KR101142130B1 - Method for Detecting Breast Cancer Using Breast Cancer Specific Methylation Marker Genes - Google Patents

Abstract

The present invention relates to a method for detecting breast cancer using a breast cancer specific biomarker. More specifically, the CHL1 gene, which is a breast cancer specific expression reducing gene {NM_006614, cell adhesion molecule with homology to L1CAM (close homolog of L1)} Or breast cancer comprising a methylated CpG island of a promoter region of a TF gene (NM_001063, iron ion-binding transport protein, transferrin), a biomarker for diagnosing the stage thereof, and a breast cancer using the biomarker, and a method for detecting the stage. .

By using the biomarker of the present invention, early diagnosis of breast cancer can be performed more accurately and faster than conventional methods, and serum, which is a non-invasive sample, can be used as a sample, so that breast cancer can be diagnosed more simply and accurately.

Breast Cancer, Methylation, Methylation Markers, Breast Cancer Diagnosis, Early Diagnosis, Prognosis Prediction

Description

Method for Detecting Breast Cancer Using Breast Cancer Specific Methylation Marker Genes}

The present invention relates to a method for detecting breast cancer using a breast cancer specific biomarker. More specifically, the CHL1 gene, which is a breast cancer specific expression reducing gene {NM_006614, cell adhesion molecule with homology to L1CAM (close homolog of L1)} Or breast cancer comprising a methylated CpG island of a promoter region of a TF gene (NM_001063, iron ion-binding transport protein, transferrin), a biomarker for diagnosing the stage thereof, and a breast cancer using the biomarker, and a method for detecting the stage. .

Breast cancer is a malignant tumor of the breast, and it is mostly known to be caused by a transition from cancer cells to epithelial cells, which are the outermost cells in the tissue from which the milk (milk) is made or the tube from which the milk comes out. Breast cancer is known to occur most frequently among women in developed countries, and Korea is the second most common cancer after stomach cancer. In addition, it is the fifth highest mortality cancer after stomach cancer, liver cancer, uterine cancer, and lung cancer.

This increase in breast cancer is likely to be due to changes in lifestyle, such as changes in diet, Western lifestyles, the first menstruation, or fewer births, avoiding lactation, and using birth control pills. This phenomenon is remarkable in neighboring Japan, and the most common cancer in Japanese women has become breast cancer, and it is not difficult to predict that breast cancer will overtake uterine cancer and occupy the level in female cancer in the near future.

In particular, the characteristics of breast cancer in Korea is that the frequency of breast cancer is relatively high in the 40's, unlike the prevalence in the 50's in the West. Therefore, a breast cancer management guideline suitable for Korea, which is different from the western breast cancer management guideline, is needed.

Breast cancer is a cancer that can be diagnosed early and treated early. Therefore, early diagnosis is important. Early detection of breast cancer is known to have a 10-year survival rate of about 85% or more. It is easy to think of cancer as symptoms such as tiredness, lack of appetite, and anemia, but there are no such symptoms in the early stages of breast cancer, and no breast or core pain. Because of this, many people do not realize, and many are found to be quite large cores. Therefore, in order to increase the survival time of patients, early diagnosis is the best method when the lesion range is small. Therefore, the diagnosis method is more efficient than the conventional methods for diagnosing breast cancer. The development of breast cancer specific biomarkers with high sensitivity and specificity is urgently needed.

Recently, methods for diagnosing cancer through DNA methylation measurement have been proposed. DNA methylation mainly occurs in the cytosine of CpG island of the promoter region of a specific gene, thereby binding of transcription factors. As a result of being disturbed, the expression of a specific gene is blocked, and detection of methylation of the promoter CpG island of a tumor suppressor gene is a great help in cancer research, which is called methylation specific PCR (MSP). Attempts to diagnose cancer screening and screening by using methods such as) and automatic base analysis have been actively conducted.

Although methylation of the promoter CpG islands directly causes carcinogenesis or causes secondary changes in carcinogenesis, tumor suppressor genes, DNA repair genes, and cell cycles in many cancers are controversial. It has been confirmed that the expression of these genes is blocked due to hyper-methylation of regulatory genes. In particular, it is known that hypermethylation occurs at the promoter region of specific genes at an early stage of cancer development.

Therefore, promoter methylation of tumor-related genes is an important indicator of cancer, which can be used in many ways, such as diagnosis and early diagnosis of cancer, prediction of carcinogenic risk, prediction of cancer prognosis, follow-up treatment, and prediction of response to anticancer therapy. . Attempts have recently 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).

Accordingly, the present inventors have made intensive efforts to develop a method for effectively detecting breast cancer. As a result, when the degree of methylation is measured using a promoter of a methylation-related gene, which is methylated specifically for breast cancer, as a biomarker, progression of breast cancer and breast cancer It was confirmed that the can be effectively detected to complete the present invention.

It is an object of the present invention to provide a biomarker for diagnosing breast cancer comprising a CpG island of a methylated promoter region of a gene that is specifically methylated in breast cancer, which can be effectively used for diagnosing breast cancer.

Another object of the present invention is to provide a method for detecting breast cancer and its progression step using the breast cancer specific biomarker.

In order to achieve the above object, the present invention provides a CHL1 gene which is a breast cancer specific expression reduction gene {NM_006614, cell adhesion molecule with homology to L1CAM (close homolog (heart))} Or it provides a biomarker for diagnosing breast cancer or its advanced stage comprising a methylated CpG island of the promoter region of the TF gene (NM_001063, iron ion binding transport protein, transferrin).

The invention also comprises the steps of (a) isolating DNA from clinical samples; And (b) CHL1 gene in the isolated DNA {NM_006614, cell adhesion molecule with homology to L1CAM (close homolog of L1)} Or it provides a detection method of breast cancer or breast cancer progression step comprising detecting the methylation of TF gene (NM_001063, transferrin).

By using the biomarker of the present invention, early diagnosis of breast cancer can be performed more accurately and faster than conventional methods, and serum, which is a non-invasive sample, can be used as a sample, so that breast cancer can be diagnosed more simply and accurately.

In one aspect, the present invention is a CHL1 gene that is a breast cancer specific expression reduction gene {NM_006614, cell adhesion molecule with homology to L1CAM (close homolog (heart)) Or breast cancer comprising a methylated CpG island of a promoter region of a TF gene (NM_001063, iron ion binding transport protein, transferrin) or a biomarker for diagnosis thereof.

In the present invention, the biomarker may be characterized by having a nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

The methylated biomarkers according to the present invention were screened by the following methods: (a) selecting a gene that has been DNA-methylated only in the transformed cell line for the transformed and non-transformed cell lines; (b) comparing the methylation tendency of the transformed breast cancer tissue cells and the non-transformed tissue cells at the junction to sort the list of hypermethylated genes in the transformed tissue cells; (c) treating the transformed breast cancer cell line with a methylation inhibitor and sorting the list of genes that express more in the transformed breast cancer cell line treated with the methylation inhibitor as compared to the untreated transformed breast cancer cell line; And comparing the gene lists obtained in steps (a), (b) and (c) to determine the methylation in the genome of the cells that are converting the genes that are common to both lists to the transformed breast cancer cell form in an untransformed state. Screening with biomarkers controlled by.

In another aspect, the invention comprises the steps of (a) isolating DNA from clinical samples; And (b) CHL1 gene in the isolated DNA {NM_006614, cell adhesion molecule with homology to L1CAM (close homolog (heart))} Or it relates to a method for detecting breast cancer or breast cancer progression step comprising detecting the methylation of TF gene (NM_001063, iron ion binding transport protein, transferrin).

In the present invention, CHL1 gene {NM_006614, cell adhesion molecule with homology to L1CAM (close homolog (heart))} Alternatively, methylation detection of the TF gene (NM_001063, iron ion-binding transport protein, transferrin) may be performed at DNA sites having base sequences of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

According to the present invention, the methylation detection is PCR, methylation specific PCR (methylation specific PCR), real time methylation specific PCR (real time methylation specific PCR), PCR using methylated DNA specific binding protein, quantitative PCR, DNA chip, pi It may be characterized in that it is carried out by a method selected from the group consisting of rosequencing and bisulfite sequencing.

In the present invention, the clinical sample may be characterized in that it is selected from the group consisting of tissue, sputum, cells, blood, plasma and urine from patients suspected of cancer or a diagnosis target.

As a screening method of the methylated biomarker gene, not only breast cancer but also various methylated genes can be found in various stages of dysplasia progressing to breast cancer, and the selected genes are screened for breast cancer, risk assessment, prediction, disease identification, and disease stage. It can also be used to select diagnostic and therapeutic targets.

Identifying genes methylated in breast cancer and at various stages of abnormality enables early and accurate diagnosis of breast cancer and can identify new targets for establishing and treating methylation lists using multiple genes. In addition, methylation data according to the present invention will be able to establish a more accurate breast cancer diagnosis system in conjunction with other non-methylated associated biomarker detection methods.

With the methods of the present invention, one can diagnose several stages or stages of breast cancer progression, including determining the methylation stage of one or more nucleic acid biomarkers obtained from a sample. The methylation of nucleic acid isolated from a sample of each stage of breast cancer can be compared to the methylation of one or more nucleic acids obtained from a sample that does not have cell proliferative abnormalities of breast tissue, thereby identifying the specific stage of breast cancer of the sample, wherein the methylation The step may be hypermethylation.

In one example of the invention, the nucleic acid may be methylated at the regulatory site of a gene. As another example, since methylation starts from the outside of the regulatory region of the gene and proceeds to the inside, detection of methylation at the outside of the regulatory region enables early diagnosis of genes involved in cellular transformation.

As another example, in the present invention, the methylation state of the CHL1 gene {NM_006614, cell adhesion molecule with homology to L1CAM (close homolog of L1)} and TF (NM_001063, Transferrin) gene promoter region is detected by using a kit. Cell growth abnormalities (dysplasia) of the existing breast tissue can be diagnosed.

According to the method of the present invention, it is possible to determine the cell growth abnormality tendency (dysplasia progression) of the breast tissue of the specimen. The method includes determining the methylation status of one or more nucleic acids isolated from the sample, wherein the methylation step of the one or more nucleic acids is compared to the methylation step of the nucleic acid isolated from the sample without cell growth abnormality (dysplasia) of breast tissue. It can be characterized by.

The method includes contacting a sample comprising one or more nucleic acids isolated from a sample with an agent capable of determining one or more methylation states. The method comprises identifying the methylation status of one or more sites in one or more nucleic acids, wherein the methylation status of the nucleic acid is identical to the methylation status of the same site in a nucleic acid of a sample that does not have cell growth abnormalities (progressive dysplasia) of breast tissue. It may be characterized by the difference

In one aspect of the invention, the detection method can be used in the form of a kit. Kits for use in the detection methods of the invention include a compartmentalized carrier means for holding a sample, a first container containing an agent that sensitively cleaves unmethylated cytosine, a second container containing a primer for amplifying CpG-containing nucleic acids, and a truncated or One or more containers including a third container containing means for detecting the presence of the uncleaved nucleic acid. Primers used in accordance with the present invention include the sequences set forth in SEQ ID NOs: 3-6, functional combinations, and fragments thereof. Functional combinations or fragments are used as primers to detect whether methylation has occurred on the genome. The carrier means is suitable for containing one or more containers such as bottles, tubes, each container containing independent components used in the method of the invention. In the context of the present invention, one of ordinary skill in the art can readily dispense the required formulation in the container. For example, a container containing methylation sensitivity restriction enzyme may be configured as one container. One or more containers may contain primers that have homology with important nucleic acid locus. In addition, one or more of the containers may contain isoxisomers of methylation sensitive restriction enzymes.

In another aspect of the invention, using the kit, the method for detecting breast cancer comprises the steps of (1) separating the genomic DNA from clinical samples (2) treating the isolated genomic DNA with methylation sensitive restriction enzyme (3) amplifying the genomic DNA treated with the methylation sensitive restriction enzyme using a primer capable of amplifying the breast cancer diagnostic biomarker of the present invention, and (4) presence or absence of a biomarker for diagnosing breast cancer in the amplified product. The biomarker fragment may be diagnosed as a sample of breast cancer or a breast cancer progression step, and the kit may be used to diagnose breast cancer for biopsy of a breast cancer in order to confirm the presence of a PCR amplification product using an included primer. It may further contain fragments capable of hybridizing under marker and stringent conditions.

In addition, as a method for determining the presence or absence of the biomarker for diagnosing breast cancer, bisulfate sequencing, pyrosequencing, methylation-specific PCR, MethyLight, PCR using a methylated DNA binding protein, and DNA chip may be used.

In another embodiment of the present invention, the methylation gene marker can be used for early diagnosis of cells likely to form breast cancer. When a gene identified to be methylated in cancer cells is methylated in cells that appear clinically or morphologically normal, the cells that appear to be normal are in progress of cancer. Therefore, breast cancer can be diagnosed early by confirming methylation of the breast cancer specific gene promoter site in cells that appear normal.

In one aspect of the present invention, whether the CHL1 gene and the TF gene is methylated may include the following steps: (a) separating the sample DNA from the clinical sample; (b) treating bisulfite with the separated DNA; (c) amplifying using a primer capable of amplifying fragments comprising CpG islands of the CHL1 and TF gene promoters; And (d) performing pyro sequencing using the result amplified in step (c) to determine methylation of the promoter region.

In another embodiment of the present invention, by examining the methylation frequency of the gene specifically methylated in breast cancer, and by determining the methylation frequency of the tissue having the possibility of breast cancer progression, the possibility of tissue development into breast cancer can be evaluated.

As used herein, "cell transformation" is characterized from one form to another, such as from normal to abnormal, from non-tumor to tumorous, from undifferentiated to differentiation, from stem cells to non-stem cells. It means to change. Additionally, the transformation can be recognized by the morphology, phenotype, biochemical properties, etc. of the cell.

In the present invention, "early identification" of cancer is to discover the possibility of cancer before metastasis, preferably before morphological changes are observed in sample tissue or cells. In addition, "early confirmation" of cell transformation refers to the likelihood of transformation occurring at an early stage before the cell is transformed.

"Hypermethylation" in the present invention means methylation of CpG islands.

In the present invention, "sample" or "sample sample" means a wide range of samples including all biological samples obtained from individuals, body fluids, cell lines, tissue culture, etc., depending on the type of assay being performed. Methods of obtaining bodily fluids and tissue biopsies from mammals are commonly known. Preferred source is biopsy of the breast.

Screening of Methylation Regulated Biomarkers

In the present invention, a biomarker gene that is methylated is screened when the cell or tissue is transformed or the cell is changed to another form. Herein, "transformed" cells means that the shape of the cell or tissue is changed from one form to another, such as abnormal form, non-tumorigenic tumor, and undifferentiated form into differentiation.

Therefore, in the present invention, the methylation regulatory marker gene in the transformation into breast cancer cells was identified by a systematic method. In one embodiment of the method (1) genomic DNA was isolated from breast cancer tissue and adjacent normal-tissue tissue samples. The genomic DNA was reacted with MDB2bt protein that binds to methylated DNAdp and then amplified methylated DNA that binds to MBD2bt protein. Amplified DNA derived from breast cancer tissue was Cy5, and amplified DNA derived from adjacent normal finding tissue was Cy3, and then hybridized to a human CpG microarray to select a gene having a large difference in methylation degree between adjacent normal tissue and breast cancer tissue. In addition, (2) a list of genes with increased expression in transformed breast cancer cell lines treated with methylation inhibitors was selected, as compared with transformed breast cancer cell lines treated with methylation inhibitors and untreated transformed breast cancer cell lines. . Comparing the list of genes obtained in steps (1) and (2), markers regulated by methylation in the genome of cells converting genes present in both lists into transformed breast cancer cell forms in an untransformed state The genes were considered and a total of six genes were selected.

In the present invention, to further confirm whether the six biomarkers are methylated, pyro sequencing was performed on breast cancer cell lines.

In other words, the whole genomic DNA was isolated from breast cancer cell lines MCF-7 and MDAMB-231 to treat bisulfite, and then PCR using bisulfite-converted genomic DNA using primers that can specifically amplify each marker. Was performed. Thereafter, the amplified PCR product was subjected to pyro sequencing to measure the degree of methylation. As a result, it was confirmed that two biomarkers of the eight biomarker genes were methylated in breast cancer cell lines.

Use of cancer cells for comparison with biomarker-normal cells for breast cancer

In this example, "normal" cells refers to cells that do not exhibit abnormal cell morphology or changes in cytological properties. A "tumor" cell refers to a cancer cell, and a "non-tumor" cell refers to a cell that is part of the diseased tissue but is not considered to be a tumor site.

In one aspect, the present invention is based on the discovery of a relationship between breast cancer and promoter hypermethylation of two genes described below: CHL1 gene {NM_006614, cell adhesion molecule with homology to L1CAM (close homolog of L1)}; TF (NM_001063)-iron binding transport protein (Transferrin); gene.

In another use of the diagnostic kit of the invention, the methylation stage of one or more nucleic acids isolated from a sample can be determined to early diagnose cell growth abnormalities of the breast tissue of the sample. The methylation step of the one or more nucleic acids may be compared with the methylation status of one or more nucleic acids isolated from a specimen that does not have cell growth potential of breast tissue. The nucleic acid is preferably a CpG-containing nucleic acid such as a CpG island.

In another use of the diagnostic kit of the invention, one can diagnose abnormalities in cell growth of breast tissue of a sample comprising determining methylation of one or more nucleic acids isolated from the sample. The nucleic acid is a CHL1 gene {NM_006614, cell adhesion molecule with homology to L1CAM (close homolog of L1)}; TF (NM_001063)-iron binding transport protein (Transferrin); And encoding the gene and combinations thereof, wherein the methylation step of the one or more nucleic acids is compared with the methylation status of the one or more nucleic acids isolated from a sample that does not have a knowledge of cell growth potential of breast tissue. You can do

The term “prescription” means a property that is susceptible to abnormal cell growth. A virulent sample is a sample that does not yet have cell growth abnormalities, but has or has increased cell growth abnormalities.

In another aspect, the present invention provides a method for cell growth of breast tissue of a sample comprising contacting a sample comprising the nucleic acid of the sample with an agent capable of determining the methylation state of the sample and confirming methylation of one or more sites of the one or more nucleic acids. It provides a method for diagnosing abnormalities. Here, the methylation of one or more sites of one or more nucleic acids can be characterized as different from the methylation step of the same site of the same nucleic acid of a sample having no cell growth potential.

The methods of the present invention comprise determining methylation of one or more sites of one or more nucleic acids isolated from a sample. Here, "nucleic acid" or "nucleic acid sequence" means oligonucleotides, nucleotides, polynucleotides or fragments thereof, single stranded or double stranded genomic origin or synthetic genomic origin or synthesis of DNA, RNA, sense or antisense strands. Refers to DNA or RNA of origin, peptide nucleic acid (PNA) or DNA amount or RNA quantity material of natural or synthetic origin. If the nucleic acid is RNA, it is apparent to those skilled in the art that, instead of deoxynucleotides A, G, C, and T, it is replaced with ribonucleotides A, G, C, and U, respectively.

Any nucleic acid capable of detecting the presence of differently methylated CpG islands can be used. The CpG island is a CpG rich site in the nucleic acid sequence.

Methylation

Any nucleic acid in purified or unpurified form may be used in the present invention, and any nucleic acid containing or suspected of containing a nucleic acid sequence containing a target site (eg, a CpG-containing nucleic acid) may be used. . A nucleic acid site that can be differentially methylated is a CpG island, which is a nucleic acid sequence having a high CpG density compared to other dinucleotide CpG nucleic acid sites. Doublet CpG is only 20% probable in vertebrate DNA when predicted by the ratio of G * C base pairs. At certain sites, the density of double CpG is ten times higher than at other sites in the genome. CpG islands have an average G * C ratio of about 60%, with the average DNA G * C ratio of 40%. CpG islands are typically about 1 to 2 kb in length and there are about 45,000 CpG islands in the human genome.

In many genes, CpG islands start upstream of the promoter and extend to the transcriptional site downstream. Methylation of CpG islands in promoters usually inhibits expression of genes. CpG islands may also surround the 3 'region of the gene coding region, as well as the 5' region of the gene coding region. Therefore, CpG islands are found at several sites including the coding sequence upstream of the regulatory region comprising the promoter region, the coding region (eg exon region), downstream of the coding region, eg, the enhancer region and the intron. do.

Typically, the CpG-containing nucleic acid is DNA. However, the method of the present invention may apply for example a sample containing DNA or RNA containing DNA and mRNA, wherein the DNA or RNA may be single stranded or double stranded, or DNA-RNA hybrids. It may be characterized by the contained sample.

Nucleic acid mixtures may also be used. The specific nucleic acid sequence to be detected may be a fraction of a large molecule, and from the outset the specific sequence may exist in the form of isolated molecules that make up the entire nucleic acid sequence. The nucleic acid sequence need not be nucleic acid present in pure form, and the nucleic acid may be a small fraction in a complex mixture, such as containing whole human DNA. Nucleic acids included in the sample used to measure the degree of methylation of nucleic acids contained in the sample or used to detect methylated CpG islands are described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY., 1989). It can be extracted by various methods described in.

The nucleic acid may comprise a regulatory site, which is a DNA site that encodes information or regulates transcription of the nucleic acid. The regulatory site comprises at least one promoter. A "promoter" is the minimum sequence necessary to direct transcription and can regulate inducibility by cell type specific, tissue specific or external signal or agent in a promoter-dependent gene. The promoter is located at the 5 'or 3' site of the gene. Nucleic acid numbers of all or part of a promoter site can be applied to measure methylation of CpG island sites. Methylation of the target gene promoter proceeds from outside to inside. Therefore, the early stages of cell turnover can be detected by analyzing methylation outside of the promoter region.

The nucleic acid isolated from the sample is obtained by biological sample of the sample. If one wants to diagnose breast cancer or the stage of its progression, the nucleic acid must be separated from the breast tissue by scrap or biopsy. Such samples may be obtained by various medical procedures known in the art.

In the present invention, "hypermethylation" or "hypermethylation" refers to a state where the degree of methylation at a specific CpG position or a site connected to CpG of tumor cells is higher than that of normal cells.

Sample

The present invention describes early identification of breast cancer and utilizes breast cancer specific gene methylation. Methylation of breast cancer specific genes also occurred in tissues near the tumor site. Therefore, the early identification method of breast cancer can confirm the presence or absence of methylation of breast cancer-specific genes in all samples including liquid or solid tissue. The sample includes, but is not limited to, tissue, cells, urine, serum or plasma.

Individual genes and panels

The present invention can be used individually as a diagnostic or predictive marker, or a combination of two marker genes in the form of a panel display, wherein the two marker genes are weighted according to the presence and extent of the methylated genes together. And the level of likelihood of developing cancer. Such algorithms belong to the present invention.

Methylation Detection Method

Methylation specific PCR

When bisulfite is treated with genomic DNA, cytosine at the 5'-CpG'-3 site remains cytosine as it is methylated and becomes uracil if unmethylated. Therefore, PCR primers corresponding to the sites where the 5'-CpG-3 'nucleotide sequence exists were prepared for the converted nucleotide sequence after bisulfite treatment. In this case, PCR primers for methylation and two primers for unmethylation were prepared. After converting the genomic DNA to bisulfite and PCR using the two kinds of primers, the PCR product is made by using the primer corresponding to the methylated sequence when methylated. In the PCR product is produced by using a primer corresponding to unmethylation. Methylation can be qualitatively confirmed by agarose gel electrophoresis.

Real time methylation specific PCR

Real-time methylation-specific PCR converts the methylation-specific PCR method into a real-time measurement method. After treating bisulfite with genomic DNA, a PCR primer for methylation is designed and real-time PCR is performed using these primers. To do. At this time, there are two methods of detection using a TanMan probe complementary to the amplified base sequence and a method of detection using Sybergreen. Thus, real-time methylation specific PCR can selectively quantitate only methylated DNA. At this time, a standard curve was prepared using an in vitro methylated DNA sample, and amplification of the gene without the 5'-CpG-3 'sequence in the nucleotide sequence by a negative control group was quantitatively analyzed.

Pyro Sequencing

The pyro sequencing method is a method of converting the bisulfite sequencing method into quantitative real-time sequencing. Similar to bisulfite sequencing, genomic DNA was converted by bisulfite treatment, and then PCR primers corresponding to sites without the 5'-CpG-3 'sequence were prepared. After treating genomic DNA with bisulfite, it was amplified by the PCR primers, and real-time sequencing was performed using the sequencing primers. Quantitative analysis of cytosine and thymine at the 5'-CpG-3 'site indicated the methylation degree as the methylation index.

PCR or quantitative PCR and DNA chips using methylated DNA specific binding proteins

In the PCR or DNA chip method using methylated DNA-specific binding proteins, when a protein that specifically binds to methylated DNA is mixed with DNA, only methylated DNA can be selectively separated because the protein specifically binds to methylated DNA. . After genomic DNA was mixed with methylated DNA specific binding proteins, only methylated DNA was selectively isolated. These isolated DNAs were amplified using a PCR primer corresponding to a promoter site, and then methylated by agarose electrophoresis.

In addition, methylation can also be determined by quantitative PCR.Methylated DNA separated by methylated DNA-specific binding proteins can be labeled with a fluorescent dye and hybridized to DNA chips having complementary probes to measure methylation. Can be. Wherein the methylated DNA specific binding protein is not limited to McrBt.

Detection of Differential Methylation—Methylation Sensitivity Restriction Endonuclease

Detection of differential methylation can be accomplished by contacting the nucleic acid sample with a methylated sensitive restriction endonuclease that cleaves only unmethylated CpG sites, thereby cleaving the unmethylated nucleic acid.

In a separate reaction, the samples were contacted with isochimers of methylation sensitive restriction endonucleases that cleave both methylated and unmethylated CpG sites, thereby cleaving the methylated nucleic acids.

Specific primers were added to the nucleic acid sample and the nucleic acid was amplified by conventional methods. If there is an amplification product in the sample treated with the methylation sensitive restriction endonuclease, and there is no amplification product in the isomerized sample of the methylation sensitive restriction endonuclease that cleaves both methylated and unmethylated CpG sites , Methylation occurred in the analyzed nucleic acid site. However, no amplification products were present in the samples treated with methylation sensitive restriction endonucleases, and the amplification products were also found in the samples treated with isosomeomers of methylation sensitive restriction endonucleases that cleave both methylated and unmethylated CpG sites. Existence means that no methylation occurs in the analyzed nucleic acid site.

Here, a "methylation sensitive restriction endonuclease" is a restriction enzyme that contains CG at the recognition site and has activity when C is methylated compared to when C is not methylated (eg, Sma I). Non-limiting examples of methylation sensitivity limiting endonucleases include Msp I, Hpa II, Bss HII, Bst UI and Not I. The enzymes may be used alone or in combination. Other methylation sensitivity limiting endonucleotides include, but are not limited to, for example, Sac II and Eag I.

Isochimers of methylation sensitive restriction endonucleases are restriction endonucleases that have the same recognition site as methylation sensitive restriction endonucleases, but cleave both methylated and unmethylated CGs, e.g., Msp. I can be mentioned.

The primers of the present invention are designed to have "alternatively" complementarity with each strand of the locus to be amplified and, as described above, include the appropriate G or C nucleotides. This means that the primers have sufficient complementarity to hybridize with the corresponding nucleic acid strands under the conditions for carrying out the polymerization. The primer of the present invention is used in the amplification process, which is an enzymatic continuous reaction in which the target locus, such as PCR, increases to an exponential number through many reaction steps. Typically, one primer (antisense primer) has homology to the negative (-) strand of the locus and the other primer (sense primer) has homology to the positive (+) strand. When the primer is annealed to the denatured nucleic acid, the chain is stretched by enzymes and reactants such as DNA polymerase I (Klenow) and nucleotides, resulting in newly synthesized + and-strands containing the target locus sequence. The newly synthesized target locus is also used as a template, and the cycle of denaturation, primer annealing and chain extension repeats exponential synthesis of the target locus sequence. The product of the continuous reaction is an independent double stranded nucleic acid having an end corresponding to the end of the specific primer used in the reaction.

The amplification reaction is preferably a PCR that is commonly used in the art. However, alternative methods such as real-time PCR or linear amplification with isothermal enzymes can also be used, and multiplex amplification reactions can also be used.

Detection of Differential Methylation-Bisulfite Sequencing Method

Other methods of detecting nucleic acids containing methylated CpG include contacting a sample containing nucleic acid with an agent that modifies unmethylated cytosine and amplifying the CpG-containing nucleic acid of the sample using CpG-specific oligonucleotide primers. It includes. Here, the oligonucleotide primer may be characterized by detecting the methylated nucleic acid by distinguishing the modified methylated and unmethylated nucleic acid. The amplification step is optional and desirable but not necessary. The method relies on a PCR reaction that distinguishes between modified (eg, chemically modified) methylated and unmethylated DNA. Such methods are disclosed in US Pat. No. 5,786,146, which is described in connection with bisulfite sequencing for the detection of methylated nucleic acids.

temperament

Once the target nucleic acid site is amplified, the nucleic acid amplification product can be hybridized with a known gene probe immobilized on a solid support (substrate) to detect the presence of the nucleic acid sequence.

Here, a "substrate" is a mixture means comprising a substance, structure, surface or material, abiotic, synthetic, inanimate, planar, spherical or specific binding, flat surface material, hybridization or enzyme recognition site Or many other recognition sites beyond the vast majority of other recognition sites or numerous other molecular species composed of surfaces, structures or materials. Such substrates include, for example, semiconductors, (organic) synthetic metals, synthetic semiconductors, insulators and dopants; Metals, alloys, elements, compounds and minerals; Synthesized, degraded, etched, lithographic, printed and microfabricated slides, devices, structures and surfaces; Industrial, polymers, plastics, membranes, silicones, silicates, glass, metals and ceramics; Wood, paper, cardboard, cotton, wool, cloth, woven and non-woven fibers, materials and fabrics, but are not limited thereto.

Some types of membranes are known in the art to have adhesion to nucleic acid sequences. Specific and non-limiting examples of such membranes include membranes for gene expression detection, such as nitrocellulose or polyvinylchloride, diaotized paper and commercially available membranes such as GENESCREENTM, ZETAPROBETM (Biorad) and NYTRANTM. have. Beads, glass, wafers and metal substrates are also included. Methods of attaching nucleic acids to such objects are well known in the art. Alternatively, screening can also be performed in the liquid phase.

Hybridization conditions

In nucleic acid hybridization reactions, the conditions used to achieve stringent specific levels vary depending on the nature of the nucleic acid being hybridized. For example, the length of the nucleic acid region to be hybridized, degree of homology, nucleotide sequence composition (eg, GC / AT composition ratio), and nucleic acid type (eg, RNA, DNA) select hybridization conditions. Is considered. Further considerations are whether the nucleic acid is immobilized, for example, in a filter or the like.

Examples of very stringent conditions are as follows: 2X SSC / 0.1% SDS at room temperature (hybridization conditions); 0.2X SSC / 0.1% SDS at room temperature (low stringency conditions); 0.2X SSC / 0.1% SDS at 42 ° C. (conditions with moderate stringency); 0.1X SSC at 68 ° C. conditions with high stringency. The washing process can be carried out using one of these conditions, for example a condition with high stringency, or each of the above conditions, each of 10-15 minutes in the order described above, all or all of the conditions described above. Some iterations can be done. However, as described above, the optimum conditions vary with the particular hybridization reaction involved and can be determined experimentally. In general, conditions of high stringency are used for hybridization of critical probes.

Label

Important probes are labeled so that they can be detected, for example, with radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelates or enzymes. Proper labeling of such probes is a technique well known in the art and can be carried out by conventional methods.

Kit

According to the present invention, there is provided a kit useful for detecting cell growth abnormality of a specimen. Kits of the invention include a compartmentalized carrier means for holding a sample, a first container containing an agent that sensitively cleaves unmethylated cytosine, a second container containing a primer for amplifying a CpG containing nucleic acid, and a cleaved or uncleaved nucleic acid. One or more containers including a third container containing means for detecting the presence. Primers used in accordance with the present invention include the sequences set forth in SEQ ID NOs: 5-21, functional combinations, and fragments thereof. Functional combinations or fragments are used as primers to detect whether methylation has occurred on the genome.

The carrier means is suitable for containing one or more containers such as bottles, tubes, each container containing independent components used in the method of the invention. In the context of the present invention, one of ordinary skill in the art can readily dispense the required formulation in the container. For example, a container containing methylation sensitivity restriction enzyme may be configured as one container. One or more containers may contain primers that have homology with important nucleic acid locus. In addition, one or more of the containers may contain isoxisomers of methylation sensitive restriction enzymes.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1 Screening of Hypermethylated Genes in Breast Cancer Tissue Samples

In order to select hypermethylated genes in breast cancer, breast cancer tissues and adjacent normal-tissue tissue samples were selected as follows.

First, genomic DNA was isolated from each tissue sample. 500 ng of genomic DNA was fragmented into 300-400 bp by ultrasonic disruption (Vibra Cell, SONICS), and methylated DNA was selectively isolated from each cell line using MethylcaptureTM (Genomictree, South Korea). After amplifying the isolated methylated DNA using GenomePlex® Complete Whole Genome Amplification Kit (Sigma, USA), amplified DNA from breast cancer tissue was labeled with Cy5-dUTP, and amplified DNA from adjacent normal-tissue tissue was labeled with Cy3-dUTP. Hybridization was performed using a CpG microarray (manufactured by Agilent, Inc.), in which probes for CpG present in about 27,800 CpG islands present in the human genome were integrated. After the hybridization, a series of washing procedures were performed, followed by scanning using an Agilent scanner. The calculation of signal values from microarray images can be found in the Feature Extraction Program v. 9.5.3.1 (Agilent) was used to calculate the relative difference in signal intensity between adjacent normal-tissue tissue and breast cancer tissue samples, and the difference in methylation level between adjacent normal and breast cancer tissues was analyzed using GeneSpringGX (Agilent). Large genes were selected. Through statistical techniques, 60 hypermethylated candidate genes were selected from breast cancer tissues, and a large number of genes in the gene group were selected (FIG. 1).

Example 2: Gene Selection Reactivated by Demethylation

To confirm whether the gene selected in Example 1 is regulated by promoter methylation, 5-aza- which is a dimethylating agent in breast cancer cell lines MCF-7 (KCLB (Korea Cell Line Bank) 30022) and MDAMB-231 (KCLB 30026)). 2-deoxycytidine (DAC, Sigma, USA) was treated for 5 days at 200 nM concentration. Untreated cell lines and treated cell lines were treated with Tri reagent to separate total RNA.

By directly comparing transcription levels between the DAC untreated cell line and the DAC treated cell line, changes in gene expression by DAC treatment were measured. As a result, it was confirmed that the expression of 103 genes was increased when the DAC was treated, compared to the control without the DAC. A list of 60 breast cancer specific hypermethylated genes obtained in Example 1 and genes re-expressed more than two times by 103 dimethylation were compared, and six common genes were selected (FIG. 1 and Table 1).

  List of Genes Gene
Symbol GenBank
No. Description Chr.
location CHL1 NM_006614 cell adhesion molecule with homology to
L1CAM (close homolog of L1) 3p26.1 TF NM_001063 transferrin 3q22.1 CGNL1 NM_032866 cingulin-like 1 15q21.3 ITGB8 NM_002214 integrin, beta 8 7p15.3 LAMA3 NM_198129 laminin, alpha 3 18q11.2 NTRK2 NM_001007097 neurotrophic tyrosine kinase, receptor, type 2 9q22.1

Example 3: Methylation Measurement of Biomarker Genes in Cancer Cell Lines

To further confirm the methylation status of the six genes selected in Example 2, bisulfite pyro sequencing was performed for each promoter.

In order to transform unmethylated cytosine to uracil using bisulfite, whole genomic DNA was isolated from breast cancer cell lines MCF-7 (KCLB 30022) and MDAMB-231 (KCLB 30026), and 200 ng of genomic DNA was isolated. Bisulfite was treated using the EZ DNA methylation-Gold kit (Zymo Research, USA). Treatment of DNA with bisulfite leaves unmethylated cytosine modified with uracil and methylated cytosine remains unchanged. The bisulfite-treated DNA was eluted with 20 µl of sterile distilled water to perform pyrosequencing.

PCR and sequencing primers for performing pyro sequencing for the six genes were designed using the PSQ assay design program (Biotage, USA). PCR and sequencing primers for methylation determination of each gene are shown in Tables 2 and 3.

PCR primers and conditions gene primer Sequence (5 '→ 3') order
number CpG location a Amplicon size CHL1

forward GTG TAA AGG GAG GGA AGA TAA G 3 -227, -224, -208, -205 70 bp

reverse Biotin-CCT CTA CTC CCT TCC TAA ATT CT 4 TF

forward AGT AAG GAA GGG GGG TTG 5 -55, -46, -37 108 bp

reverse Biotin-TCC CTT TAT TCC ATT CCC 6 CGNL1

forward AAG GGA AAG GGG TAA ATT GAG T 7 -367, -364, -358, -352, -350 82 bp

reverse Biotin-CCT CTC CAA ATT CCT AAC CTA CAA 8 LAMA3 forward GGG TAT TGG GGT TAG TAT T 9 +136, +139,
+142, +145 276 bp reverse Biotin-AAA CCC TCA CCT AAA TAA TAT AAC 10 ITGB8 forward GGG GAG GTT TTT TGA TTT TA 11 -721, -712, -709, -702 101 bp reverse Biotin-ACA AAC CCC AAT CTC TCA 12 NTRK2 forward GAG TAA GGG TTT GGG ATG TTT TA 13 +270, +285, +287 105bp reverse Biotin-TCA TTT TCT CTC ATC CTT TAA CCT 14

^a nucleotide from the transcription initiation point (+1): the position on the genomic DNA of the CpG site used to measure methylation

Sequencing Primer Sequences of Methylation Marker Genes gene order SEQ ID NO: CHL1 GTA AAG GGA GGG AAG AT 15 TF GGG AGG TTA AAG ATT 16 CGNL1 AAA GGG GTA AAT TGA GTT 17 LAMA3 GGG TAT TGG GGT TAG TA 18 ITGB8 GGG GTA GTG AGT ATT T 19 NTRK2 GGG TTT GGG ATG TTT TA 20

20 ng of genomic DNA converted to bisulfite was amplified by PCR. PCR reaction solution (20 ng genomic DNA converted to bisulfite, 5 μl of 10X PCR buffer (Enzynomics, Korea), 5 units of Taq polymerase (Enzynomics, Korea), 4 μl of 2.5 mM dNTP (Solgent, Korea), 2 μl of PCR primer) (10 pmole / μl)) was treated at 95 ° C. for 5 minutes, followed by 45 times of 40 seconds at 95 ° C., 45 seconds at 60 ° C., and 40 seconds at 72 ° C., followed by reaction at 72 ° C. for 5 minutes. Amplification of the PCR product was confirmed by electrophoresis using 2.0% agarose gel.

After the amplified PCR product was treated with PyroGold reagent (Biotage, USA), pyro sequencing was performed using the PSQ96MA system (Biotage, USA). After the pyro sequencing, the degree of methylation was measured by calculating the methylation index. The methylation index was calculated by calculating the average rate of cytosine binding at each CpG site.

As a result of quantitatively expressing the degree of methylation of six biomarkers in breast cancer cell lines using the pyro sequencing method, it was confirmed that three markers among the six biomarker genes were methylated at high levels in both cell lines (FIG. 2). ).

Example 4: Determination of Hypermethylation of Biomarker Genes in Breast Cancer Tissues

As shown in Examples 1 to 3, CHL1 and TF hypermethylated in breast cancer tissues, reactivation of marker expression under dimethylation conditions, containing CpG islands in the promoter region, and hypermethylated in breast cancer cell lines In order to verify that the CGNL1 gene can be used as a biomarker for diagnosing breast cancer, methylation assays were performed on three biomarkers using 10 healthy breast tissues and 23 breast cancer tissue clinical specimens. The methylation assay was performed by the method described in Example 3 using the pyro sequencing method.

As a result, compared to normal breast tissue CHL1 and TF genes except CGNL1 organization of breast cancer patients it showed a very high degree of methylation levels (Fig. 3). ROC curve analysis was performed to determine the methylation index cut-off for diagnosing breast cancer patients, and to determine sensitivity and specificity. As a result, CHL1 showed 36.4% sensitivity, 100% specificity, and TF showed 95.5% sensitivity and 100% specificity, thus confirming that it is an excellent biomarker for diagnosing breast cancer. In addition, when the two marker genes were used as a panel, the sensitivity and specificity were calculated as 100% and 100%, respectively, showing the potential as an ideal breast cancer diagnosis marker.

Sequence of Methylation Marker Gene Promoter gene SEQ ID NO: CHL1 One TF 2

Example 5: Determination of Hypermethylation of Biomarker Gene Using Serum

There are several reports confirming that methylation of biomarkers can be detected using serum, a non-invasive sample, and the serum convenience of CHL1 and TF can be diagnosed by using serum for early and recurrence of breast cancer. The degree of hypermethylation of the gene was confirmed. Fifteen normal serum and fifteen breast cancer sera were tested for methylation-specific PCR analysis of CHL1 and TF genes.

Because of the limitation of detection sensitivity when pyro sequencing was applied to serum, a noninvasive sample, it was confirmed that the methylation difference between normal serum sample and cancer patient sample could not be represented (Yulia). Korshunova et al. , Genome Res. , 18: 19-29, 2008). In this case, a relatively high sensitivity methylation-specific PCR assay was applied to the non-invasive serum sera.

To confirm the degree of hypermethylation of the non-invasive samples of the CHL1 and TF genes, free-DNA was purified from the serum samples using silica gel. Bisulfite was treated using EZ DNA methylation-Gold kit (Zymo Research, USA) on 200ng of purified free-DNA. The bisulfite-treated DNA was eluted with 20 µl of sterile distilled water to perform methylation-specific PCR (methylation-specific PCR) analysis.

Methylated and unmethylated specific primers for performing methylation specific PCR analysis were designed using the MethPrimer program. PCR primers corresponding to the sites where the 5'-CpG-3 'base sequence exists were prepared for the converted base sequence after bisulfite treatment. In this case, two kinds of primers corresponding to the methylated PCR primer and the unmethylated primer were prepared, and the sequence of each primer is shown in Table 3.

20ng of the bisulfite-treated genomic DNA was subjected to PCR using methylated specific primers and non-methylated specific primers, respectively. PCR was carried out a total of 40 cycles of 1 minute at 94 ° C, 1 minute at 60 ° C, and 1 minute at 72 ° C, and finally extended at 72 ° C for 10 minutes. Thereafter, the amplified products were identified by agarose gel, respectively, to confirm that methylated specific products were amplified. When methylated specific products were identified, the samples were determined to contain methylated genes.

As a result, it was found that the degree of hypermethylation of CHL1 and TF genes was higher in serum samples of breast cancer patients than in normal human serum (FIG. 4). The serum sensitivity of CHL1 and TF genes to the detection of breast cancer was 66.7% and 46.2%, respectively, and the specificity was 93.3% and 80%, respectively (Table 5). . Therefore, by measuring DNA hypermethylation of CHL1 and TF genes using sera, a non-invasive clinical sample, it can be applied to various diagnosis of breast cancer such as early diagnosis, recurrence, monitoring after drug treatment and monitoring of recurrence after surgery. It was confirmed that there is.

Sensitivity and Specificity of CHL1 and TF Genes in Serum Samples gene No. of methylation positive sample No. of methylation negative samples Sensitivity Specificity Cancer Normal CHL1 6/9 1/15 66.7% 93.3% TF 6/13 3/15 46.2% 80%

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a schematic diagram showing a process of discovering a methylated biomarker for diagnosing breast cancer through CpG microarray analysis and comparative selection of genes re-expressed by demethylation of adjacent normal-tissue tissues.

      Figure 2 shows the degree of quantitative methylation through pyro sequencing of six methylated genes in breast cancer cell lines.

Figure 3 measures the degree of methylation of CHL1 and TF genes in normal tissues and breast cancer tissues, ROC curve analysis was performed to measure the ability to diagnose breast cancer. ( ^* criterion: cut-off; Sensitivity: sensitivity; Specificity: specificity).

Figure 4 shows the positive frequency of methylation of CHL1 and TF gene promoter using serum DNA of normal and breast cancer patients ( ^* red box: methylated sample, gray box: non-methylated sample)

<110> Genomic Tree, Inc. <120> Method for Detecting Breast Cancer Using Breast Cancer Specific          Methylation Marker Genes <130> P09-B038 <160> 20 <170> KopatentIn 1.71 <210> 1 <211> 3000 <212> DNA <213> Homo sapiens <400> 1 ctttcgtaaa atttggttgc ctgggataac gtgtattgca tacaagctga cttccttact 60 gtccctagta agctatgtgt ggctattatt tcttcattta tatgcaatgc cagaagctta 120 gaagaatgtt atctcccttt cagttaaaaa ctcatagtta agaactggcc atcttcttgt 180 tttgggtatt gtggcttggt actgtctaat atatttggag ggtatttgat tttttaaaaa 240 gctactggcc atgcctattg agtttcctac tgagcccagc acccagacca gtgattgatt 300 aaatagtagc tgctcaataa atatttgttt tgtaaataat gattctattg tcaagaaaca 360 aatcaccaag aaagaaggag ctatatattt aagagtactc tgttcatctg gccaaaatgg 420 agttccgagt cccttgctca gctgatgttt gttcacccag aagatggtaa actcctgaga 480 ccaacttcgc tttgttgttc tcttgtggtt ccttgacctt ggaaatgttt ttctacaggc 540 agagtatttc cccctgttgg aattttattt aatattctat ctttgtttct actttagaaa 600 acatgccacg tgaataatga tgcctcaggc aaaagaaatc actccagcag tacaatttaa 660 acttaggaca aatcctgccc tcacttgcat tcacaatttg ccaaggcagt tggtaagaac 720 tgtgcccaca cacaaattga cagaatcgag gccccaaggt tagcgatgat taaaatttaa 780 aagtccattc attcattttc aaaatatttt attcgattgc ttctgaggca gaatcatgag 840 ttgccttcgc ttttgctgct cccggtggtg gcatccaact ctgaggatag ctcatccaac 900 tctgagggta gctctgaggg gagctaaata tcctcattgt tcaatgagct agatgaaaac 960 aaaacgaggc tgtaaggtca atctcacagc cttagtggag taaaaattag aagtcaataa 1020 ttctcttacc aacaaagagg acttgcattc aacaggattt tcaaaagtgg tatcctgtaa 1080 aaattttgga aaataccacc ataatggaaa aaaaataaag tggcatataa atcaaatgta 1140 gtgctaaaaa ttataatttt tccatatgaa ttttggaatc ctaaaatatt tttatgtgtt 1200 tgtggattca taaggcatca gaactggctt tttgcactga atatgagtac agaaggcttt 1260 cctctccaat agtgaattaa taaaggataa gtaggaaaaa cgttacgaaa cgatgtggta 1320 acattataga ctctgtatta gggcaaaaat tgtgagatag tagaagatct gtaaatttaa 1380 gcatgcacaa tttgacagcg gatggtcgta gcatattttg taaattcaat agcaagcccc 1440 accacgccct taaatgaagg aaagcaagaa gacaattttt aatacgtaca tggcaccaca 1500 cccctctaag acgcagcaat gggagaaaag cagattggcg acctgtgtgt gcaacatgaa 1560 ccgagtgaaa ccatcgggga gggggtgggg ggcgtttctt ttaaatgtcg cttttgcaga 1620 taaacgagca gggatcctac ctcttttgac cggagatgtt gaaaagcacg accaaagttt 1680 ttggcttggt ctcttgcttt ccacaacgag ataaagtctg aagcctgtaa ctctccctgt 1740 cccgcgggag cgtgcaaagg gagggaagat aagcggcgtg ggtgaggggt ggcggcgcag 1800 aacccaggaa gggagcagag gcgagagcca gcctccggca ggagggcgta gaccccggtg 1860 ccagccagaa cggctccagc cttcccgcgg ccagagagcc agcggcggga ggggacgggc 1920 gggacctccg cggacagccc cgggtgcgga ccagcgggca gcggccggcg agaccctccc 1980 tggatccgcc cagagcttac cggaccctgc gcgcccccgt cccggctccc ggccggctcg 2040 ggggagaagg cgcccgaggg gaggcgccgg acagatcgcg tttcggaggc ggcgcaggtg 2100 tgtcaggggt gggggtccgc tggcatctct cgtgccggcg ctgggggggt ctcccgtgga 2160 ggaagggctg aaggtgctcc gggaggggtc cactcgttat ggaaaggaag cggggcgcgg 2220 tcagggtccc tcgtccgcag tgggggtggt ccgggcccct cagcctgcac ccgggaggag 2280 acgccccctt ttcacgcccc gcccccggtg gccgaggccg gattcggaca gctcggggtc 2340 agtcgacgga gcacaccggg cgcccgaatc cgccgggtcc cactcacgcc cgccggccgc 2400 cggtaggttg gccctcccgc ttccagcctc cgccacccca tccctcgctg ctctctgagg 2460 aggattccag acccccccgt agatctccgc cctctctcgg gtggggctcc gaggtctagc 2520 tggtctgggg gtcagtcccg gcctctgtct cccaccgcgc ccctcgctcc cctggacacc 2580 gcctggtgtg tggggctcag acccccccag tccactgccc ctgtgtggct cctcctctca 2640 ggagaggatt ctagatccca gcccgggggg ggttccgaaa atgccgccag cctggagcgg 2700 ggaatccatg gaccgtgggg ttgtggggtt ggggagcacg aaaacccaga gaggggctag 2760 agctccacct ccccagaccg ccgaggggcg agagggcgcg ccgggggccg tggttgccat 2820 ggctcctggt agcggagccc ggggggctgg agatgccggt cgcggatggt cccttcttcc 2880 tctttgtgcc tctctctttc tctcgctttt tttttttttt tggagtgtga gtgtgcgtgg 2940 ggaggcagca cggagaaagt gattaatttg gggagcaggg attggagccg ggaggctggg 3000                                                                         3000 <210> 2 <211> 3000 <212> DNA <213> Homo sapiens <400> 2 ctttcgtaaa atttggttgc ctgggataac gtgtattgca tacaagctga cttccttact 60 gtccctagta agctatgtgt ggctattatt tcttcattta tatgcaatgc cagaagctta 120 gaagaatgtt atctcccttt cagttaaaaa ctcatagtta agaactggcc atcttcttgt 180 tttgggtatt gtggcttggt actgtctaat atatttggag ggtatttgat tttttaaaaa 240 gctactggcc atgcctattg agtttcctac tgagcccagc acccagacca gtgattgatt 300 aaatagtagc tgctcaataa atatttgttt tgtaaataat gattctattg tcaagaaaca 360 aatcaccaag aaagaaggag ctatatattt aagagtactc tgttcatctg gccaaaatgg 420 agttccgagt cccttgctca gctgatgttt gttcacccag aagatggtaa actcctgaga 480 ccaacttcgc tttgttgttc tcttgtggtt ccttgacctt ggaaatgttt ttctacaggc 540 agagtatttc cccctgttgg aattttattt aatattctat ctttgtttct actttagaaa 600 acatgccacg tgaataatga tgcctcaggc aaaagaaatc actccagcag tacaatttaa 660 acttaggaca aatcctgccc tcacttgcat tcacaatttg ccaaggcagt tggtaagaac 720 tgtgcccaca cacaaattga cagaatcgag gccccaaggt tagcgatgat taaaatttaa 780 aagtccattc attcattttc aaaatatttt attcgattgc ttctgaggca gaatcatgag 840 ttgccttcgc ttttgctgct cccggtggtg gcatccaact ctgaggatag ctcatccaac 900 tctgagggta gctctgaggg gagctaaata tcctcattgt tcaatgagct agatgaaaac 960 aaaacgaggc tgtaaggtca atctcacagc cttagtggag taaaaattag aagtcaataa 1020 ttctcttacc aacaaagagg acttgcattc aacaggattt tcaaaagtgg tatcctgtaa 1080 aaattttgga aaataccacc ataatggaaa aaaaataaag tggcatataa atcaaatgta 1140 gtgctaaaaa ttataatttt tccatatgaa ttttggaatc ctaaaatatt tttatgtgtt 1200 tgtggattca taaggcatca gaactggctt tttgcactga atatgagtac agaaggcttt 1260 cctctccaat agtgaattaa taaaggataa gtaggaaaaa cgttacgaaa cgatgtggta 1320 acattataga ctctgtatta gggcaaaaat tgtgagatag tagaagatct gtaaatttaa 1380 gcatgcacaa tttgacagcg gatggtcgta gcatattttg taaattcaat agcaagcccc 1440 accacgccct taaatgaagg aaagcaagaa gacaattttt aatacgtaca tggcaccaca 1500 cccctctaag acgcagcaat gggagaaaag cagattggcg acctgtgtgt gcaacatgaa 1560 ccgagtgaaa ccatcgggga gggggtgggg ggcgtttctt ttaaatgtcg cttttgcaga 1620 taaacgagca gggatcctac ctcttttgac cggagatgtt gaaaagcacg accaaagttt 1680 ttggcttggt ctcttgcttt ccacaacgag ataaagtctg aagcctgtaa ctctccctgt 1740 cccgcgggag cgtgcaaagg gagggaagat aagcggcgtg ggtgaggggt ggcggcgcag 1800 aacccaggaa gggagcagag gcgagagcca gcctccggca ggagggcgta gaccccggtg 1860 ccagccagaa cggctccagc cttcccgcgg ccagagagcc agcggcggga ggggacgggc 1920 gggacctccg cggacagccc cgggtgcgga ccagcgggca gcggccggcg agaccctccc 1980 tggatccgcc cagagcttac cggaccctgc gcgcccccgt cccggctccc ggccggctcg 2040 ggggagaagg cgcccgaggg gaggcgccgg acagatcgcg tttcggaggc ggcgcaggtg 2100 tgtcaggggt gggggtccgc tggcatctct cgtgccggcg ctgggggggt ctcccgtgga 2160 ggaagggctg aaggtgctcc gggaggggtc cactcgttat ggaaaggaag cggggcgcgg 2220 tcagggtccc tcgtccgcag tgggggtggt ccgggcccct cagcctgcac ccgggaggag 2280 acgccccctt ttcacgcccc gcccccggtg gccgaggccg gattcggaca gctcggggtc 2340 agtcgacgga gcacaccggg cgcccgaatc cgccgggtcc cactcacgcc cgccggccgc 2400 cggtaggttg gccctcccgc ttccagcctc cgccacccca tccctcgctg ctctctgagg 2460 aggattccag acccccccgt agatctccgc cctctctcgg gtggggctcc gaggtctagc 2520 tggtctgggg gtcagtcccg gcctctgtct cccaccgcgc ccctcgctcc cctggacacc 2580 gcctggtgtg tggggctcag acccccccag tccactgccc ctgtgtggct cctcctctca 2640 ggagaggatt ctagatccca gcccgggggg ggttccgaaa atgccgccag cctggagcgg 2700 ggaatccatg gaccgtgggg ttgtggggtt ggggagcacg aaaacccaga gaggggctag 2760 agctccacct ccccagaccg ccgaggggcg agagggcgcg ccgggggccg tggttgccat 2820 ggctcctggt agcggagccc ggggggctgg agatgccggt cgcggatggt cccttcttcc 2880 tctttgtgcc tctctctttc tctcgctttt tttttttttt tggagtgtga gtgtgcgtgg 2940 ggaggcagca cggagaaagt gattaatttg gggagcaggg attggagccg ggaggctggg 3000                                                                         3000 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gtgtaaaggg agggaagata ag 22 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cctctactcc cttcctaaat tct 23 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 agtaaggaag gggggttg 18 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tccctttatt ccattccc 18 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 aagggaaagg ggtaaattga gt 22 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cctctccaaa ttcctaacct acaa 24 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gggtattggg gttagtatt 19 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 aaaccctcac ctaaataata taac 24 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 ggggaggttt tttgatttta 20 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 acaaacccca atctctca 18 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gagtaagggt ttgggatgtt tta 23 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tcattttctc tcatccttta acct 24 <210> 15 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gtaaagggag ggaagat 17 <210> 16 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 gggaggttaa agatt 15 <210> 17 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 aaaggggtaa attgagtt 18 <210> 18 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 gggtattggg gttagta 17 <210> 19 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 ggggtagtga gtattt 16 <210> 20 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gggtttggga tgtttta 17  

Claims (6)

delete delete Detection of methylation of breast cancer or its advanced stage diagnostic biomarker containing CpG of promoter region of CHL1 gene {NM_006614, cell adhesion molecule with homology to L1CAM (close homolog (heart)) Way: (a) isolating DNA from clinical samples; And (b) detecting methylation of CHL1 gene {NM_006614, cell adhesion molecule with homology to L1CAM (close homolog of L1)} in the isolated DNA. delete The method of claim 3, wherein the methylation detection is performed by PCR, methylation specific PCR, real time methylation specific PCR, PCR using methylated DNA specific binding protein, quantitative PCR, DNA chip, pi And sequencing and bisulfite sequencing. The method of claim 3, wherein the clinical sample is selected from the group consisting of tissue, sputum, cells, blood, plasma, and urine from patients suspected of cancer or from being diagnosed.

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