KR20070098034A - Composition for cancer diagnosis containing methylated promoters of colon cancer specific expression-decreased genes and use thereof - Google Patents

Abstract

A composition for cancer diagnosis containing methylated promoters of colon cancer specific expression-decreased genes is provided to diagnose cancer at an early stage and predict prognosis of cancer. A composition for cancer diagnosis contains a methylated promoter of a colon cancer specific expression-decreased gene selected from genes of ID3(inhibitor of DNA binding 3) of SEQ ID NO:1, RGS2(regulator of G-protein signaling 2) of SEQ ID NO:2, WISP2(WNT1 inducible signaling pathway protein 2) of SEQ ID NO:3, MGLL(monoglyceride lipase) of SEQ ID NO:4, CPM(carboxypeptidase M) of SEQ ID NO:5, GABRA1(gamma-aminobutyric acid(GABA) A receptor) of SEQ ID NO:6, CLU(clusterin) of SEQ ID NO:7 and F2RL1(coagulation factor II(thrombin) receptor-like 1) of SEQ ID NO:8, wherein the promoter contains at least one methylated CpG dinucleotide.

Description

Composition For Cancer diagnosis Containing Methylated Promoters of Colon Cancer Specific Expression-decreased Genes and Use Thereof}

1 illustrates a process of selecting a methylation biomarker for colorectal cancer diagnosis by selecting genes showing a decrease in gene expression due to promoter methylation.

FIG. 2 is a diagram illustrating Hpa II / Msp I enzyme digestion assay, which is a method for measuring promoter methylation, for a promoter of a diagnostic gene for colorectal cancer discovered in the present invention.

Figure 3 is a method for measuring the promoter methylation in the colon cancer cell lines Caco-2 and HCT116 targeting 15 promoters discovered in the present invention, Hpa II / Msp I enzyme digestion assay (enzyme digestion assay) One result is shown.

Figure 4 shows that the expression of 15 genes discovered in the present invention is increased by the demethylating agent treatment.

FIG. 5 shows Hpa II / Msp I enzyme digestion assay for cancer tissues obtained from 40 colorectal cancer patients and adjacent normal and normal colorectal tissues from the promoters of 8 genes selected in the present invention. ) And then create a methylation profile.

Field of invention

The present invention relates to a composition for diagnosing cancer containing a methylated promoter of a colorectal cancer specific gene and a use thereof, and more particularly, to a composition for diagnosing cancer containing a methylated promoter of a colorectal cancer specific expression reducing gene, the composition The present invention relates to a cancer diagnostic microarray fixed on the substrate and a colorectal cancer diagnostic kit containing the composition.

Background of the Invention

Today, even with advanced medicine, the five-year survival rate is less than 50% for human cancers, especially solid tumors (most of which are blood cancers). About two-thirds of all cancer patients are found in advanced stages, most of whom die within two years of diagnosis. The treatment effect of such low cancer is not only a problem of treatment, but it is because it is not easy to diagnose the actual cancer early and to accurately diagnose the advanced cancer and follow-up after treatment.

The diagnosis of cancer in the current clinical practice is history taking, physical examination, clinical pathology, and once suspected, radiographic examination and endoscopy are performed. Finally, biopsy is confirmed. However, the current clinical test method can be diagnosed only when the number of cancer cells 1 billion cells, the diameter of the cancer is more than 1cm. In this case, the cancer cells already have metastatic capacity, and in fact, more than half of the cancer has already metastasized. On the other hand, tumor markers in the blood, which directly or indirectly produce cancer-producing substances, are used for cancer screening, which is limited in accuracy and appears to be about half normal even when cancer is present. In the absence of this, they often appear positive, causing confusion. In addition, the anti-cancer agent mainly used for the treatment of cancer, there is a problem that shows the effect only when the volume of cancer is small.

As described above, the diagnosis and treatment of cancer are both difficult because they differ from normal cells and are very complicated and diverse. Cancer continues to overgrow, frees from death, continues to survive, invades surrounding tissues and spreads (transfers) to distal organs, causing human death. It survives the attack of immune mechanisms and chemotherapy, and it is constantly evolving, and the population of cells (clones) which are most favorable for survival multiply selectively. Cancer cells are highly viable survivors caused by mutations in a number of genes that require mutations in multiple genes in order for one cell to turn into a cancer cell and develop into a malignant cancer mass seen in the clinic. Therefore, a gene-level approach is needed to diagnose and treat cancer fundamentally.

Recently, genetic testing has been actively attempted to diagnose cancer. The simplest representative method is by PCR to find the presence or absence of the ABL: BCR fusion gene, a gene marker for leukemia in the blood. This is more than 95% accuracy, and is a simple test that is useful for the diagnosis of chronic myelogenous leukemia, evaluation of the result after treatment, and follow-up. However, this method is only applicable to minor hematologic cancers.

In addition, a method of diagnosing cancer cells coexisting in blood cells by attempting to identify the presence of genes expressed by cancer cells by RT-PCR and blotting is also attempted. However, this method is applicable only to some cancers, such as prostate cancer and melanoma, has a high false positive rate, difficult standardization of test and reading methods, and its usefulness (Kopreski, MS et. al ., Clin . Cancer Res . , 5: 1961, 1999; Miyashiro, I. et al ., Clin . Chem . , 47: 505, 2001).

Genetic testing using DNA in serum or plasma has recently been actively attempted. This is a way to find cancer-related genes that are isolated from cancer cells and come out into the blood and are present in the serum as free DNA. In actual cancer patients, the DNA concentration in serum is increased by 5-10 times that of normal people, and this increased DNA is found to be mostly released from cancer cells. Cancer can be diagnosed by analyzing DNA abnormalities specific to cancer, such as mutations, loss, or loss of oncogenes and tumor suppressor genes, with DNA released from these cancers. Lung cancer, head and neck cancer, breast cancer, colorectal cancer, mutant type K-Ras oncogenes, p53 tumor suppressor genes, promoter methylation of p16 genes, and microsatellite labeling and instability were examined in serum. Diagnosis of liver cancer has been actively attempted (Chen, XQ et al ., Clin . Cancer Res . , 5: 2297, 1999; Esteller, M. et al ., Cancer Res. 59:67, 1999; Sanchez-Cespedes, M. et al ., Cancer Res . , 60: 892, 2000; Sozzi, G. et al ., Clin . Cancer Res . , 5: 2689, 1999).

On the other hand, cancer DNA can also be tested in samples other than blood. In lung cancer patients, genetic or antibody tests have been used to detect the presence of cancer cells and cancer genes in sputum or bronchoalveolar lavage (Palmisano, WA et al., Cancer) . Res . 60: 5954, 2000; Sueoka, E. et al ., Cancer Res . , 59: 1404, 1999), How to find cancer genes present in feces in colorectal and rectal cancers (Ahlquist, DA et al ., Gastroenterol . , 119: 1219-27, 2000) and methods for testing for promoter methylation abnormalities present in urine and prostate fluid (Goessl, C. et. al ., Cancer Res . , 60: 5941, 2000). However, in order to accurately diagnose cancers with multiple gene abnormalities and various mutations for each cancer, a method for simultaneously and accurately analyzing multiple genes is required, but such methods are not established yet.

Recently, methods for diagnosing cancer by measuring DNA methylation have been proposed. When the promoter CpG island of a particular gene is hypermethylated, the expression of that gene becomes gene silencing. This is a major mechanism by which genes lose their function without mutation in the protein-specific coding sequence of genes in vivo. It is interpreted as a cause. Therefore, detection of methylation of the promoter CpG island of tumor suppressor genes is very helpful for cancer research, and it can be used for diagnosis and screening of cancer by examining it by methylation-specific PCR (hereinafter referred to as MSP) or automatic base analysis. Attempts have been made recently.

Many diseases are caused by gene abnormalities, and the most common type of gene abnormality is a change in the coding sequence of a gene. Such genetic changes are called mutations. When a gene is mutated, the protein it encodes changes its structure and function, leads to disorders and defects, and these mutant proteins cause disease. However, even without a mutation in a particular gene, if there is an abnormality in the expression of the gene can cause disease. A typical example is methylation, in which the methyl group is attached to the regulatory region of gene transcription, ie, the cytosine base of the promoter CpG island, in which case the gene is blocked. This is called an epigenetic change, which, like a mutation, is transmitted to progeny cells and causes the same effect, namely loss of expression of the protein. Most notably, the expression of tumor suppressor genes is blocked by methylation of the promoter CpG island in cancer cells, which is an important mechanism of carcinogenesis (Robertson, KD et. al ., Carcinogensis , 21: 461, 2000).

In order to correctly diagnose cancer, it is important not only to identify mutational genes, but also to understand the mechanism by which the mutations occur. Previously, the research focused on mutations in the coding sequence of genes, namely, microscopic changes such as point mutations, deletions and insertions, and macroscopic chromosomal abnormalities. Recently, however, extragenic changes have been reported as important as these, and the representative one is methylation of the promoter CpG island.

The genomic DNA of mammalian cells contains a fifth base in addition to A, C, G, and T, which is 5-methylcytosine (5-mC) having a methyl group attached to the fifth carbon of the cytosine ring. 5-mC always comes only to C of CG dinucleotide (5'-mCG-3 '), and this CG is often referred to as CpG. Most of C of CpG is methylated because a methyl group is attached. This methylation of CpG inhibits the expression of repetitive sequences in the genome, such as alu or transposon, and is the site where extragenic changes occur most frequently in mammalian cells. This 5-mC of CpG naturally deaminates and changes to T, so that CpG in the mammalian genome only has a frequency of 1%, much lower than the frequency at which it should normally appear (1/4 x 1/4 = 6.25%). Indicates.

Among CpGs, there are exceptionally dense ones, which are called CpG islands. CpG islands are 0.2-3kb in length, and the distribution percentage of C and G bases is over 50%, and the distribution percentage of CpG is concentrated to 3.75% or more. About 45,000 CpG islands appear in the entire human genome, particularly in promoter regions that regulate gene expression. Indeed, CpG islands appear in promoters of housekeeping genes, which make up about half of human genes (Cross, S. et. al ., Curr. Opin . Gene Develop . , 5: 309, 1995).

On the other hand, in somatic cells of normal humans, the CpG islands of these important gene promoter regions are not methylated, but the genes imprinted and inactivated on the X chromosome are methylated so that they are not expressed during development.

During carcinogenesis, methylation occurs in the promoter CpG island, which impairs the expression of the gene. In particular, when methylation occurs in the CpG island promoters of tumor suppressor genes that regulate cell cycle or apoptosis, repair DNA, participate in cell adhesion and intercellular coordination, and inhibit invasion and metastasis. Like mutations, they block the expression and function of these genes, which in turn promote the development and progression of cancer. In addition, methylation may occur partially on CpG islands with age.

It is interesting to note that in congenital cancers, mutations cause carcinogenesis, whereas in acquired cancers, mutations cause methylation of the promoter CpG islands instead of mutations. Representative examples include promoter methylation of genes such as VHL (von Hippel Lindau) in acquired kidney cancer, BRCA1 in breast cancer, MLH1 in colorectal cancer, and E-CAD in gastric cancer. About half of all cancers show promoter methylation of p16 or mutations in Rb, and the other half show promoter methylation of p73, p14, etc.

The important fact is that such extragenic changes caused by promoter methylation cause changes in genes, ie mutations in coding sequences, and these genes and extraneous changes combine to develop carcinogenesis. For example, in the colon cancer cells, alleles of the MLH1 gene are often lost due to mutations or deletions, and the other allele has failed due to promoter methylation. In addition, if the methylation of MLH1, a DNA repair gene, is lost due to promoter methylation, it facilitates carcinogenesis by facilitating mutations in other important genes.

Most cancers have three common characteristics with respect to CpG: hypermethylation of the promoter CpG island of tumor suppressor genes, undermethylation of the remaining CpG bases, and increased activity of methylation enzymes, namely DNA cytosine methyltransferase (DNMT). (Singal, R. & Ginder, GD, Blood , 93: 4059, 1999; Robertson, K. et. al ., Carcinogensis , 21: 461, 2000; Malik, K. & Brown, KW, Brit . J. Cancer , 83: 1583, 2000).

When the promoter CpG island is methylated, it is not clear why the expression of the gene is blocked, but methyl CpG adhesion protein (MECP) or CpG adhesion domain protein (MBD), and histone di It is speculated that the attachment of acetylase (histone deacetylase) changes the chromatin structure of chromosomes and changes histones.

There is controversy over whether methylation of promoter CpG islands directly causes carcinogenesis or whether it is a secondary change in carcinogenesis, but the clear fact is that promoter methylation of tumor-related genes is an important indicator of cancer, and therefore it is important to diagnose and premature cancer. Diagnosis, prediction of carcinogenic risk, prediction of cancer prognosis, follow-up after treatment, and prediction of response to chemotherapy can be used in many ways. In recent years, attempts have been made to investigate the promoter methylation of tumor-related genes in blood, sputum, saliva, feces, and urine and use them in various cancer treatments (Esteller, M. et. al ., Cancer Res . 59:67, 1999; Sanchez-Cespedez, M. et al ., Cancer Res . , 60: 892, 2000; Ahlquist, DA et al ., Gastroenterol. , 119: 1219, 2000).

In order to maximize the accuracy of cancer diagnosis using promoter methylation, to analyze carcinogenicity step by step, and to discriminate between cancer and aging, it is necessary to accurately analyze the methylation of all cytosine bases of the promoter CpG island. The standard method for this now is the bisulfite genome sequencing method. This is done by treating the sample DNA with sodium bisulfite, amplifying the entire region of the CpG island of the target gene by PCR, and analyzing the nucleotide sequence. However, this test has a limitation in the number of genes or samples that can be tested at one time, and it is difficult to automate and takes a lot of time and money.

Research on the promoter methylation of genes related to cancer is being actively conducted at Johns Hopkins Medical Center, MD Anderson Cancer Center, and Berlin Medical University. The basic data thus obtained are exchanged in the DNA Methylation Society (DMS), and the data are stored in MethDB (http://www.methdb.de). EpiGenX Pharmaceuticals, meanwhile, is developing a drug related to methylation of CpG islands, while Epigenomics is investigating the use of DNA chips and MALDI-TOF to test promoter methylation for cancer diagnosis.

Methylation of promoter CpG islands is deeply correlated with physiological phenomena such as human development and differentiation, aging, and the development of various cancers and benign diseases.In particular, methylation of promoter CpG islands of tumor-related genes is important for cancer development and progression. It can play an important role in cancer. However, the conventional method has the difficulty of amplifying all CpG islands of numerous genes with MSP and analyzing them by sequencing (bisulfite genome sequencing), respectively. In addition, it is possible to automatically and quickly analyze the various forms of promoter methylation changes of various genes at once, and there is no method that can be immediately applied to diagnosis, early screening, and stage evaluation of various cancers in the clinic.

Many studies have also been made on the screening of new methylation-related cancer suppressor genes. Existing methods limit the genomic DNA of cancer tissues and normal tissues to methylation-restriction enzymes, and then clone all numerous DNA fragments to select DNA sequences that differ from cancer tissues and normal tissues, and sequence and screen them. Or binding columns that recognize CpG islands (Huang, TH et). al ., Hum. Mol . Genet . , 8: 459, 1999; Cross, SH et al ., Nat . Genet . , 6: 236, 1994). However, not only do these methods require a lot of time, but also the screening of gene candidates is inefficient and difficult to apply in actual clinical practice.

Therefore, the present inventors have described the colorectal cancer specific expression gene LAMA2 (Laminin merosin alpha 2), FABP4 (Fatty acid binding protein 4), GSTA2 (Glutathione S-transferase A2), STMN2 (Stathmin-like 2), NR4A2 (Nuclear receptor). subfamily 4, group A, member 2), Down syndrome critical region gene 1-like 1 (DSCR1L1), Alpha-2-macroglobulin (A2M) and methylated promoters of SEPP1 (Selenoprotein P, plasma, 1) The invention has been filed for the array and cancer diagnostic kit (Korean Patent Application No. 10-2004-0076765). Based on this, in the present invention, colorectal cancer specific expression gene ID3 (Inhibitor of DNA binding 3), RGS2 (Regulator of G-protein signaling 2), WISP2 (WNT1 inducible signaling pathway protein 2), MGLL (Monoglyceride lipase), CPM (Carboxypeptidase M), GABRA1 (Gamma-aminobutyric acid (GABA) A receptor), CLU (Clusterin), and F2RL1 (Coagulation factor II (thrombin) receptor-like 1) methylated promoters for cancer diagnosis microarray and cancer diagnosis A study on the kit was performed.

Accordingly, the present inventors have found a promoter of a gene (colon cancer specific expression reduced gene) that is specifically suppressed from tissues and cell lines of colon cancer, confirming that they are useful for the diagnosis of cancer and completing the present invention. It became.

An object of the present invention is to provide a cancer diagnostic composition containing a methylated promoter of the colorectal cancer specific expression reduction gene.

Another object of the present invention is to provide a microarray useful for early diagnosis of cancer in which the methylated promoter is immobilized.

Still another object of the present invention is to provide a cancer diagnostic kit containing the methylated promoter.

Another object of the present invention is to provide a primer mixture useful for amplifying sample DNA from clinical samples for early diagnosis of colorectal cancer.

Another object of the present invention is to provide an early diagnosis method for cancer, characterized in that using the primer mixture and / or the microarray.

In order to achieve the above object, in the present invention, eight types of colorectal cancer-specific expression reducing genes and their promoters were identified, and the methylated promoters were identified for clinical application.

Eventually, the present invention is an inhibitor of DNA binding 3 (ID3), regulator of G-protein signaling 2 (RGS2), WISP2 (WNT1 inducible signaling pathway protein 2), monoglyceride lipase (MGLL), carboxypeptidase M (CPM), GABRA1 (Gamma) for diagnosing cancer containing methylated promoters of colorectal cancer specific expression-reducing genes selected from the group consisting of -aminobutyric acid (GABA) A receptor), CLU (Clusterin) and F2RL1 (Coagulation factor II (thrombin) receptor-like 1) To provide a composition.

In the present invention, the ID3 (Inhibitor of DNA binding 3), RGS2 (Regulator of G-protein signaling 2), WISP2 (WNT1 inducible signaling pathway protein 2), MGLL (Monoglyceride lipase), CPM (Carboxypeptidase M), GABRA1 ( Gamma-aminobutyric acid (GABA) A receptor, CLU (Clusterin) and F2RL1 (Coagulation factor II (thrombin) receptor-like 1) may be characterized by having a DNA sequence of SEQ ID NO: 1 to 8, respectively. In addition, the methylated promoter may be characterized by containing at least one methylated CpG dinucleotide, and may be characterized by comprising a sequence selected from the group consisting of:

(a) the 1747-1944 region of SEQ ID NO: 9;

(b) regions 1112-1829 of SEQ ID NO: 10;

(c) regions 1668 to 1782 of SEQ ID NO: 11;

(d) regions 607 to 1701 of SEQ ID NO: 12;

(e) regions 53 to 973 of SEQ ID NO: 13;

(f) the 1663-1787 region of SEQ ID NO: 14;

(g) regions 1790-1942 of SEQ ID NO: 15; And

(h) regions 1488-1945 of SEQ ID NO: 16.

More preferably, the methylated promoter may be any one of the DNA sequences represented by SEQ ID NOs: 9 to 16.

The present invention also provides a microarray for cancer diagnosis, wherein the composition is immobilized on a solid substrate, and a cancer diagnosis kit containing the composition.

The present invention also relates to SEQ ID NO: 17/18, SEQ ID NO: 19/20, SEQ ID NO: 21/22, SEQ ID NO: 23/24, SEQ ID NO: 25/26, SEQ ID NO: 27/28, SEQ ID NO: 29/30, and SEQ ID NO: Primer mixtures containing one or more primer pairs selected from the group consisting of 31/32 are provided.

The invention also comprises the steps of (a) separating the sample DNA from the clinical sample; (b) methylation sensitivity selected from the group consisting of (i) a reagent that modifies unmethylated cytosine residues, or (ii) Msp I, Hpa II, Bss HII, Bst UI and Not I; treating with a methylation sensitive) restriction enzyme; (c) The DNA treated in step (b) is treated with ID3 (Inhibitor of DNA binding 3), RGS2 (Regulator of G-protein signaling 2), WISP2 (WNT1 inducible signaling pathway protein 2), MGLL (Monoglyceride lipase), CPM CpG islands derived from promoters of genes selected from the group consisting of (Carboxypeptidase M), GABRA1 (Gamma-aminobutyric acid (GABA) A receptor), CLU (Clusterin), and F2RL1 (Coagulation factor II (thrombin) receptor-like 1) Amplifying using a primer capable of amplifying; And (d) determining whether the promoter is methylated based on the presence or absence of the result amplified in step (c).

In the present invention, the clinical sample may be tissue, cells, phlegm, feces, urine, membranes, brain water, amniotic fluid, eye, organs or blood derived from a suspected cancer patient or diagnosis target, and the methylated The reagent for modifying cytosine residues may be characterized as bisulfite. Methods for detecting the methylation of promoters by modifying unmethylated cytosine residues with bisulfite are described in detail in the prior art (WO 01/26536; US 2003 / 0148326A1).

In addition, the amplification may be typically characterized by PCR, DNA microarray or bisulfite sequencing, and the methylation of the amplification may be confirmed by sequencing, and the sequence amplified by the method of the present invention PCR, oligomer restriction, allele-specific oliginucleotide (ASO) probe analysis (Conner, BJ et al ., PNAS , 8 0: 278, 1983 Saiki, RK et al ., Nature , 324: 163, 1986), oligonucleotide ligation assays (OLA) (Landegren, U. et. al ., Science , 241: 1077, 1988 Landegren, U., Bioassays , 15: 761, 1993) can be evaluated, detected, cloned or sequenced in solution or after binding to a solid support by detection of specific DNA sequences.

Optionally, the methylation pattern of the nucleic acid can be confirmed by restriction enzyme digestion and Southern blot analysis. Examples of methylation sensitive restriction enzymes capable of detecting 5'CpG methylation include Sma I, Sac II, Eag I, Hpa II, Msp I, Bss HII, Bst UI, Not I Etc. Restriction enzymes containing CG as part of its recognition site inhibit C methylation. Typically, methylation sensitive restriction enzymes Hpa II, Msp I, or Bss HII alone or in combination. Other methylation sensitive restriction enzymes are well known in the art.

Primers capable of amplifying the CpG island are SEQ ID NO: 17/18, SEQ ID NO: 19/20, SEQ ID NO: 21/22, SEQ ID NO: 23/24, SEQ ID NO: 25/26, SEQ ID NO: 27/28, SEQ ID NO: 29 / 30, and a primer mixture containing one or more primer pairs selected from the group consisting of SEQ ID NOs: 31/32, and in a preferred embodiment of the present invention, the primer mixtures of SEQ ID NOs: 17-32 It may be characterized by containing all the primers.

In the method of detecting promoter methylation according to the present invention, the step (d) is performed by electrophoresis of the amplification result of the step (c), the PCR result in the mock DNA, in the DNA treated with Hpa II If there is a PCR result, the promoter may be determined to be methylated, and if there is no PCR result from the DNA treated with Hpa II, the promoter may be characterized as not being methylated.

In addition, the step (d) is a hybridization of the amplification result of the step (c) with the cancer diagnostic microarray, the promoter is methylated when both hybridized in the mock DNA and Hpa II-treated DNA It may be characterized by determining.

The term 'clinical sample' used in the present invention means not only tissues and cells derived from a subject to diagnose cancer, but also sputum, feces, urine, membranes, brain water, amniotic fluid, eyeballs, organs, blood, and the like. Another term 'colon cancer specific expression reduced gene' means a gene whose expression is specifically inhibited in colorectal cancer. Another term 'mock DNA' refers to sample DNA that has not been processed and is isolated from clinical samples.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not to be construed as being limited by these examples.

Example  1: in colorectal cancer tissue Methylation  Selection of gene groups whose expression is reduced

Using five colorectal cancer tissue samples and five normal colorectal samples adjacent thereto, a human 17K cDNA microarray experiment was performed by indirect comparison method to select a group of colorectal cancer-specific reduced genes.

100 μg of total RNA extracted from 5 colorectal cancer tissue samples and 5 normal colorectal samples was labeled with Cy5-dUTP fluorescent dye, and common reference RNA mixed with total RNA extracted from 11 cell lines (Korean Patent Application No. 10-2003 100 μg was labeled with Cy3-dUTP fluorescent dye, and then hybridized to a human 17K cDNA microarray (GenomicTree, Inc., Korea), and the expression changes were analyzed. ANOVA test (p <0.05) was performed to select 1,312 genes that significantly reduced expression in colorectal cancer tissues compared to normal tissues adjacent to it. To verify that 1,312 genes are regulated by methylation of the promoter moiety, DAC (5'-aza-deoxycytidine), a demethylating agent that inhibits the methylation of the promoter moiety, was treated. Colon cancer cell lines Caco-2 and HCT116 were incubated, treated with DAC (final concentration 1 μM), and total RNA was extracted with a control (non-drug group), and a human 22K oligo microarray experiment was performed. First, the cell lines were suspended in DMEM (10% FBS, 1% penicillin, 1% streptomycin) medium and incubated at 37 ° C. and 5% CO ₂ . The cell line was treated with DAC (final concentration 1 μM), and total RNA was extracted with a control (non-drug group) to perform human 22K oligo microarray experiments. 100 μg of total RNA of the DAC-only group was labeled with Cy5 fluorescent dye, and 100 μg of the corresponding control was labeled with Cy3 fluorescent dye, and hybridization was performed on human 22K oligochips (GenomicTree, Inc., Korea). Hybridization at 65 ° C. for 16 hours, followed by washing, scanning with an Axon Scanner 400B and signal intensity of each probe with a GenePixPro 4.0 program.

In this experiment, the genes inhibited by methylation were expected to be re-expressed due to the promoter methylation phenomenon disappeared by the above experiment. As a result of the experiment, the expression of 280 genes was increased by 1.5 times or more in the cell line treated with methylation inhibitors, compared to the control (Fig. 1). Among the 280 genes, 43 genes whose expression was reduced in colorectal cancer tissues compared to normal tissues were selected as gene candidate groups whose expression is reduced by methylation of promoters in colorectal cancers. The presence of CpG islands in the promoter region of 43 genes was examined using the MethPrimer program. As a result, it was confirmed that CpG islands exist in the promoters of 33 genes.

Example  2: 20 genes Colorectal cancer cell line  Methylation Verification

Genomic DNA was extracted from two colon cancer cell lines cultured by the method of Example 1, and 400 ng of DNA was treated with 20 U of Hpa II and 40 U of Msp I, respectively. After purifying the DNA treated with the restriction enzyme, PCR [(94 ° C. 1 min → 66 ° C.) was performed on 33 genes using the primers of Table 1 together with sample DNA (mock DNA) not treated with the restriction enzyme. 1 min → 72 ° C. 1 min) 30 cycles].

Each PCR result was electrophoresed to verify the methylation of the promoter. Since the gene in which the promoter is methylated is not cleaved by Hpa II, the PCR result is obtained, and the gene in which the promoter is not methylated is cleaved by Hpa II and thus is not PCR. Msp I restriction enzyme was used to verify whether the activity of Hpa II restriction enzyme was inhibited by the methylated promoter. Msp I, so cutting to recognize the same nucleotide sequence as the nucleotide sequence of the Hpa II restriction enzyme recognition regardless of methylation can be methylated verify the specific cleavage reaction of Hpa II restriction enzyme.

Electrophoresis of the amplified PCR product, in the sample treated with Hpa II determined that the PCR product is methylated and marked with a black block, the case where the PCR result is not seen in the sample treated with Hpa II because it is not methylated Shown as gray blocks (FIG. 2). As a result, as shown in Figure 3, it was confirmed that the promoter of the selected 15 genes are methylated in at least one of the two colorectal cancer cell lines, in order to confirm the promoter methylation of the eight genes by another method The promoters of the eight genes were also identified by bisulfite sequencing. Treatment of bisulfite on DNA turns unmethylated cytosine into uracil and methylated cytosine does not change. The genomic DNA of the colorectal cancer cell line HCT116 1㎍ using MSP bisulfite modification kit (In2Gen, Inc., Korea) bisulfite modification (Sato, N. et al . , Cancer Research , 63: 3735, 2003). Bisulfite-treated HCT116 genomic DNA was amplified by PCR using primers SEQ ID NOs: 25/26, 29/30, and 31/32, and then analyzed for the nucleotide sequence of the PCR product, resulting in dog genes (NR4A2, A2M). Cytosine is detected in all parts of the nucleotide sequence of the SEPP1 promoter including the 8 genes (ID3 (Inhibitor of DNA binding 3), RGS2 (Regulator of G-protein signaling 2), WISP2 (WNT1 inducible) signaling pathway protein 2), monoglyceride lipase (MGLL), carboxypeptidase M (CPM), gamma-aminobutyric acid (GABA) A receptor (GABRA1), CLU (Clusterin), and Coagulation factor II (thrombin) receptor-like 1 (F2RL1) It was confirmed that the promoters were all methylated.

In order to verify whether the promoters of the 15 selected genes were methylated in the normal colon tissues, it was confirmed whether they were methylated in 3 types of normal tissue DNA (Biochain). It was confirmed that no methylation at all in the normal tissue (Fig. 4). 4 shows that expression of 15 selected genes is increased by 1.5 times or more in at least one of two colorectal cancer cell lines by demethylating agent.

Example  3: diagnosis of colorectal cancer of 8 genes Biomarker  Verification Experiment

In order to confirm the clinical applicability of the methylation promoter to the eight selected genes in the present invention, the same patient's colorectal cancer tissue sample and the normal tissue DNA adjacent to the verification method for methylation in the same manner as in Example 2 It was.

As shown in FIG. 5 and Table 2, it was confirmed that eight gene promoters were methylated at high frequency in most colorectal cancer tissues.

Gene name SEQ ID NO: function ID3 One inhibitor of DNA binding 3, dominant negative helix-loop-helix protein RGS2 2 regulator of G-protein singalling 2, 24kDa WISP2 3 WNT1 inducible signaling pathway protein 2 MGLL 4 monoglycerine lipase CPM 5 carboxypeptidase M 12q14.3 GABRA1 6 gamma-aminobutyric acid (GABA) A receptor, alpha 1 CLU 7 clusterin (complement lysis inhibitor, SP-40, 40, sulfated glycoprotein 2, testosterone-repressed prostate message 2, apolipoprotein J) F2RL1 8 coagulation factor II (thrombin) receptor-like 1

Example  4: of eight gene promoters Cancer diagnosis As a marker  Usability Verification Experiment

In order to verify the usefulness as an early diagnosis biomarker for the eight kinds of genes selected in the present invention, it was confirmed whether methylation was performed in the same manner as in Example 3 with respect to normal tissue adjacent to colorectal cancer tissue. As a result, the promoters of these eight genes were confirmed to be methylated in tissues adjacent to cancer tissues (Table 2).

These eight genes are not methylated at all in normal human DNA, not colon cancer patients, and the methylation of the promoter in normal tissue adjacent to colon cancer tissues indicates that methylation proceeds from the early stage of colon cancer development. It is very useful for early diagnosis.

Methylation Frequency of Methylation Biomarkers for Diagnosing Colorectal Cancer in Eight Colon Cancers, Tissues Adjacent to Colon Cancers, and Colon Cancers Biomarker Methylation Frequency (%) Colon normal tissue Normal tissue adjacent to colon cancer tissue Colorectal cancer tissue ID3 0 20 20 RGS2 0 7.5 7.5 WISP2 0 52.5 52.5 MGLL 0 25 42.5 CPM 0 50 52.5 GABRA1 0 22.5 57.5 CLU 0 52.5 55 F2RL1 0 25 32.5

As described in detail above, the present invention has identified eight colon cancer specific expression genes and their promoters, and found that they have a function as a cancer diagnostic biomarker that is widely applied to various clinical samples. . The cancer diagnostic composition containing the methylated promoter of the colorectal cancer specific expression reduction gene according to the present invention is expected to contribute to the improvement of the cure rate of the cancer through not only early diagnosis but also prognosis prediction.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> GENOMICTREE <120> METHYLATED PROMOTERS OF COLON CANCER SPECIFIC          EXPRESSION-DECREASED GENES AND USE THEREOF <160> 32 <170> KopatentIn 1.71 <210> 1 <211> 1830 <212> DNA <213> Homo sapiens <400> 1 gatctggggt gctgccagga aaaagcaaat tctggaagtt aatggttttg agtgattttt 60 aaatccttgc tggcggagag gcccgcctct ccccggtatc agcgcttcct cattctttga 120 atccgcggct ccgcggtctt cggcgtcaga ccagccggag gaagcctgtt tgcaatttaa 180 gcgggctgtg aacgcccagg gccggcgggg gcagggccga ggcgggccat tttgaataaa 240 gaggcgtgcc ttccaggcag gctctataag tgaccgccgc ggcgagcgtg cgcgcgttgc 300 aggtcactgt agcgggactt cttttggttt tctttctctt tggggcacct ctggactcac 360 tccccagcat gaaggcgctg agcccggtgc gcggctgcta cgaggcggtg tgctgcctgt 420 cggaacgcag tctggccatc gcccggggcc gagggaaggg cccggcagct gaggagccgc 480 tgagcttgct ggacgacatg aaccactgct actcccgcct gcgggaactg gtacccggag 540 tcccgagagg cactcagctt agccaggtgg aaatcctaca gcgcgtcatc gactacattc 600 tcgacctgca ggtagtcctg gccgagccag cccctggacc ccctgatggc ccccaccttc 660 ccatccaggt aagcctcgaa gtcgggacag ggctgaacac ccaggcaagg atgctgcggg 720 accctcggag ctcccgattg cctcgcgtaa ctcttccctc ttttcctcta atcagacagc 780 cgagctcact ccggaacttg tcatctccaa cgacaaaagg agcttttgcc actgactcgg 840 ccgtgtcctg acacctccag gtgagtatct cctctcttgg agagggaggt ttaaacggca 900 agtcctggag ttggcagacg ttttgaaaaa ttgccactca ctcggtttag ggaaactgag 960 gccagagagg gacaagtgac ttgcccatgg ttgcatcaaa tgaatggcag agtcagtttc 1020 catgtgatgt gcatttaagc cttaaatgcg cctgggccct gccctcccgc agtggccgag 1080 ggtctggcaa agtagacatg ggtccgacta aatacaagtc tttctgttcc atgttgtata 1140 gggagctgtc ttcggcagcc ccctcccagc tagtgtcaat tccaagtagg aggggtagcg 1200 caagctccgc ctgtggtctt tggcgccaac tgggtggggg cagcgtgggg cgcggagtta 1260 tcagctggag gtacagacca agtttcctcc ctggcgccgg ccagtctgcg gacggccccc 1320 gcctcggcac gctcggcgga aactgactgc tccttggtct tctttcctcc cccgcccaga 1380 acgcaggtgc tggcgcccgt tctgcctggg accccgggaa cctctcctgc cggaagccgg 1440 acggcaggga tgggccccaa cttcgccctg cccacttgac ttcaccaaat cccttcctgg 1500 agactaaacc tggtgctcag gagcgaagga ctgtgaactt gtggcctgaa gagccagagc 1560 tagctctggc caccagctgg gcgacgtcac cctgctccca ccccaccccc aagttctaag 1620 gtctcttcag agcgtggagg tgtggaagga gtggctgctc tccaaactat gccaaggcgg 1680 cggcagagct ggtcttctgg tctccttgga gaaaggttct gttgccctga tttatgaact 1740 ctataataga gtatataggt tttgtacctt ttttacagga aggtgacttt ctgtaacaat 1800 gcgatgtata ttaaactttt tataaaagtt 1830 <210> 2 <211> 1868 <212> DNA <213> Homo sapiens <400> 2 aaacagccgg ggctccagcg ggagaacgat aatgcaaagt gctatgttct tggctgttca 60 acacgactgc agacccatgg acaagagcgc aggcagtggc cacaagagcg aggagaagcg 120 agaaaagatg aaacggaccc tgtgagtatg gctttcttcc ctctcccgcc accccctgcc 180 ccacactgca agctgcaaac gcggtacttt cgggctcgcc tttgacgtta ggaaactagc 240 ctgagcctat gcagggaaaa aaaatcgaaa aggtcaattt gttaagtaag gttaaatctg 300 ggtgatgctc gggtacagtt taagaaccga gggagacagt tgatatgagg gcggtggttg 360 atgcgctaag aaattgcggg ttggcttttt gtcctcctgc attcaaaatg acatcagaat 420 cctgcggctg aagcgcgtcc ccagcattca tacgttgcat gatgagttct catcagctta 480 cacagctact ggaaggtgat gctcttgctg gttctgaata tactcgttta aaatccattt 540 ttgtttttta attatagagc agatctcacc cagtccgaat gtggaacaaa taattgttat 600 gcagcgtctg cttaaaagaa gtgtcgtagg tggggaagga agagcgcagg ggaatcagtc 660 acccacctct ttgtacagtc tctggcgtgg tccagaacct cctgctctaa agagagaagc 720 gtgggccggc tccagacagt tccatgtctg tccttttcat taaagtgcaa aacgtctcgg 780 aattgtaatt aaccttgcaa acaaactgat gccctttgtg agccagaaat agtgtctgcc 840 ttttgaacta aattcattaa caattcttta aaatacccta gtgattatag gtagccctgc 900 ccttagttgt aaaactagta gatacggtca gattaatgga cgaaactgct cagtacatga 960 ggtttaaatg ttaggtggat aagacttatt tgaagagttc ttgctttgcc ttatgcggtt 1020 tgtctctagt tactgggtga ctttatttgg taaaaatgcg ttcagctgca gtagcatatt 1080 caagtgttgc tagttagtaa ttatcttttt aattttttgt tttagtttaa aagattggaa 1140 gacccgtttg agctacttct tacaaaattc ctctactcct gggaagccca aaaccggcaa 1200 aaaaagcaaa cagcaagctt tcatcaagta agttgagaat cctgtgcttg caaatatcaa 1260 tagttagctg ctgaactgaa aaggggaact ctgatgtgcg taagctaaca tacagaacct 1320 ctcttgcagg ccttctcctg aggaagcaca gctgtggtca gaagcatttg acgagctgct 1380 agccagcaaa tgtaagttaa ctcttgagct tgagccattg ctaacatcgc aaaagcctgg 1440 aaaggctgcg tccacctaac aaaagagcag cttgcctgag ggggattaga ctgcagtcac 1500 tataggataa agcctgtttt tctttccttt atttcccagg gtttaacaaa taaatgcata 1560 tattctctcc aggtggggaa gaaaatcagc ctaaacaaat taaagtggca gttgcttata 1620 gttaaggttg agtcagtttt tccattgcat acaatgtttt caagaggctt agttcccaga 1680 gaatttatgg ctcccctata aatatttact ttgcattgac agcaaagtac ttatattttt 1740 gcagcagagt gccaacataa gccttttgcc tactggtatc ttagtcttta aaaagctaaa 1800 ttttgagaat tacaggtttg agcgaaatct agaaaattca tttagaaata attaaaatgt 1860 gagggata 1868 <210> 3 <211> 1870 <212> DNA <213> Homo sapiens <400> 3 tgtgtgtgtg tgtgtgtgag cgcgcgcgcg cgcgcgcgtg tgtactcgtg cgtgtgcctg 60 tgtgtgcctg ggagtgacct cacagctgcc ggaacataaa gactcacagg tccgcctccc 120 aggctcaaag ctggctctgc aggggacatg agaggcacac cgaagaccca cctcctggcc 180 ttctccctcc tctgcctcct ctcaaaggta aggaggcccg ggccctggaa tgcactgctg 240 actatttggg tgtggagggg gtggggatgt aaaatcgcag ccaaggaccc ccttggcaga 300 attcctccag ggccccacat tgggcacagt ctggccccca tgcctaagca gggcttctct 360 ccctggaact cagagctgcc tccggccagc cctgctgtgt gacctcagat atgtgtcttg 420 ccttctctgg gcctcagcat cttcccaggt gaagggtata cagtttgtga ggcctctgtg 480 aagaccattc atgatcattc atcaaactta ccaggcaaat tgaagaggac tttaatgtat 540 cctcattatg aatgggggca ctcaagacat gacatgcttg gcctaactct gagccagcat 600 ttggatggat gcctagctga ttggaaggtt cattctgcaa gggcatcaat gctgttggac 660 tcttaggcag tgagtgaccc gcccgcccta gctagagggg aacaggagac acctctgtgg 720 agtaatggcc ttatccctga atgctgcatg ggacattcac caaagagcct gtgggacatc 780 taccttgtct aacatgcaca acaatcccat aaggaatttt cagttaagtg aggctcagaa 840 agggtaagca acttgcaacc ccagttttca gtcaagtgag gctcagaaag ggtaagcaac 900 ttgcccaaag ccacacagca aagaaaggtc agagcaatag aggagagcca tggtggagcc 960 cagaatgaca tggatgtagg gggagaagca ctccagtctg tgcctcccca agcccaaact 1020 ggaccaaggc cacagctgct aagccttgtc atcccctcgg caaatgctga gttcccagag 1080 ccatggaatc ggccccacca caagccacga tctcactgta ttttcacgct cctttgagga 1140 gcacgggcag gagcttctct ccctcaggct ccaagtcttc gtgcctggga agcctgagaa 1200 ccacgctggc atcctgtcct gctgtttgtt cttccaaagg ggatgggtag ggcatgatgc 1260 cagtgtgggt ggcttgggta gttggaatag gggacttggg gctctaggag ggagaagggg 1320 cccaggatta tacgagaaca aagtaaagct gtcctagact tgccattagg gtgagactta 1380 cgggaccatt cttccctggt cccaggaagc agggactgca cagaaacaca gggacttagt 1440 caccacaaca tggaaagcca cagcaaccct cagagggaga actgtaagtc ttccaggctg 1500 gccgttggct ccggggctcc tggctgtgga ccggggcagg aggcagacac ctggacacaa 1560 gcaaaatcac attgatcaag gactggccag aacctcattt aatctccatc atcactcaga 1620 aacaggttgt ctctactcca gtttacagag gagaaaactg agcttcacct ccaccttcca 1680 aagccgtttg tccacatgtt cagtcacgta gcaagtcact ggcagaactt gaatctcaac 1740 tcttgcccaa aaaactccat gcccagtact cctgccccag atatcatgag ccttgccccc 1800 caccaccaga tatcacagca aggccctggg gaaactgagg cacaatggga caacttgagt 1860 ttgggccttg 1870 <210> 4 <211> 1834 <212> DNA <213> Homo sapiens <400> 4 tggcggcgca gacgccgcac cctgtcgccg cgaagccggt cgcgcgcagc tcgtcccggc 60 cctggcccgc cgcaaacgag gatccgctgc gctcggggaa cgcgacagcg gcgctcgtgg 120 ccccggtaac tgcctcccgg cccctgcccc agcccgccag tgccgccgtc aggggtgggg 180 agcagcactg gggtcccgga cacgcccggc gtgggctccc gctacccatg tgtgcgtgtg 240 tgtgtgtgca cggtgtgtgt gcgcgcgtgt ccgagtgtgc gcgcgtgtgc gcgcgcgcgc 300 gccctccctg ctccttcgca ccagccccca aaccaacttg tccccatcaa ctccatcttt 360 ctagtcccca ccttccccgg tgcagacacc cggcgaagcc cacccggttt tcccagcggc 420 atttccgatg acagcttcgg ggctacgtgt cctgtgctgt cggagacgca caggaagcaa 480 agtttgtgag aagccttggg ggcgactttg ccttgggcac ccgcatttgt gcgtctgcga 540 ggtgcctcgg tgtgcgcgga gctagtttcc cagtttcccg ggcccctccc ttctccgagc 600 ccctctagcg atttgtttag gaaaagtgat gacatgaact agtagtggag aatcgcagcg 660 ccgctccccg ccctggggag ggaggggagc cccggagagc ctgccggtgg gagctggaag 720 caggctcccg gctgagcgcc ccagcccgaa aggcagggtc tgggtgcggg aagagggctc 780 ggagctgcct tcctgctgcc ttggggccgc ccagatgagg gaacagcccg atttgcctgg 840 ttctgattct ccaggctgtc gtggttgtgg aatgcaaacg ccagcacata atggaaacag 900 gtttgtcatg tatccttgga atttcagatg ggaggaataa tctgagtatc atccacctca 960 ttattgtgcc cacttgaaac tgttccttgg agattttaag atagatgggt cttcttgcag 1020 ctgggtcttt gtacccatag agctgactat aatttccact tgattacagc taaaatagtc 1080 tttaagatga agtttcctga aatgatttaa aataaacaca gatgggaagg gggaaaaact 1140 gtaatgaggc acttaaaaaa taaagtgagc attttcttaa gaacaaatgg gatttttcag 1200 tcttggagag gtttgcaaga aatgtatgta tgttttaaac ctttgaaaca gaggggtttt 1260 ttaaactgct tttaaaaaaa tgttactgac tctgaactag acttttctct cctatccata 1320 catgttcttg agatatttct gactttggaa tatgtcactt ttccttctag gacctgaaga 1380 cccttccagc atgccagagg aaagttcccc caggcggacc ccgcagagca ttccctacca 1440 ggacctccct cacctggtca atgcagacgg acagtacctc ttctgcaggt actggaaacc 1500 cacaggcaca cccaagtaag tcttccccag aaagtcaccc caggtgacag gggagcatgg 1560 cccctgcttc tcctctggtc gttatcatgt gccatctggg gcggggccgc tttctctatg 1620 acctacttcc cctgccgttt taatgttagg ctgagcaatg ttaacaaagc taaaatgtat 1680 actttataat ttttaaattt aagaagcagg aattcttttt tcattataac aactcagata 1740 tggatcctac cgtatggcaa ttttcaaaaa ttccacatga ctttattttt tgtactggag 1800 ttgatttccg gttgtacagg cattcattta aaat 1834 <210> 5 <211> 1028 <212> DNA <213> Homo sapiens <400> 5 gcatttcttc cttctgcgta tgggacagga ccctttctgg aatgggggtc ttatgaccta 60 caatcaaaca aggtaggtcc gatcatttcc ttatgaccag tttttataca gaatgtaaac 120 caaaagtata tgagacaggt cccgatcaat gtagaaagtt tactttgcca aggttaagga 180 cacacccatg acacagcctc aagaggccct gcctatgtgc ctaaggtggt caggccacga 240 ctttgtttta tacattttag ggaaacatga gacatcaatc aatacatgta agatgtacat 300 tggtttggtc cagaaaggtg ggaccactca aagtggggac ttccaggtca cagggagatt 360 taaagatttt ctgatttgca attggttgaa agagttatta tcaatagaaa ggaatgtctg 420 ggttatgata aaggattgtg gagacgaagg ttttatcgtg caggttaagc cttcagagag 480 aacagattgt aaatgtttct tatcagactt aaggagtctg ttctatcaac aattctaaaa 540 ggaagagggt acaatgaggc atgtctggct cccccttccc atcatggcct gagcttgttt 600 ttcaggttaa ctttgggatg cccttgccaa gaggaggagt ctgttcagat ggttggagta 660 cttagaattt tatttttggt ttacaagaaa ggcggatgaa aagtttgata atatttttag 720 gttttatagc tggctttcgg gaaaaggggt tctggtttct aggacccacg tggggaagag 780 ggattctagt ttctatggct gccctcgggg aagagtggga ctgagagaca ggaggcgagc 840 aggagaaggt cagagaaaac cttttgcttc tcaggctgct tctgaggcct tcattttagg 900 gtgttgtttt ccatgtccca acagtgacac aacagtgtga atgtacttaa aactactgaa 960 ctgtaggttt aaaaatggtt atgatggtaa attctgtgtt atgtgtgtat tatcacactt 1020 ggtttttt 1028 <210> 6 <211> 1827 <212> DNA <213> Homo sapiens <400> 6 gcacaattca gaggtaacag cgcctgcgtt ttctccatga taacatagac aaacagttgc 60 ctccaaaggt aaaaacccgg ttccggaggg tattcttgct gtcctctaag ctacagaaga 120 aggtggggtg gaaagcgctc ccaacctgag aaagtgcgaa agactctcta aatccagtag 180 ccaatttcct acctgccaga actctttcct accatccgtc accattcgtg actctgcaag 240 acacggggac ttagacatat gtgtgggagt gtgtgtgtga gggagagagg cgagacgagt 300 gctctcccca cacgtgtaac cctgcatctc ccagtttcag agcttgcgtt cattcatatg 360 caggcagtgg tttaatattt gatcaaaggt agaatagttg cactaaatag ttgtattttt 420 aatgggctca ccttaaaatc gggtgttcat aaattgcaag tgtgaaaaga ctctaagtgc 480 agtccgtatg tctctcaccc tttctaccct tccctccccc attgccgctc cccccaaaaa 540 gagaagcttg cagcagattg tagaaggatt tgagcctgca gctagagaga aggggattag 600 ggccagagtg gtgcaagtta aattgtgctg catataaaaa atgggcggat tggtctccag 660 atccagaggc tggtaccacc ttcctttcta aaataaaatc tctctggcat gaagtcaccg 720 cctatttcac atccggtttg ccctgggacg tattactact gtcttggtaa agagaaatct 780 tttgttgtat agctgcagat tggatattgg gaagcaaatt tgggtgtgaa atcttcagca 840 aaggagcacg cagagtccat gatggctcag accaagtgag tgagaggcag agcgaggacg 900 cccctctgct ctggcgcgcc cggactcgga ctcgcagact cgcgctggct ccagtctctc 960 cacgattctc tctcccagac ttttccccgg tcttaagaga tcctgtgtcc agagggggcc 1020 ttaggtaagt gcgactttgg accacgatac acagacagag ctttgaacgt ggcttttcct 1080 agctggagag acaggaatgt tatagtcggg gggggggggc ggggggagac ggggggagag 1140 agagagagag agaagaagga gagcgagcga gcgagcaagg gagcgagcag gacaggagcc 1200 tgatcccaca gcaggtagaa acaacgaaat gtttttcttt cagggtgaaa ccgcagcaat 1260 ataagagcat tattattatt tttttcccgc tgtctttctt cttttcttct cctctttctt 1320 aggcatattt gaatgtattc cgcagggtgg gtggtggcat gttgttttgg tcttttattt 1380 atttatttat ttgcaagggg gagctggggt gagttgggga ggggcgtcgg gggccatcat 1440 ctaaagttaa gacgctggga cggaaaacct gtctcactgt tccatcatcc tagggaaggc 1500 acgctcttgt cctggctgca aaattgcctg tttttccctt tccagtggac gctggtgaaa 1560 tgccctcttg tgtaggggtc tctcccattc tgaacacgaa aactcaactt agtctttgat 1620 atggatggca tgggatggta acgtttggag cgtgtgtctg ggggaggggg aggttgaagg 1680 tagaaagtgg taagactgca gcttagcctt tcttttaaac tattatgttt gtatttgcaa 1740 gaaactttct taacaactga agtgccatga taggtttgtt tttctctttt aaaaatgtca 1800 gaagagggca aaaatctatg gtgcctt 1827 <210> 7 <211> 1913 <212> DNA <213> Homo sapiens <400> 7 ctttccgcgg cattctttgg gcgtgagtca tgcaggtttg cagccagccc caaagggggt 60 gtgtgcgcga gcagagcgct ataaatacgg cgcctcccag tgcccacaac gcggcgtcgc 120 caggaggagc gcgcgggcac agggtgccgc tgaccggtga gatgtccccg tcttccctac 180 ccttgagcag agccacacca ggacggatgg gcgggcaggg gatggcagcc aggcagagag 240 ggatgacaca gctcgcagtc acaacccctg cgctttcgac ggagcccagg aagccaggga 300 ggggaggtgg ccggagcccc atcaccaggc agctgagcca ggggccccgg cgcaaccgcc 360 gcctgagagc acgagctcca accacaattc tgtggtgggg gggtaaatag aacagatata 420 atgatcatcc tttcgcaaag atggggaaac tgagacctgg agacctgccg cgttgcggga 480 gacccaggct agcaggtgac agagctggcc tgcaccgagc tccttcctgc agcatatcct 540 ctgcgaagat gcggatctct cagttgtggc tttcggcttg catgcatgag tcatctagtt 600 ttcttctaaa ttctctagct ctctggacac tgttgcctgt aagtatgagg ctgcggattt 660 cagtatatgc tgcaaccacc gaaatccgac tttttctgcc tcctaatgca tctgaggtgc 720 atcagagaaa agtcacacaa gatccaccag gcctcagacc tctgattcca cagtctcatt 780 ttacagatga taatctgagg cctggagagg tttaggactg gtgccaacac taaacagcaa 840 ataagtatca gaattgggat tcgagccaaa gcctcttgac cttccagaat ttctggacct 900 agttaaaaaa aatatgattt ttattattat tttttaaacg gagaggttag gaatttaaag 960 gaaagtacag atactatata aaaaaagatg cccatgaaaa tgttaagtta taataatagt 1020 ggagcattgg gcacaactga aatggccaat cttgtgagaa tggtaaaata aacttaggtc 1080 cgtgagtaag tggagtatta catagccata aaagtatgcc cttaaagaat atttgaagat 1140 ggtgaatgtg aagaatcttg tataaactgc atggaagaca gaaggaaata taccacagtg 1200 ctaacctttg cctctgggtg atatgaatta ccggtgatta tttttcttat tttccttttg 1260 gtttagtttt ctccatttga agaagcagat aggagccggg gctttgggat tgaaaccctc 1320 accatctgtg tgccctcttc actgtcttcc catcctcccc acggctccct gttcacagtc 1380 attgattttc tttctttctt ttctctcttt tttttttttt ttcctgagac caagtctcac 1440 tctgttgccc aggctggagt agagtagcgc catctcggct cactgcaacc tccgccatcc 1500 gggttcaagc agttctcatg cctcagcctc tgagtagctg ggactacagg cgcatgctgc 1560 tacatccggc taatttttgt atttttagta gagacatggt ttcaccacct tggccaggct 1620 ggtctcgaac tcctgatctc aagtaatccg cctgtcttgg cctcccaaag tgctggggtg 1680 acaggtgtga atcaatgcgc cctgccaggt cattgatttt cttaagcctc cagccctgcc 1740 ctgcttggaa acgttttggg aagctgctca gttcaaagtt cccaggaggg tgtgcctgga 1800 ggggagttgc tcccaaagtc tgcctgctcc ccccgccccc cctgcccccc accccccgcc 1860 atcttctcct cctcctcttc ccctgagcag cccctttgtc cacagaaccg gcc 1913 <210> 8 <211> 1706 <212> DNA <213> Homo sapiens <400> 8 tgaaacctaa cccgccctgg ggaggcgcgc agcagaggct ccgattcggg gcaggtgaga 60 ggctgacttt ctctcggtgc gtccagtgga gctctgagtt tcgaatcggc ggcggcggat 120 tccccgcgcg cccggcgtcg gggcttccag gaggatgcgg agccccagcg cggcgtggct 180 gctgggggcc gccatcctgc tagcagcctc tctctcctgc agtggcacca tccaaggtga 240 gaaacctggc caaggagggc tcttatctct gaggagctgg ggtcctgggc acgctgggca 300 gacggtggga tccgggcagg tgtgcgaagg ctgttctgct gccggcaccc atctctacga 360 atcccttagc ctccccttgg tggctttgat gtgaggtctc tgcgccaagg aggcacccag 420 gtgggacatg cgggagcagc tgaagtcagc ggagccgggc agaggcagag atttccccaa 480 agaccccact tcccgtagag gctgtcatgc tgggcacctc caggccccag cgtgggttcg 540 gggaaaggga gggaaggaag gagtagctgt gtctaaaggg tacgcagagg acctagtccc 600 ctccccgggc ttggttgctt tgtaaacact aagtcattaa gagctttttg ccaactccgg 660 agacttcctc cgctggcccc gcggggccgg tggggttagc gagctgattg cgcagggcag 720 ggcggagtgg gtgggaaccc gcggcccggg agccttctag gttgtttttt gcgtccctct 780 ccaactgctg gaagcctttc ttcgggttct ggcccctgcg ttcgagaccc acccgggtcc 840 cgctgctcct ggcacccaca gtggaaggcc cggggtatga aagtcagggc cggtccctga 900 tgggttctgg tggtaatgag cgctcaggcc cgttgggcgc cacgaggatt tacgagcgga 960 actttttgga ccctgcctta tatttgacca gttgaccctc tgggcctggg gttccgggac 1020 tttcctagtg tgccacccgg agcggccact ggctgccacc caggtctgca ggccagaggc 1080 tggctggcct gggaacagag cagcacttgc gcccggccgt cgcgttcctt ggatgtgctg 1140 ctccgggtgt gctcgaactt gtattgctgg gagtgtgttc ttccgggccc agcaggcctc 1200 gctgtggccg tgggggatgt ttgacctgcg atcgcgccac tgtgatccta accgaggccc 1260 ctctgtttgg ggcagcctgg gtagcaaagg gagcttttaa tttccctcag aaatgcgcat 1320 ttctcacttt tatcctcctc ttcataaaga cgaagtaaaa gagcaagtca cagtgggttg 1380 gaattagatg gaagaatgtg attcttttcc ctctgcagtt agggaaaagg gtgttaatgg 1440 tgtaagcttg aaaactgtgc accgctggta tatttttttt tcttaatcag catcagaata 1500 aaggcattta gttccaggaa gtattgaaaa ctgtaatgga cacctctcct ctatgtcctt 1560 taaagttgga aacttaaaat tcaggagcaa aggttagtat gtgcaaactt taaaaaaata 1620 catgttggta tagctgattc aacatcttta tgattcagag atccctgtag gaagcatgcc 1680 ccgattattt gtcttgttct tgttta 1706 <210> 9 <211> 1700 <212> DNA <213> Homo sapiens <400> 9 gcatttgcac agaaatctgc ctggcccgtg atttttctag aaccctctgg acttgaccgt 60 tttgccacag aagtctgcaa gataggaaaa gaaggatctt ctcttcggtt attcaggagt 120 tcctacagcc attattttgc ctttcctgac caaatagctg gcacttgacg gctgtagcat 180 gtaaagactg acttgcccac tcacaggttt tcttcctctt ttcttactga agagcctgtc 240 cttgaaaaaa acattccagg aagaaatctt tcttgggcca cagagccctg tggtttgatg 300 gaaaagaacg aggcttcaag actggctctg ctattacttg tagtgtgaca ttgggcatgt 360 cttaaatgcc ttccttgggc ccctcagcat ctttcaccta catatccaaa tttcacacaa 420 ccttggactt ctacaattgg aaaggatttt aaataatcac ttagtaccat cgtctatttt 480 actcacaagg caattaaagc cagagtacct gggcttgttc aagattacat gacaaattat 540 tgtcagggct agcatgatga acccagggct ctcaattccc agtcagtgtc cttttcaacc 600 gttttgcgac tctctttggc tctgagcccc cttgtcctgt taatcagtgc cacacattat 660 cgaactcttg tttcctgtgt taatcttgtc tccccaaatt gtgagattct tgaaagcagg 720 agccactgtt ttatgcggag ggactccata aatattcacc aatgattcat taggaaagca 780 actattgggg gaatcccact tggtcatttc caaaatggaa agcagcatct tggaagtgca 840 ggtccccaaa tgtctaggga ggtgacaaga aggcaagcaa agaatcaaag aatactgagc 900 acagtgtcct gttgcaatac aggacttagt ttgccggccc tctggttcct ttcctggtct 960 ttcagatgga gctggagcta cttcagcagg gctaagaata atcaagtcca ggtggcctgc 1020 ccttccatag cagaaaggga cagacaatag agcaatgaaa ttaccactgt tttctacaca 1080 gaggtgtgtg gaaagtgaag agaggggaag ttaagaaatt tgctgaccca attgggtggg 1140 ggggggctct taaaaaggtt tatgtagaaa ttgtcccatc tgtgcctcca tatttaaaac 1200 cttgtccaaa attctaattg aactcacaaa gcacaactgt gcatatattt agttcgttat 1260 ggagtttatg gggcagaggg tccaggctcc ttttttcccc atgcaaaaag catgggctca 1320 agctttcttc ttttcccctg ttgctcaaat aaatagtgtt ctttgctcaa accccctttc 1380 cctcctcctt ctgcaatctc agcgcctagc gcaaatctgt tttcttcatt gtaacctcag 1440 cttcaccgca attaattttt tttccctctg gtcacaagat aattcctgac gccagtgagt 1500 ctggaggtca gacgaacagc aaattgggga acaaggcggc actaattcct tacaagtttc 1560 ccttgaaaaa tctttcgctt aaaaaaaacg gggggtgggg ggagcttctt tgctgttcag 1620 ggatttatga cctcggagga gctgtggctc gaaccagtgt tgggctaaag gcggactggc 1680 agggggcagg gaagctcaaa 1700 <210> 10 <211> 1700 <212> DNA <213> Homo sapiens <400> 10 cctctttgtc agaactcctg gcctgccagc gaatttaact gtctatgagt aaattctaac 60 atctaaggtt ctaagtataa aatatattga ggtttacagt ggtgagaaaa agcaagtgtc 120 tgcgtattac agacagctga taaatacgta ttgagtcatc agaaagtgaa aatagatgta 180 ggactattat gcaaagcata ggagaggaag agcatatgct taagaaaaaa tggcattcct 240 agagttaaca agcatgtttc aaagtatcta ttactgaaat ctacacgatt ttcctttcca 300 caataaagtc cacaactgtt gtcttaaata ataactgcag gttttgccct ccaagtcaaa 360 cttggagctc caactactgg cagctcaccg taagaatata ttaattattt ccaatttcct 420 tttaagaagt cgaatacaaa gtagagagaa tatctgcagc ttggttgcaa agccgcttaa 480 acatttcact ggctagttct ggtaatatta tccgaaatca actcattttg tggttgaggt 540 aatctatttg tctttaccaa tcaagtcgtt ttaaaacctt caggagtaga gatgatactt 600 gaagaattaa caaataaaac ttaatcaagg aaactgttaa ctcattctcc cccctacttt 660 ttgccagtta ttaaatgttc tggtaaatcc taactcaaca agaatgactc atgaggaaat 720 cctgactcct tgaactcact gattctggtg cattacagct atgaaatctc tatgtctgca 780 gctttctctc ccaccataag gaaatctgtg atttctattt tggcaggaga tgtgaccaag 840 gactcctgag gaatgaccct atagacagac ccagccacgt gcctagtagt gatcaaaaat 900 gcaaacccaa ttaaagcact gtcttctgat aaagtcactt tctcttgttc aatccctaat 960 gttcattctc aaagtgttcc aagacgcccc agcaagctgc agagcagatc ccaccctgcc 1020 cctgggcaat caacgtttca ggttactcct tccttaagtt aagtggcaga gagaaacggg 1080 cgagggtctc cccattgcct cagttcacag accagggggt gtcgaatgag tcctacagca 1140 ggacagcaaa caagaaatga ggcgcggggt cagggaacgc gctcaaaaaa ggaagaaaaa 1200 tcccactctt cattcgaaat caggccactg cactccggcc tcgtggcggg cgacctcctc 1260 ctgccaggga atcgccgctc tggcctcggg ctggggagcc cgtcagggtg ggtgagggag 1320 cgccggcgcc cccgccgggc cgccgccaac ttcggctccc tccctccgtc gcaaagccct 1380 cgagcccgcc cggccgccac gcttcagcaa aaggtcgtgc gcagcggccc acactgaaga 1440 ctctccatct gctcccacac ctgccccagc tggccgctgc tatgtggccc gagtgcgcaa 1500 gaagccgggg ccgcagacgt cagcagcgcc ccggcttcga gacccttcgg cagcagccgt 1560 gactgccgcc ggcgggcgct gacccatccc cgtgccagtc tgcagccgac caatccgcgt 1620 cctcttgagg cggggccgga gccgcgaggc cccgccccca agccgaggcc tcataaatgc 1680 tgcgacgcac gcccagccgc 1700 <210> 11 <211> 1700 <212> DNA <213> Homo sapiens <400> 11 tcaattttgt cattttacga aactacttgg agttttgatt tgtatttaca gtttttaaaa 60 gagtaaaaca gtgtgaactg tgaatggtaa tgattttgtt tggtaagtgc aaattttaat 120 tcatacatga aaaatatttg ttttgtttca tgattattac tgaaaataat tttgtactat 180 gaaggaagag gggtgttaaa aatgatccac tgtggatgta aattattcta ggcatgcttc 240 tgactaaaac attagcattt ccacaagaat cccaaaaaat aaaacaaaat ttaaaagatc 300 ccttctgttt cttttcctcc aagtttccag acccccatct ccaccctggc cacctcttcc 360 tgccataaat aatcaaatcc cacactttca tcttcacctt ccgtttccat tgcctcttcc 420 gatgggacag cacccgtggg gaatctcctc catgctcaca atccccgtct tgccactata 480 tcaagtcaca cttcttcctc accaatcgcc cattctggaa cctctgctat ccagctagat 540 cactttgagg tttggacaac tactggtgga ggtaatgggg gccccagaca gcccttgaca 600 tcacctcttt tatctttgcc aaattctaca aacaaagccc atgtctttct gtcccccgcc 660 cccatggctg gcacatagta gatgtcccca aatcttggtg atgtgaactt gtgtgtctca 720 cccttcacag acatcaccat ttcccgttgc acagaaggct ccactcatac caggcgcttt 780 ggctcccaga ataagcaggc tatcatgcta tgtccgacat ctaggcgttg gtacgtactg 840 ttcccttggc ctggaacatt ctgccctctc tcaccctctc ttttcttggc tttctgaaat 900 ggcacctcca tcagaaagtg tccctcactc ttggtctgag caagggacct tgcctggtat 960 tcctgaactt ccacctgttt tccctctgtt ttctccctct ctgggccttg tcctcaggtc 1020 agtgttgtgc ctccagctcc tgcagaggcg aactcagagg cggccgaatg aaaccaaatg 1080 gatttggctt gaccccatca tctagcggtg cccagcaagc gctggcacat agtaggtcca 1140 gcctgcccgg actcgcccac cacgggcccc cttccagagc cgggaggcag gccaagggag 1200 gccccttatt gccaagagca aacaggccgc ccagggaagt tttgctctgg gtcacaccca 1260 cctctgggtg gctgccaatc tgcctgccag aaaccgcagg ctgggccgcg gctacccaga 1320 gtcagggcca cggagcttag gagaccttgg gtcagctctg caaaggggtt gtttactgaa 1380 tgcttctggt atctgcagcc ccgctggggt tcggacaggg ggtctggaat agggcttcac 1440 aactacagca gggctaggac cctcttgggc tggaaggagc tggagaggcc tctgcaagct 1500 gtcacaggct cttggtgctg gcactggctc aggctttcac acacacacac gcgcacacac 1560 acacacacac acacacggac aggcaccccc ttggtggcct tcacagtttc accttcaggt 1620 aaatgggctc atcctttgag ccatgaggat gggaagcgaa gcaaggaatg aaaaagctag 1680 tgtgtttgtg tgtgtgtgtg 1700 <210> 12 <211> 980 <212> DNA <213> Homo sapiens <400> 12 atactggaca gagctcagac tgtgccctgg cctgtgtaga tgcaccagag gttttatctg 60 ggggtcacag ccgctgggaa cgggagagtt gctggggatt gccaggaagg gacaaggcat 120 ttgtagccat gcaccatctg cttttacact accctagagt caagtggacc ccctccagca 180 tttttttttc cccgagatgg agtctcactc tgtcacccag gctgaagtgc aatggtgtgg 240 tatcggctca ctgcaacctc tgcctcccgg gttcaagcga ttctcctgag tagctgggac 300 tacaggcgca tgccaccacg accggttaat tgcccctcca gcattgtatg tgtctgactg 360 caatgaagtg tgagtgtgtc tgtgtgcgac tgtgtgtgta tgatgtgagt gtgtctgtgt 420 ctatgtgggg gccaggtgta tagtctgcat atgtctgtgc acctctgtgt ctctgtgggt 480 tgtctgtgcc tatgtctcgg cttgtgtgtg tgcatgcatg tgtggtctgt gtgtgtctgt 540 atgcatgtgg tatctctggg atgtgtgtct gtatgtccct gcgtgcagtg tagtgacttt 600 gggagtgcac atgtgtacac gtgtggtgag tgtgcggccc tgcgcgagtt gtctgtgtac 660 gtgtggtgcc tgtgggtctg tgtgcgcgcg tgtgcccgtg ggtgtggtgt gtcctcccgc 720 gctgaggctc ccatgaaccc agctcagttc aggcctccct gacagtcctc gggcggccac 780 cccggggcgc ggctgtccac gaggcgcgga ggagaggagt gcgtggctgg gtcggcgggc 840 ggcgcccccg ctcctccccg cgggccgggc acacgtgggc cccgggcgcc gcctccccgc 900 gccgcccgcc gcccgccggg tcccgcgcgt ccccacccac ctcgggccgc ccccgccgcc 960 gagccggccc agggataaag 980 <210> 13 <211> 2000 <212> DNA <213> Homo sapiens <400> 13 tctaatttac acaaatccca cccactatgt aaacttgttg gaaaatgtcc tgcactctgc 60 acttcgtggc atttaaaact tccacacacg cgcgcgttct ttctcgaagc cccgtgattg 120 cttagcctcg ctgggcagct tggcactgct gggagcttgg ctcgccctgc cggggccgac 180 gccgcccgtc ccgcaggagc ccgcgcgggg ctcagggcac tcaggactcc gcatgcgtcc 240 cggctccagg tgggccccgg caccgccaac cgcaggaaac ccgccgagcc ctaaacgtct 300 cccaagcggc tgcagtctgc gacagagagt gtccctcggt ggagcgccct gtggctgccc 360 aggctacagc cgtggccgag gcgaggacac acttctgacc tggggctcca gcaaagactg 420 tccgcgagcg gcgactccat gcccgcagcc ctccgcccag ctcagccgcc cggccgcggg 480 caccagcagc cgcgccacga aagggcgcac cgcgcgggcg ccgtctctcc taggtgcgaa 540 ggcggctgag gccccgcccg ggaggcaccc gcgcggctcc ggagtgggcc ggagggacgt 600 ccgggggcgg ggcccgggcg cgcccgccct ctgaccgggc tataacaccc ggccccgccg 660 ggcggccgcg ggtgggtaga ggtgcgcgcc tgggacctgg tgaggctggg ggtgcgcggg 720 gccgggcgca gctgtggcag ctgccggacg gcggaggcgc caggaggagg aggagaggga 780 ggcgcgggcg gctgggtcga gggcaccgag gctgcccgtg ctcccggtct ctggttgcac 840 ggctcactcc cgaaggtgtt gcttccagct tttgcctcct taggaggcag ggagcgtcag 900 tgtcgggaga ccctgagacc ggagtaccga gacgtagctg gtgatgcccc cgcctgccct 960 catgtgttct caggttcttc ttatttttat tcatctctag aacatggact tcccgtgcct 1020 ctggctaggg ctgttgctgc ctttggtagc tgcgctggat ttcaactacc accgccagga 1080 agggatggaa gcgtttttga agactgttgc ccaaaactac agttctgtca ctcacttaca 1140 cagtattggg aaatctgtga aaggtagggt ccgtctcgtg aacactttgc caaaccctca 1200 gtcctccctt tcagtattca ttaaatatgc cccagcttcc tgtctgctct tccacgcacc 1260 tactctgagt ggcacagaac aagtcaaccg gtaccgtgcg tgttggttgt tttctgcttt 1320 tgttgggagg aatagtagga agaactgaat tttactggac ttgtccattg taattcagtg 1380 tcactgagtc ctttccatta ttggagttct tctgtctttt tggatcttgc agacattggt 1440 tatttgggat gtatgtttta gttccttttc aagataaact cccaagtaag tccgtttatc 1500 cgtttcagtt cccctttgtg tgggcttctt tatatatgac ttggactgtt aatgtcattt 1560 cttcatgtct cttttaaact gaaataatgc agttttgttg gtaagatttc tgtgtcatct 1620 gtagttagcc ttttatttaa agttatgcaa aactatcatt tctgcaagtt tcttttaatc 1680 taagtagtac agttctgttg gttagatttg tgtcgtgtat aattagccct atggcttaaa 1740 gttatgcaaa aaagtggttc tatgattaaa ggctgttttt aaaatgtatc catttgaaga 1800 agacaatgct agataatgaa tatatattag tagtgattga aactcttccc agcattttca 1860 tatttatcat taataattta ttgttctaag ttagaaacta cataaagtta ttttcatttt 1920 tatagacagc aagtttgaat cagataaatt aaataatttg ttcaaggtct cccagatggt 1980 gaattttata gccaggactg 2000 <210> 14 <211> 800 <212> DNA <213> Homo sapiens <400> 14 ctctaaaatc acttcccagt tgacacttga ccacaaaact cagaaggtaa ccaagcccaa 60 cccctaatgt ggtcttcagt tttatattcc ttattattgc aatcacctta tttaagtttt 120 aatcattaag ctgattgcat tgctgcacaa ggtcaagaag cacttgtaga atcagaaacc 180 ccaaagtggt tatggactgg ctactcagat ttttttcttc acttgctcag gctccgcccc 240 atctgaaacg tcttctctgc taggaactca ctaggagaaa atgaccagtg agcttaccaa 300 agctcagacc ttatccacca gaaacattgt atggggcacc tcaccttttc agcacctcct 360 gccataacag gacggaagtg ctgaagggac agctcccagt gttttaggtc ttagtaagcg 420 ctcccaggaa gacagcaccc atcccctctc tgctacaaac agccattcac aaaatccgag 480 gaggaaacca agtcatccct gattccttgg aaatttagtt agactgatgt tgaaacacac 540 ccccctttcc tagtgtataa ttatttgtgg gtggagagct accagggagg gtaacagaag 600 atgccggaga agggggggga aaagtagatg cggatttcgt cctgacttct aaaaattcct 660 cctctccctc tcccattttc ctaatccgag aatgatggag ctcgaggcaa aggaatgatt 720 ccggaaatgg agatatgatt ctcaaaccta gaaatgatcg gagtgattta ttagttaaat 780 attcttcgtc caggaaccca 800 <210> 15 <211> 2000 <212> DNA <213> Homo sapiens <400> 15 ttgcatggca ggtagtgagc tccctgttat tggtggcatt caaacatggg caggatgacc 60 accgggtgca gatgctgggg atggggtagg aatcaagcag tgtgtgagtc ctgaaccaat 120 caacgtctat gatctgctta gaatatgaaa catctggtga tgcatttatg gcgctttcaa 180 aatttacact tttctttcct caaagtttga ccttctgaac cttgtttgct tagtagtggg 240 aataatcaag taattattcc caagggatca cagtcatgac tgtgaatctg tgaatcatgc 300 tattttctcc caagtccttt cttcctctct tcccttcccc taattcagct ggctaaggcg 360 gatctcactg actgacctcc aaagtctaat ttttaaaaat ataactagat ttatgccttc 420 tgttccggat gctgagggag ctacagaaca gaaaagttac aaggagcgag ctcaaaaaca 480 attcttctaa cctcaggtat tgataaggag caggcactga taaggagcag gcactgtcca 540 cagaaaatgt agctgttact gataattaga catttttttt ttttaagaaa aataaagtgt 600 agcctcttct gggacatttt tttcttcttc tgggttaatg gccttctaat acttggcttg 660 gctctaggtt aggttgcagg gagccgtgga gatgatccat tcccgattcc tcatcgtcca 720 gatggaagaa actgaggccc aagggcaaag tgattagtcc gaggtcaccc agtgtctagg 780 ggcacaccta ggactgtaat cagactttca tggacctggt ctgggttctc ccacttagtc 840 atgggccttg aagattcccc gaggctgcct cctgaaaagg actggggtcc tagtggcccc 900 tggacgttgg gcaagcaagg gactgggcct ccatgttgtg cctccatagt cctgatcctg 960 aactggaaaa ctcagcccct gaccacgcag ctctccttta agcccctttg tttcacatgg 1020 ttttcaaagt ctgccaccca cagtggggct gcctgtaccc gccctgtcca cccattgccc 1080 cagctgtcag ccccttgact tctctcctgg ggcttaaaca tccctggctc caaaatgggc 1140 agctcacttt cttccccaag aagtagctgc acctccaggg ttcctagatt tgcccctcct 1200 tgccaggggg aggggtggct gcgacaggag attctccctg ctctcagcag aaggaactcc 1260 agcagttgga gaccagcaaa cccctctgga cacagatctg atttcctaac tgggaaggct 1320 cagggcaaaa taaaaattca ggtccactgg ttcaaaaact atgaagaatt tcaagaccgt 1380 cacagtagcc cattaaacca aacgtggatc tgcaagggtc ccacagccat gaagcccacc 1440 ctgcttggtt gggttccaaa aagatgggga cagtgattgc ttaagctctg tggatcaagg 1500 accccggaga ggccttctgg ctctccacat atctgctctg atcactccta aacacaattc 1560 tgtttcctcc aggcctggcg ggtcagtcca gggaccccca tcagtgtgat gtttccagga 1620 gtaggcgttt caatacttcc tgtgctctct tctccagcac aaggcccctc tccatcccac 1680 cctcattatg tctgactctt tactatttaa atgggtcaag agaagtggcg cttgtgtaat 1740 gtgaaggtta aggtcagtag ggccagggaa ctgtgagatt gtgtcttgga ctgggacaga 1800 cagccgggct aaccgcgtga gaggggctcc cagatgggca cgcgagttca ggctcttccc 1860 tactggaagc gccgagcggc cgcacctcag ggtctctcct ggagccagca cagctattcg 1920 tggtgatgat gcgccccccg gcgcccccag cccggtgctg caccggcccc cacctcccgg 1980 cttccagaaa gctccccttg 2000 <210> 16 <211> 2000 <212> DNA <213> Homo sapiens <400> 16 tcccagatga tagatgtaat attaataagc tacagatgag gaaatggatg cttgaaaagt 60 ttgaagggac atcttaggaa tcagttatct cccctgggga gggaccttca ggaatggcct 120 gatccctatc aggagagctg tccgctgttc aggatgtcaa aggttcctgt tctgcttcta 180 gaagtcagct tttcctggga ggaaaagtgg gcatgggctg aggtgagagc tggggtgcag 240 gtggagtatg aagactcctc agagaaagga aataaacaaa aaatatttat tgagcttcta 300 ccatgtatca aacccagtca ctagtcacta ttttatcctc agacagacac ctataaacct 360 ttaaaccacg tgtgtgtgta tatatatata tattataaga aagcttagag aggataagta 420 aacttgggat cacacagatg tcaacaaggg gagaagcctg tccgggtgtg gtggctcatt 480 cctgtaatcc cagcactttg ggaggctgag tggggaggat cacatgaagc caggagttcc 540 agaccagact ggacaagata gggagacccc acttctattt tctaatagaa attaaaataa 600 aaaataaaga aaatttcttt atacattgag ctgaaattaa accccactgt ggcttccacc 660 aatttttgct ctctggagca actcaaaaca catctaaatc ctcttctaca agcgaatcct 720 ttaaatattt gaaaatacct atgtctctct tctgtgctct tctggtacca atagtttaaa 780 tgggtaacag tagactttgt tttattcaat cttttaaaaa gcaaataaaa agtaagacat 840 gctgggtgcg gtggctcacg cctgtaatcg cagcactttg ggaggcggag gtgggctgat 900 cacgaggtca ggagttcgag actagccaga ccaacatggt gaaaccccgc ctctactaaa 960 aatacaaaaa ttagccagat gtggtgcacg cctgtaactc cagctactcg ggaggctgcg 1020 gccagagaat tgcttgaacc caggagacaa agtttgcagt gagccgagat cacgccactg 1080 cactccagcc tgggagacag agcgagactc cgtctcaaaa acaaaaagta agacattacc 1140 aatatgatgc caaccctcat gcactcatcc ccaaccccac acttctcttc tcccacccct 1200 tctatctgct tttgtattta tttagcctat gggatcccaa tgcagaggag ccctggtgga 1260 cgcatttata gtgcagcgat gaatagaaga cagtgatagt cttatactgg agttgaaatg 1320 tgataaacat ttatacactg tgtttataag tcgtgtaact ctggccaatt tatctggtct 1380 ttctaagctc actttcctcg tctgtaaaat gggaagaata atcatactta ttgcgctggt 1440 tgttatgggg attaagcaag ataattctct aaagctttgg cacttggcgc tgaaagtagc 1500 cattccatgt cttctttccc gccccgcctc ttgtgctccc caccgctttc gtgatgtccg 1560 cagttgccca cctgcctcta caataaaaaa cgcatccctc ctcctgcagg gtccaccgca 1620 ccgggaagcc ctgtctgtat cagttaccaa ccacaattgc agtgagtacg aatcgtggct 1680 ttcccacagt caggaaaggc aagggagacc gacgacccgc ttctctagga gtaagtaaag 1740 attaaaggta gttcgcggta tagcctaggc agggattaac ccgtggtccc agcgctcctg 1800 ctatttgcat tccaaagcag acacctcatg cgctcaaccc cgcccgcagg cggctcccgc 1860 agtctaaggg acctggcgcg agtccgggaa gcggagggcg cagctgcgca gggaaggggg 1920 ccgggggcgg gaccagggcg cgcgttccgg tcccggggcg tggcctcccg caggtgagta 1980 cgctgctcct tcggtttccc 2000 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 tatgacctcg gaggagctgt gg 22 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 cgttcacagc ccgcttaaat tg 22 <210> 19 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 aagccgaggc ctcataaatg ct 22 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 cgggagaggg aagaaagcca ta 22 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 ctggctcagg ctttcacaca ca 22 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 ggaggcggac ctgtgagtct tt 22 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 cgagcccctc tagcgatttg tt 22 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 aaatcgggct gttccctcat ct 22 <210> 25 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 25 tcactcccga aggtgttgct tc 22 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 26 ctagccagag gcacgggaag tc 22 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 27 aggagcacgc agagtccatg at 22 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 28 tacctaaggc cccctctgga ca 22 <210> 29 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 29 cagtagggcc agggaactgt ga 22 <210> 30 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 30 ctgtgctggc tccaggagag ac 22 <210> 31 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 31 ttggcgctga aagtagccat tc 22 <210> 32 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 32 tcccttgcct ttcctgactg tg 22

Claims (28)

Inhibitor of DNA binding 3 (ID3), Regulator of G-protein signaling 2 (RGS2), WNT1 inducible signaling pathway protein 2 (WISP2), Monoglyceride lipase (MGLL), Carboxypeptidase M (CPM), Gamma-aminobutyric acid (GABARA1) A cancer diagnostic composition comprising a methylated promoter of a colorectal cancer specific expression reducing gene selected from the group consisting of A receptor), CLU (Clusterin) and F2RL1 (Coagulation factor II (thrombin) receptor-like 1). According to claim 1, Inhibitor of DNA binding 3 (ID3), Regulator of G-protein signaling 2 (RGS2), WNT1 inducible signaling pathway protein 2 (WISP2), Monoglyceride lipase (MGLL), Carboxypeptidase M (CPM), GABRA1 (Gamma-aminobutyric acid (GABA) A receptor), CLU (Clusterin) and F2RL1 (Coagulation factor II (thrombin) receptor-like 1) each has a DNA sequence of SEQ ID NO: 1 to 8, characterized in that the composition for cancer diagnosis. The cancer diagnostic composition of claim 1, wherein the promoter contains at least one methylated CpG dinucleotide. The composition for diagnosing cancer according to claim 1, wherein the promoter comprises a sequence selected from the group consisting of: (a) the 1747-1944 region of SEQ ID NO: 9; (b) regions 1112-1829 of SEQ ID NO: 10; (c) regions 1668 to 1782 of SEQ ID NO: 11; (d) regions 607 to 1701 of SEQ ID NO: 12; (e) regions 53 to 973 of SEQ ID NO: 13; (f) the 1663-1787 region of SEQ ID NO: 14; (g) regions 1790-1942 of SEQ ID NO: 15; And (h) regions 1488-1945 of SEQ ID NO: 16. According to claim 4, wherein the promoter is a cancer diagnostic composition, characterized in that any one of the DNA sequence represented by SEQ ID NO: 9 to 16. A microarray for cancer diagnosis, wherein the composition of any one of claims 1 to 5 is immobilized on a substrate. 7. The microarray of claim 6 wherein the cancer is colorectal cancer. A cancer diagnostic kit containing the composition of any one of claims 1 to 5. The diagnostic kit of claim 8, wherein the cancer is colorectal cancer. The group consisting of SEQ ID NO: 17/18, SEQ ID NO: 19/20, SEQ ID NO: 21/22, SEQ ID NO: 23/24, SEQ ID NO: 25/26, SEQ ID NO: 27/28, SEQ ID NO: 29/30, and SEQ ID NO: 31/32 Primer mixture containing one or more primer pairs selected from. A method for detecting whether or not promoter methylation of a gene derived from a clinical sample, comprising (a) isolating sample DNA from clinical samples; (b) methylated sensitivity selected from the group consisting of (i) a reagent that modifies unmethylated cytosine residues, or (ii) Msp I, Hpa II, Bss HII, Bst UI and Not I Treating with restriction enzymes; (c) The DNA treated in step (b) is treated with ID3 (Inhibitor of DNA binding 3), RGS2 (Regulator of G-protein signaling 2), WISP2 (WNT1 inducible signaling pathway protein 2), MGLL (Monoglyceride lipase), CPM CpG islands derived from promoters of genes selected from the group consisting of (Carboxypeptidase M), GABRA1 (Gamma-aminobutyric acid (GABA) A receptor), CLU (Clusterin), and F2RL1 (Coagulation factor II (thrombin) receptor-like 1) Amplifying using a primer capable of amplifying; And (d) determining the methylation of the promoter based on the presence or absence of the amplified product in step (c). The method of claim 11, wherein the clinical sample is tissue, cells, sputum, feces, urine, membranes, cerebrospinal fluid, amniotic fluid, eyeballs, organs or blood derived from a suspected cancer patient or a diagnosis subject. The method of claim 11, wherein the reagent for modifying the unmethylated cytosine residue is bisulfite. 12. The method of claim 11, wherein said amplifying is PCR, DNA microarray or bisulfite sequencing. The method of claim 11, wherein the primers capable of amplifying the CpG islands are the primer mixture of claim 10. The method of claim 15, wherein the primer mixture contains all the primers of SEQ ID NOs: 17-32. The method according to claim 11, wherein the step (d) is performed by electrophoresis of the amplification product of the step (c), and the PCR product is present in the DNA treated with Hpa II while the PCR product is displayed in the mock DNA. Wherein the promoter is determined to be methylated and the promoter is not methylated if there is no PCR product in the DNA treated with Hpa II. 12. The method according to claim 11, wherein step (d) hybridizes the amplification product of step (c) with the microarray for cancer diagnosis of claim 6, thereby hybridizing both mock DNA and DNA treated with Hpa II. Determining if the promoter is methylated. Inhibitor of DNA binding 3 (ID3), Regulator of G-protein signaling 2 (RGS2), WNT1 inducible signaling pathway protein 2 (WISP2), Monoglyceride lipase (MGLL), Carboxypeptidase M (CPM), GABRA1 (Gamma) detecting methylation of the CpG-containing promoter of a gene selected from the group consisting of -aminobutyric acid (GABA) A receptor), CLU (Clusterin) and F2RL1 (Coagulation factor II (thrombin) receptor-like 1) How to diagnose cancer. 20. The method of claim 19, wherein the detection is performed through the following steps: (a) isolating sample DNA from clinical samples; (b) limiting methylation sensitivity selected from the group consisting of (i) a reagent to modify unmethylated cytosine residues, or (ii) Msp I, Hpa II, Bss HII, Bst UI and Not I Treating with an enzyme; (c) The DNA treated in step (b) is treated with ID3 (Inhibitor of DNA binding 3), RGS2 (Regulator of G-protein signaling 2), WISP2 (WNT1 inducible signaling pathway protein 2), MGLL (Monoglyceride lipase), CPM CpG islands derived from promoters of genes selected from the group consisting of (Carboxypeptidase M), GABRA1 (Gamma-aminobutyric acid (GABA) A receptor), CLU (Clusterin), and F2RL1 (Coagulation factor II (thrombin) receptor-like 1) Amplifying using a primer capable of amplifying; And (d) determining the methylation of the promoter based on the presence or absence of the amplified product in step (c). The method of claim 20, wherein the clinical sample is tissue, cells, sputum, feces, urine or blood from a suspected cancer patient or a diagnosis subject. The method of claim 20, wherein the reagent for modifying the unmethylated cytosine residue is bisulfite. The method of claim 20, wherein said amplifying is PCR, DNA microarray or bisulfite sequencing. The method of claim 20, wherein the primer capable of amplifying the CpG islands is a primer mixture of claim 10. The method of claim 24, wherein the primer mixture contains all of the primers of SEQ ID NOs: 17 to 32. The promoter of claim 20, wherein the step (d) is performed by electrophoresis of the amplification result of step (c), and the PCR result is present in the DNA treated with Hpa II while the PCR result is shown in the mock DNA. Wherein the promoter is determined to be methylated and the promoter is not methylated if there is no PCR product in the DNA treated with Hpa II. The method of claim 20, wherein step (d) hybridizes the amplification product of step (c) with the microarray for diagnosing cancer of claim 6, where both mock DNA and Hpa II-treated DNA are hybridized. Determining that the promoter is methylated. The method of claim 20, wherein the cancer is colorectal cancer.

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