or more cytosine residues are selected from:
Chr12:52400748-52409671 (GRASP)
Chrl2:52399713 Chrl 2:52399731 Chrl 2:52399749 Chrl 2:52399783
Chrl2:52399796 Chrl 2:52399808 Chrl2:52399823 Chrl2:52399835
Chrl2:52399891
Chr 12:52400847 Chrl2:52400850 Chrl2:52400859 Chrl 2:52400866
Chr 12:52400869 Chrl2:52400873 Chrl2:52400881 Chrl 2:52400886
Chrl 2:52400893 Chrl2:52400895 Chrl2:52400899 Chrl2:52400902
Chrl 2:52400907 Chrl2:52400913 Chrl 2:52400919 Chrl2:52400932
Chrl 2:52400938 Chrl2:52400958 Chrl 2:52400962 Chrl 2:52400971
Chr12:52400973 Chrl2:52400976 Chrl2:52400998 Chrl 2:52401008
Chrl2:52401010 Chrl 2:52401012 Chrl2:52401016 Chrl2:52401019
Chrl2:52401025 Chrl2:52401041 Chrl2:52401044 Chrl 2:52401053
Chrl 2:52401060 Chrl2:52401064 Chrl 2:52401092 Chrl2:52401118
Chr8:97505882-97624037 (SDC2)
Chr8:97506843 Chr8:97506845 Chr8:97506861 Chr8:97506876
Chr8:97506902 Chr8:97506909 Chr8:97506911 Chr8:97506913
Chr8:97506924 Chr8:97506932 Chr8:97506934 Chr8:97506940
Chr8:97506977 Chr8:97506993 Chr8:97507005 Chr8:97507008
Chr8:97507014
Chr8:97507038 Chr8:97507061 Chr8:97507064 Chr8:97507078
Chr8:97507082 Chr8:97507084 Chr8:97507089 Chr8:97507119
Chr8:97507128 Chr8:97507136 Chr8:97507144 Chr8:97507l53
Chr8:97507162 Chr8:97507214 Chr8:97507216
Chrl3:l 10959094-110959330 (COL4A1).
Chrl 3:97507038 Chrl 3:97507061 Chrl 3:97507064 Chrl 3:97507078
Chrl 3:97507082 Chrl 3:97507084 Chr 13:97507089 Chrl3:97507119
Chrl 3:97507128 Chrl 3:97507136 Chrl3:97507144 Chrl 3:97507153
Chrl 3:97507162 Chrl 3:97507214 Chr13:97507216
Chr5:3594663 Chr5:3594679 Chr5:3594684 Chr5:3594697
Chr5:3594703 Chr5:3594711 Chr5:3594725 Chr5:3594738
Chr5:3594745 Chr5:3594758 Chr5:3594768 Chr5:3594774
Chr5:3594778 Chr5:3594794 Chr5:3594799 Chr5:35948l8
Chr5:3594840
Chrl3:95361879-95364389 (SOX21)
Chrl 3:95364016 Chrl3:95364019 Chrl 3:95364042 Chrl 3:95364050
Chrl 3:95364054 Chrl 3:95364061 Chrl 3:95364064 Chrl 3:95364074
Chrl 3:95364080 Chrl 3:95364082 Chr 13:95364106 Chrl3:95364119
Chrl 3:95364123 Chrl 3:95364125 Chrl3:95364128 Chrl3:95364l41
Chrl3:95364163 Chrl3:95364171
Chrl 3:95364543 Chrl 3:95364545 Chrl3:95364548 Chrl 3:95364551
Chrl 3:95364560 Chrl3:95364571 Chrl 3:95364582 Chrl 3:95364584
Chrl3:95364587 Chrl3:95364602 Chr 13:95364623 Chrl 3:95364627
Chrl3:95364640 Chrl 3:95364649 Chrl 3:95364653 Chrl 3:95364656
Chrl3:95364665 Chrl3:95364684 Chrl 3:95364696 Chrl3:95364712
Chrl3:95364729 Chrl 3:95364732 Chrl 3:95364734 Chrl 3:95364748
Chrl 3:95364751
Chr4:81187742-81212171 (FGF5)
Chr4:81186919 Chr4:81186947 Chr4:81186964 Chr4:81186968
Chr4:81186973 Chr4:81186975 Chr4:81186983 Chr4:81187001
Chr4:81187011 Chr4:81187041 Chr4:81187056 Chr4:81187062
Chr4:81187093 Chr4:81187116 Chr4:81187125 Chr4:81187161
Chr4:81187191 Chr4:81187196 Chr4:81187198
Chrl9:57019212-57040269 (ZNF471)
Chrl 9:57018990 Chrl9:57018994 Chrl 9:57019003 Chrl 9:57019010
Chrl9:57019018 Chrl9:57019020 Chrl 9:57019025 Chrl9:57019029
Chrl 9:57019044 Chrl9:57019047 Chrl 9:57019067 Chrl 9:57019073
Chrl9:57019084 Chrl 9:57019101 Chrl9:57019118
Chrl9:57019315 Chrl9:57019321 Chrl 9:57019346 Chrl9:57019351
Chrl 9:57019355 Chrl 9:57019361 Chrl 9:57019364 Chrl9:57019366
Chrl 9:57019371 Chrl 9:57019373 Chrl9:570l9385 Chrl9:57019387
Chrl 9:57019405 Chrl9:57019428 Chrl 9:57019433 Chrl 9:57019435
Chrl9:57019437 Chrl 9:57019443 Chrl 9:57019451 Chrl 9:57019456
Chrl 9:57019463 Chrl 9:57019465 Chrl9:57019470 Chrl 9:57019483
Chrl9:57019487 Chrl 9:57019492 Chrl 9:57019502 Chrl 9:57019505
Chr3:33191537-33260707 (,SUSD5)
Chr3:33260601 Chr3:33260621 Chr3:33260631 Chr3:33260640
Chr3:33260651 Chr3:33260665 Chr3:33260676 Chr3:33260694
Chr3:33260698 Chr3:33260711 Chr3:33260715 Chr3:33260732
Chr3:33260742 Chr3:33260748 Chr3:33260755 Chr3:33260760
Chr3:33260769 Chr3:33260776 Chr3:33260778 Chr3:33260780
Chr3:33260788 Chr3:33260806
Chr2:56093097-56151298 (EFEMP1)
Chr2:56150376 Chr2:56150389 Chr2:56150394 Chr2:56150415
Chr2:56150419 Chr2:56150423 Chr2:56150433 Chr2:56150473
Chr2:56150475 Chr2:56150478 Chr2:56150499 Chr2:56150537
Chr2:56150549 Chr2:56150580 Chr2:56150601
Chr6:134210259-134216675 (TCF21)
Chr6:134210556 Chr6:134210598 Chr6:134210615 Chr6:134210640
Chr6:134210649 Chr6:134210667 Chr6:134210692 Chr6:134210694
Chr6:134210697 Chr6:134210720 Chr6:134210745
Chr6:134210720 Chr6:134210745 Chr6:134210776 Chr6:134210781
Chr6:134210784 Chr6:134210790 Chr6:134210792 Chr6:134210794
Chr6:134210800 Chr6:134210806 Chr6:l34210812 Chr6:1342l0868
Chr6:134210894 Chr6:134210906 Chr6:134210919 Chr6:134210946
Chr7:94023873-94060544 (COL1A2)
Chr7:94024172 Chr7:94024191 Chr7:94024214 Chr7:94024230
Chr7:94024254 Chr7:94024266 Chr7:94024268 Chr7:94024272
Chr7:94024288 Chr7:94024291 Chr7:94024310
Chrl5:60296421-60298142 (FOXB1)
Chrl 5:60296555 Chrl 5:60296561 Chrl 5:60296563 Chrl 5:60296578
Chrl5:60296585 Chrl 5:60296598 Chrl 5:60296601 Chrl5:60296614
Chrl 5:60296616 Chrl 5:60296619 Chrl 5:60296627 Chrl 5:60296633
Chrl 5:60296639 Chrl 5:60296643 Chrl 5:60296647 Chrl 5:60296654
Chrl 5:60296665 Chrl 5:60296668 Chrl 5:60296670 Chrl5:60296675
Chrl 5:60296679 Chrl 5:60296684 Chrl 5:60296689 Chrl 5:60296694
Chr15:60297035 Chrl 5:60297050 Chrl 5:60297053 Chrl5:60297109
Chrl5:60297118 Chrl 5:60297121 Chrl5:60297126 Chrl 5:60297128
Chrl 5:60297130 Chrl 5:60297152 Chrl 5:60297169 Chrl 5:60297174
Chrl 5:60297178 Chrl 5:60297185 Chrl 5:60297192 Chrl 5:60297203
Chrl 5:60297212 Chrl 5:60297221 Chrl5:60297228 Chrl 5:60297252
Chrl 5:60297266 Chrl 5:60297273 Chrl 5:60297298
Chr16:86544133-86548070 (FOXF1)
Chrl 6:86544268 Chrl 6:86544273 Chrl 6:86544295 Chrl6:86544298
Chrl 6:86544305 Chrl 6:86544308 Chrl 6:86544312 Chrl 6:86544321
Chrl 6:86544337 Chrl 6:86544339 Chrl 6:86544346 Chrl6:86544377
Chrl 6:86544384 Chr16:86544391 Chrl 6:86544416 Chrl6:86544431
Chrl6:86544460 Chrl 6:86544464 Chrl 6:86544477 Chrl6:86544484
Chrl 6:86544518 Chrl 6:86544523 Chrl 6:86544547 Chrl 6:86544552
Chrl 6:86544559 Chrl 6:86544571
Chr13:28494168-28500451 (PDX1)
Chrl3:28502109 Chrl 3:28502153 Chrl 3:28502161 Chrl3:28502177
Chrl3:28502179 Chrl 3:28502191 Chrl 3:28502198 Chr 13:28502205
Chrl3:28502207 Chrl3:28502210 Chrl 3:28502245 Chrl 3:28502309
Chrl 3:28502442 Chrl 3:28502449 Chrl 3:28502461 Chrl 3:28502464
Chrl 3:28502475 Chrl 3:28502507 Chrl3:28502512 Chrl3:28502538
Chrl 3:28502544 Chrl 3:28502549 Chrl 3:28502559 Chrl 3:28502564
Chrl3:28502585
Chr7:96649702-96654143 (DLX5)
Chr7:96653596 Chr7:96653605 Chr7:96653607 Chr7:96653617
Chr7:96653620 Chr7:96653623 Chr7:96653644 Chr7:96653656
Chr7:96653683 Chr7:96653686 Chr7:96653692 Chr7:96653698
Chr7:96653701
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 (eg. blood), faeces, 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 homogenisation 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 permeabilisation 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 mobilise 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 faecal (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'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'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 characterised 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 utilised 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 tumour resection in order to determine whether the full margin of the tumour 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 ongoing monitor of those individuals thought to be at risk of neoplasia development or as a monitor of the effectiveness of therapeutic or prophylactic treatment regimes 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 etal. 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 tumours 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 die 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 qnd 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 etal. 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 el ai, 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-labelled 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-labelled 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, Μ. 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. Common. 1247,1998. While, probes comprising peptide-nucleic acid (PNA) are synthesized as described, for example, in Egholm etal.,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, Hhal or Hpalf. 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 Hpall.
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 etal.,Mol. Cell. Biol. 14:7421-7427, 1994; Gonzalgo etal., 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 x 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 ai, 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 ai, 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 labelled 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, IN, 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 labelled 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. 32P).
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., W098/49557; WO 96/17958; Yodot et al., Science 767-773, 1991; U.S. Pat. No. 5,143,854; and U.S. Pat. No. 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 DNasel 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 1.
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 1 digestion in a healthy/normal control or healthy/normal test sample are used to amplify the DNase l-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 ai, Proc. Natl. Acad. Sci. USA 89, 1827-1831,1992). 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.,
Proc. Natl. Acad. Sci. USA 89: 1827-1831, 1992 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, Nucl. Acids Res., 25:2529-2531 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 labelled with a detectable marker (e.g. a fluorophore). Following primer extension, unbound labelled nucleotide diphosphates are removed, e.g. using size exclusion chromatography or electrophoresis, or hydrolyzed, using for example, alkaline phosphatase, and the incorporation of the labelled 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 etal., 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, Nucl. Acids Res., 25:2532-2534, 2001. 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 Taql 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.
Hvbridization-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., Mol. Cell. Biol. 14:7421 -7427, 1994; Gonzalgo et al., Cancer Res. 57:594-599,1997). 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 labelled 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 labelled, 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 el 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 etal., Science, 286:531-537, 1999). Preferably, for each CpG dinucleotide analyzed two different oligonucleotides are used. Each oligonucleotide comprises a sequence N2-16CGN2-16 or N2-16TGN2-16 (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 labelled 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 el al., Nucl. Acids Res. 32: elO, 2003 (HeavyMethyl PCR), Rand et al. Nucl. Acids Res. 33:el27, 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., Nucl. Acids Res. 18,687, 1990), 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 labelled with a S' 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 labelled 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 unmutatcd sites (i.e. methylated nucleotides) in a nucleic acid.
Fluorescently labelled locked nucleic acid (LNA) molecules or fluorescently labelled 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, rhodomine 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 labelled 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., Nucl. Acids Res. 32: elO, 2003) is used to determine the presence or level of methylation of nucleic acid in a test sample. Essentially, this method uses one or more nonextendible 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 labelled 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 heatthy/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.
Hvbridization-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., Mol. Cell. Biol. 14:7421-7427, 1994; Gonzalgo et al., Cancer Res. 57:594-599, 1997). 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-complementaiy 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. IThese 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 defined by any one or more of Hg 19 coordinates and 2kb upstream of the transcription start site: (1) chrl2:52400748..52409671 (34) chrl0:101292690..101296281 (2) chr5:3596168.,3601517 (35) chr4:4190530..4228621 (3) chrl3:95361876..95364389 (36) chrl 2:54943404..54973023 (4) chr4:81187742..81212171 (37) chr5:176047210.. 176057557 (5) chrl9:57019212..57040270 (38) chrl2:22346325..22487648 (6) chr3:33191537..33260707 (39) chrl 9:56894648..56904889 (7) chrl 5:60296421 ..60298142 (40) chr20:21491648..21494664 (8) chrl3:28494168..28500451 (41) chrl :50883225..50889141 (9) chr7:96649702..96654143, (42) chr7:27180996..27183287 (10) chr8:140,811,770-141,537,860 (43) chrl 1:2016406..2019065 (11) chr5:2746279..2751769 (44) chrl4:57267425..57277184 (12) chrl 8:55102917..55158530 (45) chr4:126237567.. 126414087 (13) chr20:37353101..37358016 (46) chr8:23559964..23563922 (14) chr8:2792875..4852328 (47) chrl 0:131633547.. 131762091 (15) chrl6:66613351 ..66622178 (48) chr4:62362839..62938168 (16) chr5:37815753..37839782 (49) chrl :47901689..47906363 (17) chrl :63788730..63790797 (50) chrl 7:77768176..77770890 (18) chrl5:37156644..37178734 (51) chrl7:93598762..93604831 (19) chr7:27139973..27142394 (52) chrl :33789224..33841194 (20) chr20:21686297..21696620 (53) chr9:124,004,679-124,030,840 (21) chrl 6:51169886..51185183 (54) chr4:l 58141736.. 158287227 (22) chrl2:85253267..85306606 (55) chrl2:9445136..9462559 (23) chr8:6357172..6420784 (56) chrl2:24964278..25102308 (24) chrl 4:85996488..86094270 (57) chrX:21542357..21690352 (25) chr2:182541194..182545381 (58) chr20:52769988..52790516 (26) chr7:30951468..30965131 (59) chr3:172162951.. 172166203 (27) chr8:131792547..132052835 (60) chrl 3:28366780..28368089 (28) chr3:128749292..128759583 (61) chr7:50344378...50472799 (29) chr10:101088856.. 101154087 (62) chr7:149412148.. 149431664 (30) chr7:27282164..27286192 (63) chr7:24323809..24331477 (31) chrl 0:129535538.. 129539450 (64) chr4:30722037. .31148421 (32) chrl 9:49316274..49339934 (65) chrl 0:47083534..47088320 (33) chr6:391752..411443 (ii) the gene region, including 2kb upstream of any one or more of: (1) GRASP (18) ANGPT2 (35)NKX2-6 (52) HOXA5
(2) IRX1 (19) LHX6 (36) PAX 1 (53) GDNF (3) SOX21 (20) NEUROD1 (37)FOXD2 (54) FAT4 (4) FGF5 (21) AC 149644.1 (38)SLC6A15 (55)HOXA2 (5) ZNF471 (22) CCDC48 (39) PHC2 (56) LPHN3 (6) SUSD5 (23) EVX1 (40)FLRT2 (57)ADCYAP1 (7) FOXB1 (24) GHSR (41)GATA2 (58) GRIA2 (8) PDX1 (25) HSD17B14 (42) ADCY8 (59)AQP1 (9) DLX5 (26) KRBA1 (43)CNNM1 (60) BCAT1 (10) ONECUT2 (27) OTOP1 (44)IKZF1 (61)CYP24A1 (11) DMRTA2 (28) PPYR1 (45)NKX2-3 (62) FOX12 (12) CMTM2 (29) SRMS (46) PCDH7 (63)GSX1 (13) OTX2 (30) ZNF582 (47) SNCB (64) 1RF4
(14) LOC145845 (31)IRX2 (48)ST8S1A1 (65)NPY
(15) EBF3 (32) CSMD1 (49)TRAPPC9 (66)PDE1B (16) SALL 1 (33) MIR675,H 19 (50) NK.X2-2 (17) CBX8 (34) FOXD3 (51) SLC32A1 in a biological sample from said individual wherein a lower level of expression 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.
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 analyse 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 therefore 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 region defined by any one or more of Hg 19 coordinates and 2kb upstream of the transcription start site: (1) chrl2:52400748..52409671 (6) chr3:33191537..33260707 (2) chr5:3596168. .3601517 (7) chr 15:60296421 ..60298142 (3) chrl 3:95361876..95364389 (8) chrl 3:28494168..28500451 (4) chr4:81187742..81212171 (9) chr7:96649702..96654143, (5) chrl 9:57019212..57040270 (ii) the gene region, including 2kb upstream of any one or more of: (1) GRASP (4) FGF5 (6) SUSD5 (8)PDX1 (2) IRX1 (5) ZNF471 (7)FOXBI (9)DLX5 (3) SOX21 in a biological sample from said individual wherein a lower level of expression of the gene or transcripts 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. , 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 characterised 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 utilised to indicate reversal or other form of improvement of the subject individual’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 regimes 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 tumour biopsy or a cellular population which has been enriched from a heterogeneous starting population) or a tissue section, one may utilise a wide range of techniques such as in situ hybridisation, 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: (i) 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 at., BBA, 1402:239-249, 1988; Weisslederef 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. (ii) 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). (iii) 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 DMA 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. Mo. 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' 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. (iv) 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 singlesite 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 labelled antibody to a target. (v) Determining altered expression of protein neoplastic markers on the cell surface, for example by immunohistochemistry. (vi) 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 one 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 hybridising 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 molecule; or (iii) nucleic acid probes or oligonucleotides comprising a nucleotide sequence capable of hybridising 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 molecule; or (iv) probes capable of binding to any one 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) 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 Tm = 69.3 + 0.41 (G+C)% (Marmur and Doty, J. Mol. Biol. 5:109,1962). However, the Tm of a duplex DNA decreases by l°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 x SSC buffer, 0.1% w/v SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x 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, 5X 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 minimises the incidence of nonspecific 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
Bisulfite-tag technology was applied to produce methylated and unmethylated genome fractions as described below. Briefly, DNAs were digested with methylation insensitive enzymes Mspl and TaqI and treated with sodium bisulfite under non-denaturing conditions, such that the cytosine in the 5’-CG single-stranded overhang left by each restriction enzyme would be converted to uracil if it were unmethylated but would remain unconverted if methylated. Separate linkers with either 5’-CG or 5’-CA overhangs were ligated to provide linkered methylated and unmethylated fractions respectively. After incorporation of a second primer by random-primed copying of a reverse strand this common primer was used in combination with the appropriate forward primer to amplify the methylated and unmethylated fractions.
In detail, tumour and matched normal DNA samples from eight patients were processed and analysed as described below. 1. DNAs from cancer and normal tissues (1 to 2 ug) were sheared by sonication using a Bioruptor UCD-200 sonicator (Diagenode, Belgium) in 300 pL of 10 mM Tris, 0.1 mM EDTA, pH 7.5 at a power setting of “high” for 60 minutes on ice, with alternating cycles of 30 seconds “on” or “off’. A small part of each sample was then applied to a 2% agarose gel, to confirm that a mean size range of 200 to 300 bp had been reached. DNA was precipitated and resuspended in water. 2. The volume was adjusted to 20 pL in Antarctic Phosphatase reaction buffer (New England Biolabs) and DNA de-phosphorylated using 5 units of Antarctic phosphatase at 37°C for 60 min. The enzyme was then inactivated by heating to 65°C for 10 min. 3. Sequential restriction enzyme digestion: 3 pL of 10X New England Biolabs Buffer 4 and 2 pL, 20 units, of Mspl enzyme were added and incubation continued at 37°C for 2 hr. 20 units, 2 pL, of TaqI and BSA to 100 pg/mL were added to give a volume of 28 pL and incubation continued at 65°C for a further 2 hr. Incubation was stopped by addition of 2 pL of 500 mM EDTA. 4. The cut DNA (1 μg) was reacted with sodium bisulfite, using the MethylEasy kit from Human Genetic Signatures (North Ryde, Australia), but omitting any prior heating or alkali denaturation step. Briefly: (a) 25-30 uL of restriction digested DNA was combined in a 2 mL tube with 250 μΐ of sodium bisulfite, then incubated at 37°C for 4 hours while omitting mineral oil. (b) 20 μg of blue-dye-labelled glycogen was added and DNA precipitated with 750 pL of MethylEasy solution 4 and 1 ml of isopropanol. Following centrifugation and washing of the pellet with 70% ethanol DNA was dissolved in 12 pL of MethylEasy Solution 3. Alternatively excess bisulfite was removed using a small silica column, and sample eluted from the column in 16 pL of MethylEasy Solution 3. (c) Desulphonation was achieved by heating in MethylEasy solution 3 for 60 minutes at 72° C, while spinning down several times to reduce evaporation. 5. Ligation of CG and CA linkers. Separate ligations were set up with Pl-CG or Pl-CA linkers (Table 1) as described below. Both Pl-CG and Pl-CA linkers (Table 1) were modified by incorporation of a biotin at the 3’ end of the upper strand to allow subsequent capture.
Table 1. Sequences of detection facilitation and competitor linkers
Mixes of combined linkers and competitors were prepared as in Table 2 below. These amounts, and ratios, are for lOOOng of DNA of average length 200 bp.
Table 2
Ligation reactions with 1 pg of bisulfite treated DNA were set up in 50 pL with 0.6 pL (12 units) of T4 DNA ligase and incubated at room temperature for 5 hr. After ligation, DNA was purified through a Promega Wizard column and eluted with 2 x 50 pL of 10 mM Tris.HCI, 0.1 mM EDTA, pH8. This step removes the unligated linkers. 6. Bead capture and release of single-strand: 25 pL of Dynal M280 streptavidin beads were first washed twice with 500 pi of 2X salt wash buffer (2M NaCI, lOmM Tris pH7.5, ImM EDTA, 0.2% Tween 20) and resuspended in IOOul of 2x wash buffer.
The lOOpL of eluted DNA was added and mixed on a shaker at room temperature with the beads for 15min at 500rpm. Beads were magnetically captured for 1 min and the supernatant removed. This was followed with two washes in 500 pL of IX wash buffer, once with 500 pL of 1 x SSC and then with 500 pL of 0.5 x SSC at 65°C for 3 min with shaking. 7. The beads were then resuspended in 50 pL of 10 mM Tris.HCI, 0.1 mM EDTA, pH8 and transferred to a new tube. After incubation at 98°C for 5 min and chilling for 1 min on ice, the beads were magnetically captured for one minute on ice and the supernatant (50 pL) transferred to a fresh tube. After drying, the DNA was resuspended in 10 pL of water. 8. Random priming .The P2 primer sequence was incorporated by random priming using a P2-N6 primer (Table 1). The 10 pL of eluted single-stranded DNA was mixed with 500 ng (5 pL) of P2-N6 primer, heated at 98°C for 2 min and chilled on ice. To this mix was added, 2.5ul lOx New England Biolabs buffer 2, 0.625ul lOmM dNTPs, 0.625ul Klenow fragment of DNA polymerase (5000u/ul) and the volume adjusted to 25 pL. After incubation on ice for 10 min, then at room temperature for 60 min, the reaction was terminated by heating at 75°C for 20 min. 9. Amplification . After trial reactions using 1 pL of random primed DNA, large to estimate amplification cycles, 300 pL amplification reactions for each of the methylated and unmethylated fractions were set up as follows: 10 pL random primed DNA sample ISO pL Promega GoTaq 2x Hot start mastermix 18 pL Pl-CA or PI-CG primer (Table 1, 5uM stock) 18 pL P2-long primer (Table 1, 5uM stock) 7.5 pL Sybr Green (1:3000 dilution of lOOOOx concentration) 96.5 pL water
Samples were run on a Roche LightCycler 480, SYBR green programme: 95°C 2min xl cycle, and 95°C 15sec, 60°C Imin x 40 cycles. Fluorescence was monitored and reactions stopped at the top of the exponential phase. DNA was purified using Promega Wizard columns, eluted in 30 pL water and quantified by spectrophotometry. 10. Labelling and hybridisation. 20 pg samples of each fraction were fluorescently labelled using the Nimblegen Dual-Color DNA labelling kit but with the Cy3 and Cy5 random nonamers being substituted with Cy3 and Cy5 random hexamers (Geneworks,
Adelaide, Australia). Methylated DNA fractions were labelled with Cy3 and unmethylated with Cy5 dyes.
Methylated fractions of cancer and normal DNAs were co-hybridised with a Nimblegen 720K promoter tiling array following Nimblegen protocols and the unmethylated fractions co-hybridised to a separate array according to manufacturer’s protocols. Arrays were scanned on an Axon scanner
In addition to individual samples, four pools of DNA were prepared for fluorescent labelling (as well as four additional pools in which Cy3 label was used for the cancer DNAs and Cy5 for normal)-
Pool 1 combined the methylated DNA fractions from the cancer tissues: cy5 label
Pool 2 combined the methylated DNA fractions from matched normal tissues: cy3 label
Pool 3 combined the unmethylated DNA fractions from the cancer tissues; cy5 label
Pool 4 combined the unmethylated DNA fractions from matched normal tissues: cy3 label
Identification of differentially methylated genes
Data from microarrays for the eight individual cancer and normal pairs as well as the analysis of pooled DNA and the dye-swap hybridisation with pooled DNAs were combined as described below to identify the most differentially methylated genes. Differential methylation is assessed via a difference-difference score, DD. This defined for each probe as DD = (Y.meth.tumour - Y.unmeth.tumour) - (Y.meth.normal - Y.unmeth.normal) where each Y value is the base-2 logarithm of the raw probe response value for the given combination of methylation status (meth/unmeth) and disease status (tumour/normal). Larger values of DD mean methylation is increased in the tumour samples relative to normal samples. For each tiled region probes were ranked in order of highest DD as DD1, DD2, DD3 etc.
Two gene lists were created, one that was based on the differential methylation for the two most differential probes in a tiled region (DD2) and one that was based on the top four differential probes (DD4).
For the DD2 list data for where the probe quality as scored by Nimblegen is beyond the threshold of 1.5 were discarded. This threshold accepts about 17% of all probes and minimises the potential for “noise’ when only two probes are considered. For each tiled region, the DD2 values for the eight individual samples were combined via a median. Then an overall DD2 value across all samples obtained from the average of three values: this median, and the two pooled sample values, forward and dye-swap. All tiled regions were then ranked by this score, and the top 30 returned.
For the DD4 list where a greater number of probes per tiled region were considered, a quality score threshold of 4 (that accepts about 40% of probes) was used. The overall DD4 value for each tiled region was determined as for DD2 except using the individual DD4 values in each case.
Table 3 shows a list of 44 genes shown to be differentially methylated between cancer and normal DNAs, based on either the level of the differential signal based on the DD2 or DD4 analysis. The list is ordered by the difference based on 4 probes, and then 2 probes. The top 4 ranked probes (and 9 in all) appear in the top 30 gene list derived by both methods. The hgl 8 coordinates of the tiled regions on the Nimblegen arrays showing differential methylation are given in the first column. Corresponding gene names where available linked to tiled regions and the coordinates of the genes (hgl 9) are shown in the right two columns. DD plots across the tiled regions for each gene are shown in Figure 1.
Table 3
The double-difference plots of these differentially methylated genes ate shown in Figure 1. In these plots, low signals are expected where probes lie >300 bp from a restriction site (because of the sheared length of fragments that all have one end located at a restriction site), or where a restriction site lies within a probe, shortening the size of the hybridising region. Extensive differential methylation is seen across the tiled regions of some genes, PDX1 and TRAPP9C for example, while in some cases increased methylation is confined to part of the tiled region, for example the right hand half of ZNF471. EXAMPLE 2
Methylated DNA fractions from bi sulfite-treated DNA of the colorectal cancer cell lines HCT116, HT29 and SW480 and from DNA isolated from whole blood were prepared using a biotin capture method described below and libraries of methylated DNA were sequenced using the Applied Biosystems SOliD sequencing system. Briefly, DNAs were sheared and modified SOLiD P2 linkers ligated to the sheared DNAs. DNAs were then cut with Csp61 (cut site G’TAC) and ligated with modified SOLiD PI linkers. DNA was then denatured and treated with sodium bisulfite to convert all unmethylated cytosines to uracil, The bisulfite treated DNA was then copied using a modified P2 primer and the original, uracil-containing bisulfite-treated DNA strand removed. The PI forward primer was then used to prime forward strand synthesis in the presence of biotin-dCTP. Thus biotin dCTP was incorporated in positions that that contained methylated cytosine in the original DNA, and hence were not converted to uracil. The biotin containing fraction, representing molecules containing methylated cytosines and lying adjacent to Csp61 sites, was captured on magnetic beads and subsequently amplified for high throughput sequencing. Details of the protocol are as below: 1. Genomic DNAs, 2 ug, were sonicated as in Example 1 to produce DNA fragments ranging in size from 90 to 1000 bp, with most molecules ranging between 100 - 230 bp. DNA was end-repaired to ensure flush ends and 5’ phosphorylation for efficient ligation using the End-it kit (Epicentre Biotechnologies). Reactions were done in lx End-it buffer, 1 mM ATP, 0.25 mM of each dNTP and 1 pL of End-it enzyme mix (T4 DNA polymerase and polynucleotide kinase). After incubation at room temperature for 45 min reactions were heated to 70°C for 10 min to inactivate the T4 DNA polymerase. 2. Repaired DNA was purified and concentrated with a Qiagen MinElute reaction clean up kit. DNA was eluted in 2 x 16.5 pL of Qiagen Elution Buffer (EB). The eluate was adjusted to 50 μΐ of NEBuffer 2 (50 mM NaCI, 10 mM Tris-HCl, 10 mM MgCL, 1 mM dithiothreitol, pH7.9 @ 25°C) containing 200 μΜ dATP and incubated with 50 units of 3’—»5' exonuclease N-terminal truncated Klenow fragment of DNA polymerase (new England Biolabs). The reaction was stopped by heating at 75°C for 20 min. 3. Repaired, A-tailed DNAs were ligated with annealed linkers SOLP2-AB/SOLP2-BP. The linker is adapted from the Applied Biosystems P2 linker used in its SOLiD high throughput sequencing system, the underlined bases in SolP2-BP having replaced cytosines present in the original sequence. The SolP2-BP sequence contains no cytosines so that subsequent incorporation of biotin dCTP will be restricted to the insert sequence.
SolP2-AB 5’ CCTACCCCACATTCCTCATTCTCT SEQ ID NO: 11
SolP2-BP 3’ TTGG ATGGGGTGTAAGG AGTAAGAGp SEQ ID NO:12
Complementary oligonucleotides were combined in a 0.2 mL PCR tube with Quick ligase buffer (New England Biolabs) to give a final 500 μΜ concentration. The oligonucleotides were annealed by using a thermal cycler as specified in the Applied Biosystems SOLiD library preparation appendices. DNA ligations were done in Quick Ligase buffer (66 mM Tris-HCl, 10 mM MgCh, 1 mM dithiothreitol, ImM ATP, 7.5% w/v polyethylene glycol 6000, pH7.5 @ 25°C), using I pL of Quick ligase per 40 pL of reaction (New England Biolabs). The ratio of linkers to DNA fragment ends was approximately 10 or 15 to 1. Linkers were removed using a QiaQuick PCR purification kit (Qiagen) and DNA eluted in 40 or 50 pL of EB. 4. Subsequent to the first linker ligation, the DNA was cut with the restriction enzyme Csp61 (G’TAC) that leaves a 5'-TA-3‘ overhang. DNAs were cut overnight in restriction enzyme buffers provided by the supplier (Fermentas, buffer B) with 10 U of Csp6I.
These ends were then ligated with the hemimethylated SolPl linker in which the cytosines on the upper strand SolPl-AM had been replaced with 5-methyl cytosines (shown as “M”) and the a 5’-TA-3’ overhang, underlined, added to the original SolP2-B sequence.
SolPl-AM 5’ MMAMTAMGMMTMMGMTTTMMTMTMTATGGGMAGTMGGTGAT SEQ ID NO: 13
SolPl-BC 3’ CGGTGATGCGGAGGCGAAAGGAGAGATACCCGTCAGCCACTAAT SEQ ID NO:14 5. Following PI linker ligation, DNA was denatured and treated with sodium bisulfite using a MethylEasy kit (Human Genetic Signatures) and resuspended in 30 pL of HGS reagent 3. In the upper strand, the methyl cytosines in the PI linker are protected from conversion and the P2 linker strand contains no cytosines, restricting C to U conversion to the insert region. Due to the single stranded nick, after denaturation only the upper strand will have adaptors at both ends. 6. The primer SolP2-AB was used to prime and extend to synthesise a copy of the converted DNA. Briefly, bisulfite-treated DNA (1 pg) was incubated with Invitrogen Platinum Taq DNA polymerase (1 pL, 5 units) in 25 pL Platinum Taq buffer containing 50 pM dNTPs, 2.5 mM MgCb and 500 nmol SolP2-AB primer. After heating at 94°C for 3 min the reaction was incubated for 15 min at 65°C. The 25 pL of primed strand reaction had 2.5 pL of Arctic phosphatase buffer added along with 5 U of Arctic phosphatase (to remove dNTP pool). Incubation was for 30 min at 37°C with subsequent denaturation of the Antarctic phosphatase for 8 min at 65°C. 7. Following synthesis of the complementary strand, the original bisulfite-treated strand was degraded by treatment with USER enzyme mix that cleaves DNA at positions of uracil bases. The remaining strand was then used as a template for synthesis of the strand corresponding to the original bisulfite treated strand, with incorporation of biotin-dCTP for specific tagging of methylated sites. The reaction tube was cooled and a mixture of reagents was added to bring the volume to 50 pL with a final concentration of 100 pM deoxyadenosine-triphosphate, 100 pM deoxyguanosine-triphosphate, 100 pM deoxythymidine-triphosphate and 50 pM biotin-14-deoxycytidine-triphosphate along with 500 nM of SolPl-A primer, 1 U of hot-start Taq, 2 U of uracil-specific excision reagent, USER enzyme mix (New England Biolabs) and 1 * Taq buffer, The reaction was incubated for 10 min at 37°C to allow uracil DNA glycosylase to degrade uracil containing bisulfite treated DNA before denaturation for 2 min at 94°C and extension of the template for 10 min at 65°C. A final aliquot containing 2.5 nmol of unlabeled deoxycytidine-triphosphate was then added, with a further 5 min incubation at 65°C. The product was purified with a QiaQUICK PCR purification kit (Qiagen) and eluted in200pLofEB. 8. The DNA was then incubated with streptavidin beads in order to bind material labelled with biotin. A series of wash steps selectively enriched for this bound material. Heat denaturation in water allowed the enriched material to be recovered. Bovine serum albumin was used as a blocking agent and a surfactant in the wash steps used to increase the stringency of enrichment. Briefly, Invitrogen M-270 streptavidin magnetic beads were prepared according to the manufacturer's specifications then washed in 500 pL of 0.1 mg/mL bovine serum albumin before resuspension in 200 pL of 2x binding and wash (B&amp;W) buffer (2 mol/L NaCl, 20 mmol/L Tris-HCl [pH 7.2], 2 mmol/L EDTA and 0.2% v/v Tween80) and the addition of equivolume purified labelled DNA solution. In some instances 10 pg of each control annealed oligonucleotide DNA was also 'spiked-in'. The 400 μΐ, mixed solution was incubated with gentle rocking for 30 min at 37°C. The tube was placed on a magnet for 3 min before the supernatant was aspirated and kept. Two extra washes were performed - the first with 500 μΐ. lx B&amp;W buffer and the second with 550 pL water. Then beads were resuspended in 45 pL of water and the tube heated to 90°C for 2’ and placed immediately on a magnet. The heated water was aspirated as soon as the magnetic beads cleared from solution, 5 pL of 100 mM Tris-HCl pH 8.0, 1 mM EDTA solution was added and the tube. The captured and released fraction was aspirated, fractions were ethanol precipitated and resuspended in 50 pL of 10 mM Tris-HCl pH 8.0,0.1 mM EDTA. 9. Captured DNA was amplified for a limited number of cycles with a hot start Taq polymerase in the presence of 1000 nM of the standard SOLiD PI and P2 primers. The reverse primer was used to modify the amplicon sequence to match that of the original Applied Biosystems primer 2 sequence. The illegitimate mismatch priming in the first few amplification cycles required a lower annealing temperature. The amplification used 10 sec denaturation steps at 94°C and 30 second extension steps at 72°C with annealing steps at 50°C for the first four cycles before moving to a 62°C annealing temperature for subsequent cycles. PCR products were purified and quantitated then another few final rounds of PCR were performed with 2 U Phusion Taq (Finnzymes) in 100 pL Phusion Taq High-Fidelity buffer, 1000 nM primers and 10 sec denaturation steps at 98°C, 30 second annealing steps at 62°C and 30 second extension steps at 72°C. 10. Reaction products were run on Low-Range agarose (Biorad) gels, stained with SYBR Gold (Invitrogen) and 125-200 nucleotide DNA was cut from the agarose with a scalpel under blue light on a Safe Imager (Invitrogen). Cut agar was processed with a Wizard SV Gel extraction and PCR clean up kit (Promega) according to the manufacturer’s instructions, excepting that the agarose was dissolved at room temperature.
The PCR products were purified using a Qiagen MinElute kit, quantitated with a spectrophotometer (Nanodrop) and sequenced on a Applied Biosystems SOLiD apparatus using SOLiD version 3 chemistry.
The bisulfite sequencing reads were aligned to the human genome and the number of reads of a CpG at each potential position within the 50 base reading distance on each side of a Csp6l site was determined for each DNA sample. Each read of a CpG in he bisulfite sequencing reads corresponds to a methylated CpG in the original DNA. For each DNA sample the sums of methylated CpG sites read in the region from 2 kb upstream to 1 kb downstream of the most upstream annotated transcription start site of a gene was computed. This allowed identification of genes and promoter regions where there was significant differential methylation comparing each of the colon cancer cell line DNAs with blood DNA. The bisulfite-tag methylation profiles of these genes obtained from analysis of 8 pooled cancer DNA and 8 pooled normal DNA samples (Figure 2) were then used to develop a list of genes identified through the biotin capture method that also showed significant differential methylation in clinical samples (Table 4). In Table 4, the gene or locus name is shown in the first column. The chromosomal locations of the associated genes are shown in the next two columns and the fourth and fifth columns give the positions of the first and last CpG sites sequenced in the biotin capture analysis. The number of CpG sites analysed and the total number of methylated CpG counted in each DNA sample are provide in the subsequent columns. The clear differences in methylation levels between the colon cancer cell line DNAs and blood DNA in these regions are evident.
Notably, five genes identified with an asterisk in Table 4, IRX1, DLX5, SALL1, SLC32A1 and ZNF471, had been also identified as highly differentially methylated genes using the bisulfite-tag method for primary screening.
Also notably, related members of the same gene families were found by one or both methods to be differentially methylated in colon cancer compared with normal tissue. This included genes of the IRX family, IRX1 and 1RX2, of the FOXD family, FOXD2 and FOXD3, of the HOXA family, HOXA2 and HOXA5, and of the NK.X2 family, NKX2-2, NK.X2-3 and NKX2-6.
EXAMPLE 3 DNA METHYLATION PROVILES OF SELECTED GENES IN COLORECTAL CANCER AND NORMAL TISSUE DNA Primers were designed for inethylation status independent amplification of gene and/or promoter regions for a set of genes identified in the previous Examples. The genes, primers and chromosomal co-ordinates of amplicons are shown in Table 5,
The primers were used for PCR from bisulfite treated DNA of 10 colorectal cancer specimens, their matched normal tissue and normal blood DNA. Amplification was done using Promega GoTaq master mix (without SybrGreen), 4mM MgCl2 and with primers at 200nM and lOng of input DNA. Cycling conditions were 95°C, 2 min (1 cycle), followed by 50 cycles of 95°C 15 sec, NeC 30 sec; 72°C 30 sec. where the annealing temperature N for each amplicon is shown in Table 5. For some amplicons an additional 200μΜ of dATP and dTTP was added to enable comparable amplification of both methylated and unmethylated DNA sequences. Amplified bands of DNA were purified and equivalent amounts of the separate amplicons derived from each DNA sample were pooled and ligated with linkers for sequencing on the Roche 454 Titanium FLX system. Samples from individual patient’s cancer or normal DNA and the blood DNA sample were separately ligated with bar coded “MID” linkers (Roche Cat No 05619211001) so that sequence reads could later be assigned to individual samples for sequence alignment and scoring. Libraries were prepared following protocols provided with the Roche Library preparation kit and reagents and sequenced on two halves of a flow cell; one half contained all the cancer samples and one the equivalently bar-coded normal samples. The bisulphite sequencing reads were segregated to individual samples using the bar-code sequences and aligned with the bisulphite converted sequence of each amplicon. After best alignment, the fraction of cytosines at each potential CpG methylation site was determined for each sample.
Profiles across individual amplicons are shown in Figures 3-27. The data for 57 amplicons representing 24 genes or regions is summarised in Table 6. The table shows the approximate range of methylation levels at CpG sites across each amplicon for the individual cancer samples, A-J and combined data for the 10 matched normal DNAs. Columns show headed m/10 and p/10 the number of cancer samples out of 10 showing high leyel (>60%) or partial (30-60%) methylation across the amplicon. Methylation of two of these genes, ADAMTS1 and TMEFF2 has been previously reported in colorectal cancer and they show partial or high level methylation in 9 or 10 cancer samples respectively. Among the 23 new genes tested, only the FGFR2 gene did not show differential methylation between cancer and normal samples. Most genes showed differential methylation in a high proportion of samples. 9 genes - DLX5, FOXD2, IRX1, MEIS1, MMP2, NPY, PDX1, SUSD5 and TCF21- showed high or partial methylation in all 10 samples, 9 genes - COL 1A2, COL41, EFEMP, FGF5, FOXF1, GRASP, SDC2, SOX21 and ZNF471 - in 9 samples, FOXB1 in 8 samples, PPP1R14A in seven, FBN1 and EDIL3 in six and MEIS in three samples. In some cases, eg EDIL3, FBN1, GRASP Region 2, MEIS1 and SDC2, the level of methylation in matched normal colonic tissue was consistently very low. For other genes or regions, eg. DLX5, GRASP Region 3, IRX1, MMP2, NPY, PDX1 and TCF21, significant levels of methylation were evident in the matched normal tissue but methylation was always significantly increased in the cancer tissue. The data also demonstrate that for a given gene, not all regions show equivalent cancer-specific methylation. For example, for the COL4A gene(s) Regions 1 and 5 show high or partial methylation in 9 of 10 cancer samples, while Regions 2 and 3 are methylated in only 4 or 2 samples respectively. COL4A Region 1 lies within the COL4A1 gene, while COL4A Region 5 lies within the neighbouring, divergently transcribed COL4A2 gene.
The sequencing data thus demonstrates colorectal cancer-specific DNA methylation for 23 novel genes and specific regions that may be used for development of assays to distinguish cancer from normal DNA. EXAMPLE 4
ANALYSIS OF DNA METHYLATION IN CLINICAL SAMPLES USING METHYLATION SELECTIVE PCR DNA was extracted from colon tissue specimens comprising 10 adenomas, 15 Stage 1, 18 Stage B, 28 Stage C, 7 Stage IV, 6 matched normal colon specimens and 7 other normal colon tissue. Isolated DNA was bisulphite converted using the Zymo EZ Gold bisulphite conversion kit as recommended by manufacturer. PCR assays were done in 15uL reaction mixtures containing a final concentration of 200nM of oligonucleotides and MgCI2 as shown in Table 7 and a 1:120,000 dilution of Molecular Probe SYBR Green (Invitrogen). GRASP and NPY amplifications were done lx Platinum Buffer (Invitrogen) using 0.15 pL Platinum Taq DNA polymerase (Invitrogen). COL4A and SDC2 amplifications were done in Amplitaq, Taqman Buffer A using 0.1 pL (0.5 units) of AmpliTaq Gold (Appllied Biosystems). The PCR amplifications were performed in a Roche LightCycler 480 real-time PCR instrument using 384-well plates using the cycling conditions as shown in Table 7 for each amplicon. Levels of
methylation were quantified using a standard curve of fully methylated DNA, 40 pg to 5 ng mixed with peripheral blood leukocyte DNA to give a total input of 5 ng. The proportion of methylated DNA in a sample was determined by dividing the amount of methylated DNA, calculated from the standard curve for fully methylated DNA, by the amount of input DNA TABLE 7
DNA Samples were scored as positive if the fraction of methylated DNA was >1 % for GRASP or >2% for NPY, SDC2 and COL4A1. The number and % of positive samples for each biomarker assay are shown in Table 8. For cancer DNA samples a high proportion of samples, ranging from 88 to 94% were positive for methylation of each marker. By contrast the proportion of positive samples among matched normal controls was low (0-25%). Within the normal DNA samples, the level of methylation measured by the MSP assays was quantitatively low compared with that of the cancer DNA samples. TABLE 8
EXAMPLE 5
ANALYSIS OF METHYLATION OF THE GRASP AND COL4A1 GENES IN DNA
ISOLATED FROM PLASMA
Assays for GRASP and COL4A1 genes were also done on plasma DNA samples from patients with colorectal cancer, colorectal adenomas or patients without colorectal neoplasia as determined by colonoscopy. DNA was extracted 4mL of human blood plasma using the QIAmp Isolation of free circulating nucleic acids from serum/plasma (QIAGEN). Isolated DNA was bisulphite converted using the Zymo bisulphite conversion kit as recommended by manufacturer. A total of 36uL of bisulphite converted DNA was retrieved from 4mL of plasma. The presence of methylated COL4A1 sequences was determined using the MSP assay described in Example 4 except that a specific detection probe COL4A1 Probe BS1, HEX-5’CTAAACCCGTCCGCCTACCCCTC-BHQ (SEQ ID NO:80) was included at lOOnM,
Detection of methylated GRASP sequences was done using a semi-nested PCR. First round PCRs was done in 30 μΙ. ( 2.5uL of bisulphite converted DNA input) consisting of a final concentration of lx Platinum Taq Buffer, 0.1 pL Platinum Taq DNA polymerase (Invitrogen), 3.3mM MgCI2,200uM dNTPs (New England BioLabs) and 33 nM of oligonucleotides. The first round amplification used the forward primer GrspA-Fl 5’-CGGATTTTCGATTCGGAAGT (SEQ ID NO:81) and GRASP MSPR1 from Table 7, Example 4.
Cycling conditions were 95’C for 2min, followed by eleven cycles of 92’C, 15sec; 60’C, 30 sec and 72’C 30 sec. The PCR amplifications were performed in PALM end-point PCR cycler using 96-well plates. Second round PCR used the primers and reaction conditions from Example 4, Table 7 and was performed on luL of material from PCR round 1 into a total PCR reaction of 15 pL. Levels of methylation were quantified using a standard curve of ful ly methylated DNA, 40 pg to 5 ng mixed with peripheral blood leukocyte DNA to give a total input of 5 ng. Standard curve were based on input into the single round for COL4A1 or the first round for GRASP.
The proportion of plasma DNA samples scoring positive in at least 2 or 3 triplicate PCRs is shown in Table 9. The data show that both TABLE 9
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
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Claims (24)

  1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:
    1. A method of screening for the onset or predisposition to the onset of a large intestine neoplasm or monitoring the progress of a neoplasm in an individual, said method comprising assessing the methylation status of a DNA region selected from: (i) the region defined by Hgl9 coordinates chrl2:52400748..52409671 and 2kb upstream of the transcription start site; or (ii) the gene region, including 2kb upstream, of GRASP in a biological sample from said individual wherein a higher level of methylation 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.
  2. 2. The method according to claim 1, said method comprising assessing the methylation status of one or more DNA regions selected from the DNA regions defined by Hgl9 coordinates: (1) Chr 12:52399672-52399922; (2) Chrl2:52400821-52401119; and (3) Chr 12:52401407-52401664
  3. 3. The method according to claim 1, said method comprising assessing the methylation of one or more cytosine residues selected from: Chrl2:52399713 Chrl2:52399731 Chrl2:52399749 Chrl2:52399783 Chrl2:52399796 Chrl2:52399808 Chrl2:52399823 Chrl2:52399835 Chrl2:52399891 Chrl2:52400847 Chrl2:52400850 Chrl2:52400859 Chrl2:52400866 Chrl2:52400869 Chrl2:52400873 Chrl2:52400881 Chrl2:52400886 Chrl2:52400893 Chrl2:52400895 Chrl2:52400899 Chrl2:52400902 Chrl2:52400907 Chrl2:52400913 Chrl2:52400919 Chrl2:52400932 Chrl2:52400938 Chrl2:52400958 Chrl2:52400962 Chrl2:52400971 Chrl2:52400973 Chrl2:52400976 Chrl2:52400998 Chrl 2:52401008 Chrl2:52401010 Chrl2:52401012 Chrl2:52401016 Chrl2:52401019 Chr 12:52401025 Chrl2:52401041 Chrl2:52401044 Chrl2:52401053 Chrl2:52401060 Chrl 2:52401064 Chrl2:52401092 Chrl2:52401118 Chrl2:52401438 Chrl2:52401448 Chrl2:52401460 Chrl2:52401465 Chrl2:52401474 Chrl2:52401477 Chrl2:52401479 Chrl2:52401483 Chrl2:52401504 Chrl2:52401514 Chrl2:52401523 Chrl2:52401540 Chrl2:52401553 Chrl 2:52401576 Chrl2:52401588 Chrl2:52401595 Chrl2:52401599 Chrl2:52401604 Chrl2:52401606 Chrl2:52401634 Chrl2:52401640 Chrl2:52401644 Chrl2:52401659 or a corresponding cytosine at position n+1 on the opposite DNA strand.
  4. 4. The method according to any one of claims 1-3 wherein said neoplastic cell is an adenoma or an adenocarcinoma.
  5. 5. The method according to any one of claims 1-4 wherein said control level is a non-neoplastic level.
  6. 6. The method according to any one of claims 1-5 wherein said control level is the level of a previously screened biological sample from said individual.
  7. 7. The method according to claim 6 wherein a decrease in the level of methylation relative to said control level is indicative of the clearing of the neoplasm.
  8. 8. The method according to any one of claims 1-7 wherein said neoplasm is a colorectal neoplasm.
  9. 9. The method according to any one of claims 1-8 wherein said biological sample is a faecal sample, enema wash, surgical resection, tissue biopsy or blood sample.
  10. 10. The method according to claim 9 wherein said blood sample is whole blood, serum or plasma.
  11. 11. The method according to any one of claims 1-10 wherein said control level is determined from a corresponding biological sample.
  12. 12. 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 defined by Hgl9 coordinates chrl2:52400748..52409671 and 2kb upstream of the transcription start site; or (ii) the gene region, including 2kb upstream of GRASP: in a biological sample from said individual wherein a lower level of expression 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.
  13. 13. The method according to claim 12 wherein said level of expression is assessed by screening for changes to the chromatin protein with which said gene is associated.
  14. 14. The method according to claim 12 wherein said level of expression is mRNA expression or protein expression.
  15. 15. The method according to any one of claims 12-14 wherein said neoplastic cell is an adenoma or an adenocarcinoma.
  16. 16. The method according to any one of claims 12-15 wherein said control level is a non-neoplastic level.
  17. 17. The method according to any one of claims 12-15 wherein said control level is the level of a previously screened biological sample from said individual.
  18. 18. The method according to claim 17 wherein an increase in the level of methylation relative to said control level is indicative of the clearing of the neoplasm.
  19. 19. The method according to any one of claims 12-18 wherein said neoplasm is a colorectal neoplasm.
  20. 20. The method according to any one of claims 12-19 wherein said biological sample is a faecal sample, enema wash, surgical resection, tissue biopsy or blood sample.
  21. 21. The method according to claim 20 wherein said blood sample is whole blood, serum or plasma.
  22. 22. The method according to any one of claims 12-21 wherein said control level is determined from a corresponding biological sample.
  23. 23. The method according to any one of claims 1-11 wherein said DNA region is the promoter region.
  24. 24. The method according to any one of claims 1-23 wherein said individual is a human.
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