Method for detecting lung cancer

Abstract

The present invention relates to a method for providing information for lung cancer diagnosis, a composition for diagnosing lung cancer, and a kit comprising the same, and more particularly, to a method for providing information for lung cancer diagnosis by using a primer for specifically amplifying a plurality of methylated lung cancer marker genes, the invention relates to a composition for diagnosing lung cancer and a kit comprising the composition.

Description

Technical Field

[0001] The present invention relates to a method for providing information on lung cancer diagnosis, a composition for diagnosing lung cancer, and a kit comprising the composition, and more particularly, to a method for providing information on lung cancer diagnosis by using primers that specifically amplify multiple methylated lung cancer marker genes, a composition for diagnosing lung cancer, and a kit comprising the composition. Background Art

[0002] In the genomic DNA of mammalian cells, in addition to A, C, G and T, there is a fifth base, 5-methylcytosine (5-mC), in which a methyl group is attached to the fifth carbon of the cytosine ring. 5-mC is always located only at the C of the CG dinucleotide (5'-mCG-3'), and this CG is usually marked as CpG. Most of the C of CpG is methylated by attaching a methyl group. Methylation of this CpG inhibits the expression of repetitive sequences in the genome, such as alu and transposons, and is the most common site of extragenomic changes in mammalian cells. The 5-mC of this CpG is spontaneously deaminated and becomes T, resulting in a frequency of only 1% of CpG in the mammalian genome, which is much lower than the normal frequency (1 / 4x 1 / 4=6.25%).

[0003] Some CpGs appear abnormally dense, and these are called CpG sites (CpG islands). CpG sites are defined as regions with a length of 0.2-3kb, a distribution percentage of C and G bases greater than 50%, and a high CpG concentration and a distribution percentage of at least 3.75%. There are about 45,000 CpG sites in the entire human genome, and they are particularly concentrated in promoter regions that regulate gene expression. In fact, CpG sites appear in the promoters of housekeeping genes, which account for about half of all human genes (Cross, S. et al., Curr. Opin. Gene Develop., 5:309, 1995). It is known that abnormal DNA methylation occurs mainly in the 5' regulatory region of a gene, resulting in reduced expression of the gene.

[0004] In normal human somatic cells, CpG islands in the promoter regions of housekeeping genes are not methylated, but genes that are imprinted and not expressed during development and genes inactivated on the X chromosome are methylated.

[0005] During carcinogenesis, promoter CpG islands are methylated and the expression of corresponding genes is disturbed. In particular, CpG island methylation in the regulatory regions of tumor suppressor genes that regulate cell cycle and apoptosis, repair DNA, participate in cell adhesion and cell-cell interaction, and inhibit invasion and metastasis will block the expression and function of these genes, such as mutations in coding sequences, and thus promote the occurrence and progression of cancer. In addition, partial methylation of CpG islands occurs with aging.

[0006] Methylation of tumor-related gene regulatory regions is an important marker of cancer and can be used for a variety of purposes, including diagnosis and early detection of cancer, prediction of carcinogenic risk, prognosis of cancer, follow-up after treatment, and prediction of chemotherapy response. In fact, recently, some people have tried to study the promoter methylation of tumor-related genes in blood, sputum, saliva, feces, urine, etc., and use it for various cancer therapies (Ahlquist, DA et al., Gastroenterol., 119: 1219, 2000).

[0007] Under this technical background, the inventors of the present invention found that the methylation of lung cancer marker genes can be detected with high detection sensitivity and accuracy by using primers that specifically amplify multiple methylated lung cancer marker genes, and completed the present invention. Summary of the invention

[0008] An object of the present invention is to provide a method for providing information on lung cancer diagnosis by using primers that specifically amplify a plurality of methylated lung cancer marker genes.

[0009] The object of the present invention is to provide a method for diagnosing lung cancer by using primers that specifically amplify a plurality of methylated lung cancer marker genes.

[0010] The present invention aims to provide a composition for diagnosing lung cancer by using primers that specifically amplify multiple methylated lung cancer marker genes.

[0011] The object of the present invention is to provide a kit for diagnosing lung cancer comprising the composition.

[0012] In order to achieve the above-mentioned purpose, the present invention relates to a method for providing information for lung cancer diagnosis, comprising the following steps: (a) treating a sample with one or more reagents that differentially modify methylated PCDHGA12 (protocadherin gamma subfamily A, 12) gene and CDO1 (cysteine ​​dioxygenase type 1) gene, unmethylated PCDHGA12 gene and CDO1 gene; and (b) treating primers that specifically amplify the methylated PCDHGA12 gene and the CDO1 gene.

[0013] The present invention also provides a composition for diagnosing lung cancer, comprising (a) one or more reagents for differentially modifying methylated PCDHGA12 (protocadherin gamma subfamily A, 12) gene and CDO1 (cysteine ​​dioxygenase type 1) gene, unmethylated PCDHGA12 gene and CDO1 gene; and (b) primers for specifically amplifying the methylated PCDHGA12 gene and the CDO1 gene.

[0014] The present invention also provides a kit for diagnosing lung cancer, comprising the composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The results of methylation-specific amplification of PCDHGA12 and CDO1 genes using methylation-specific primers are shown.

[0016] Figure 2 The methylation status of PCDHGA12 and CDO1 genes in bronchial lavage fluid of patients with benign lung disease and lung cancer and the results of receiver operating characteristic (ROC) analysis are shown.

[0017] Figure 3 Shown are the results of diagnosing lung cancer by detecting methylation of PCDHGA12 and CDO1 genes in bronchial lavage fluid of patients with benign lung diseases and lung cancer. DETAILED DESCRIPTION

[0018] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by those skilled in the art. Generally, the terms used herein are well known and commonly used in the art.

[0019] The present invention relates to a method for detecting methylation of a lung cancer-specific methylation gene CDO1 to detect lung cancer, and more specifically, provides information for diagnosing lung cancer by measuring the methylation of a specific methylation gene in lung cancer cells. In addition, when combined with the methylation of a lung cancer-specific methylation gene PCDHGA12, the present invention provides improved lung cancer detection capabilities.

[0020] The inventors of the present invention have found that by using primers that specifically amplify multiple methylated lung cancer marker genes, the sensitivity and specificity of methylated DNA detection can be improved compared with methylation detection based on a single lung cancer marker gene. In a specific example according to the present invention, by detecting the methylation of the PCDHGA12 (protocadherin gamma subfamily A, 12) gene and the CDO1 (cysteine ​​dioxygenase type 1) gene, the sensitivity and specificity of lung cancer diagnosis are improved compared with the methylation detection of the SDC2 gene, confirming its usefulness for lung cancer diagnosis.

[0021] In order to achieve the above-mentioned purpose, the present invention relates to a method for providing information for lung cancer diagnosis, comprising the following steps: (a) treating a sample with one or more reagents that differentially modify methylated PCDHGA12 (protocadherin gamma subfamily A, 12) gene and CDO1 (cysteine ​​dioxygenase type 1) gene, unmethylated PCDHGA12 gene and CDO1 gene; and (b) treating primers that specifically amplify the methylated PCDHGA12 gene and the CDO1 gene.

[0022] Therefore, the present invention relates to a method for diagnosing lung cancer, comprising the following steps: (a) treating a sample with one or more reagents that differentially modify methylated PCDHGA12 (protocadherin gamma subfamily A, 12) gene and CDO1 (cysteine ​​dioxygenase type 1) gene, unmethylated PCDHGA12 gene and CDO1 gene; and (b) treating primers that specifically amplify the methylated PCDHGA12 gene and the CDO1 gene.

[0023] In the present invention, step (a) is to treat the sample with one or more reagents that differentially modify the methylated PCDHGA12 gene and CDO1 gene, and the unmethylated PCDHGA12 gene and CDO1 gene.

[0024] As used herein, "methylation" refers to the attachment of a methyl group to the fifth carbon of the cytosine base ring to form 5-methylcytosine (5-mC), wherein 5-methylcytosine is always located only at the C of a CG dinucleotide (5'-mCG-3'), and such a CG is generally referred to as CpG. This methylation of CpG inhibits the expression of repetitive sequences in the genome, such as alu and transposons, and is the most common site of extragenomic changes in mammalian cells. The 5-mC of this CpG is spontaneously deaminated and becomes T, resulting in a frequency of only 1% for CpG in the mammalian genome, which is much lower than the normal frequency (1 / 4 x 1 / 4 = 6.25%).

[0025] Some CpGs appear unusually dense, and these are called CpG sites (CpG islands). CpG islands are defined as regions with a length of 0.2-3 kb, a distribution percentage of C and G bases greater than 50%, and a high CpG concentration with a distribution percentage of at least 3.75%. There are about 45,000 CpG islands in the entire human genome, and they are particularly concentrated in promoter regions that regulate gene expression. In fact, CpG islands appear in the promoters of housekeeping genes, which account for about half of all human genes.

[0026] The nucleic acid isolated from the sample is obtained from the biological sample of the sample. In order to diagnose lung cancer or advanced lung cancer, nucleic acid must be isolated from lung tissue by scraping or biopsy. Such samples can be obtained by various medical procedures known in the art.

[0027] The degree of methylation of nucleic acid of a sample obtained from the sample is measured by comparing the same nucleic acid portion of a sample without abnormal cell growth of lung tissue. Hypermethylation means the presence of methylated alleles in one or more nucleic acids. Samples without abnormal cell growth in lung tissue do not show methylated alleles when the same nucleic acid is tested.

[0028] A "normal" cell is a cell that does not exhibit abnormal cell morphology or changes in cytological properties. A "tumor" cell is a cancer cell, and a "non-neoplastic" cell is a cell that is part of a diseased tissue but is not identified as a tumor site.

[0029] According to the present invention, the methylation stage of one or more nucleic acids isolated from a sample can be determined for early diagnosis of abnormal cell growth in the sample lung tissue. The methylation stage of one or more nucleic acids can be compared with the methylation state of one or more nucleic acids isolated from a sample that does not have abnormal lung tissue cell growth. The nucleic acid is preferably a CpG-containing nucleic acid, such as a CpG island.

[0030] According to the present invention, the methylation stage of one or more nucleic acids isolated from a sample can be determined to diagnose abnormal cell growth in the sample lung tissue. The methylation stage of one or more nucleic acids can be compared with the methylation state of one or more nucleic acids isolated from a sample without abnormal cell growth in lung tissue.

[0031] "Susceptibility" means susceptibility to the above-mentioned cell growth abnormalities. A susceptible sample is a sample that does not yet have a cell growth abnormality but has an increased tendency to have or develop a cell growth abnormality.

[0032] The presence of CpG methylation in the target DNA can be an indicator of a disease, for example, by measuring CpG methylation at any of the promoter, 5' untranslated region, and introns of the target DNA.

[0033] Genes containing CpG are usually DNA. However, the method of the present invention can be applied to samples containing, for example, DNA or RNA containing DNA and mRNA, wherein the DNA or RNA can be single-stranded or double-stranded, or to samples containing DNA-RNA hybrids.

[0034] Mixtures of nucleic acids can also be used. As used in the present invention, "multiple" includes multiple specific nucleic acid sequence sites to be detected in a single gene and multiple target DNAs in a tube (single reactor). The specific nucleic acid sequence to be detected can be a part of a larger molecule, or it can exist as an isolated molecule, wherein the specific sequence constitutes the entire nucleic acid sequence from the beginning. The nucleic acid sequence does not need to be a nucleic acid existing in a pure form, and the nucleic acid can be a small part in a complex mixture (such as a complex mixture comprising a complete human DNA).

[0035] Specifically, the present invention relates to detecting methylation of multiple target DNAs in a sample in a single reactor, wherein the sample may include multiple multiple target DNAs, wherein the target DNA may be any gene that affects the development or progression of lung cancer when it is abnormally methylated and its expression is inhibited without any limitation.

[0036] In the present invention, the sample may be derived from the human body, for example, the sample may be lung cancer tissue, cells, feces, urine, blood, serum or plasma.

[0037] One or more reagents that differentially modify methylated and unmethylated DNA can be used without limitation, as long as they can distinguish between unmethylated and methylated cytosine bases, such as, but not limited to, sulfite, bisulfite, disulfite, and combinations thereof. Specifically, methylated cytosine bases will not be converted by the above reagents, while unmethylated cytosine bases can be converted to bases other than uracil or cytosine.

[0038] In the present invention, step (b) is a step of treating primers for specifically amplifying the methylated PCDHGA12 gene and the CDO1 gene.

[0039] Primers can be used simultaneously, for example, by pairing forward and reverse primers for PCR. In addition, the design of the primers makes it necessary for the methylated "C" of the CpG site to be located at the 3' end of the primer, which makes them highly specific for methylation detection. A forward primer for specifically amplifying the methylated PCDHGA12 gene may include, for example, a sequence of SEQ ID NO: 1. A reverse primer may include, for example, a sequence of SEQ ID NO: 2. Primers for specifically amplifying the methylated PCDHGA12 gene may include a primer pair of SEQID No: 1 and 2.

[0040] The forward primer for specifically amplifying the methylated CDO1 gene may include, for example, the sequence of SEQ ID NO: 4. The reverse primer may include, for example, the sequence of SEQ ID NO: 5. The primer for specifically amplifying the methylated CDO1 gene may include a primer pair of SEQ ID NOs: 4 and 5.

[0041] The present invention may further include the following step: treating probes that can complementarily hybridize with the methylated PCDHGA12 gene and the methylated CDO1 gene specifically amplified by the primers in the above step (b), respectively.

[0042] In hybridization reactions, the conditions used to achieve a stringent specific level vary depending on the properties of the hybridized nucleic acids. For example, the length of the nucleic acid sites being hybridized, the degree of homology, the nucleotide sequence composition (e.g., GC / AT ratio), and the type of nucleic acid (e.g., RNA, DNA) are considered when selecting hybridization conditions. Additional considerations are whether the nucleic acid is fixed, e.g., fixed on a filter.

[0043] Examples of very stringent conditions include 2X SSC / 0.1% SDS at room temperature (hybridization conditions); 0.2XSSC / 0.1% SDS at room temperature (low stringency conditions); 0.2X SSC / 0.1% SDS at 42°C (medium stringency conditions); 0.1XSSC at 68°C (high stringency conditions). The washing process can be performed using any of these conditions (e.g., high stringency conditions) or each of the above conditions, and can be repeated in whole or in part in the above order for 10 to 15 minutes each time. However, as described above, the optimal conditions vary depending on the specific hybridization reaction involved and can be determined experimentally. In general, high stringency conditions are used for the hybridization of important probes.

[0044] The probe capable of complementarily hybridizing with the amplified methylated CDO1 gene or methylated PCDHGA12 gene may include, for example, a sequence of SEQ ID NO: 3 or 6. The probe capable of complementarily hybridizing with the amplified methylated CDO1 gene may include, for example, a sequence of SEQ ID NO: 3. The probe capable of complementarily hybridizing with the amplified methylated PCDHGA12 gene may include, for example, a sequence of SEQ ID NO: 6.

[0045] In some cases, the probe is labeled for detection, and can be labeled, for example, with a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme. Suitable labels for such probes are well known in the art and can be accomplished by conventional methods.

[0046] The amount of amplified product can be detected by fluorescence signal. The intercalation method uses a reagent called an intercalator that binds to the double-stranded DNA of the amplified product to which the probe binds and emits fluorescence, or uses an oligonucleotide labeled with a fluorophore at the 5' end and a quencher at the 3' end.

[0047] Amplification according to the present invention can be carried out by real-time quantitative amplification, for example by real-time polymerase chain reaction (real-time PCR), wherein the amount of PCR amplification product can be detected by fluorescent signal. Along with the progress of real-time polymerase chain reaction, the intensity of fluorescent signal increases with the increase of polynucleotide amount, thereby producing the amplification spectrum curve showing that the intensity of fluorescent signal depends on the number of amplification cycles.

[0048] The amplification spectrum curve is usually divided into a baseline region, an exponential region, and a plateau region. The baseline region shows a background fluorescence signal that does not reflect the actual amount of the polynucleotide, the exponential region shows that the fluorescence signal increases with the increase in the amount of the polynucleotide product, and the plateau region shows that when the PCR reaction reaches saturation, the fluorescence signal intensity does not increase.

[0049] Generally speaking, the point where the baseline region turns into the exponential region, that is, the point where the amount of PCR amplification product reaches the fluorescence signal intensity that can be detected by fluorescence, is called the threshold, and the amplification cycle number corresponding to the threshold in the amplification spectrum curve is called the threshold cycle (Ct) value.

[0050] By measuring the Ct value and analyzing a standard curve in which the concentration is determined based on the Ct (threshold cycle) value of the standard substance, the concentration of the amplified gene can be determined, thereby determining the sensitivity and / or specificity of the methylation specificity.

[0051] In one example, methylation detection can be performed by a method selected from the group consisting of: PCR, methylation-specific PCR, real-time methylation-specific PCR, PCR using a methylated DNA-specific binding protein, PCR using a methylated DNA-specific binding antibody, quantitative PCR, gene chip, sequencing, sequencing by synthase, and sequencing by ligation.

[0052] (1) Methylation-specific PCR (methylation-specific PCR): When the above-mentioned methylation-specific PCR is used for detection, the cytosine at the 5'-CpG'-3 site remains as cytosine when methylated, and is converted into uracil when unmethylated. Therefore, primers corresponding to the site where the 5'-CpG-3' sequence exists can be prepared for the sequence converted after sulfite treatment. When PCR is performed using primers, if the sequence is methylated, a PCR product is generated by the primer corresponding to the methylated sequence, and the methylation can be confirmed by agarose gel electrophoresis. The methylation detection probe can be, but is not limited to, TaqMan, a molecular beacon, a self-reporting probe, or a probe with an energy transfer labeling function.

[0053] (2) Real-time methylation-specific PCR: Real-time methylation-specific PCR is to convert the methylation-specific PCR method into a real-time measurement method, that is, to treat genomic DNA with sulfite, design PCR primers corresponding to the methylation sites, and use the primers for real-time PCR. At this time, there are two detection methods: detection using a TaqMan probe complementary to the amplified sequence and detection using Sybergreen. Therefore, real-time methylation-specific PCR can selectively quantify only methylated DNA. In this case, an in vitro methylated DNA sample can be used to construct a standard curve, and genes that do not contain a 5'-CpG-3' sequence in the sequence can be amplified as a negative control for standardization, and the degree of methylation is quantitatively analyzed.

[0054] (3) PCR or quantitative PCR or DNA chip using methylated DNA-specific binding protein: PCR or DNA chip method using methylated DNA-specific binding protein. The DNA-specific binding protein can selectively separate only methylated DNA because the protein will specifically bind to methylated DNA when mixed with DNA.

[0055] Methylation can also be measured by quantitative PCR, in which methylated DNA is isolated using a methylated DNA-specific binding protein that is labeled with a fluorescent dye and hybridized to a DNA chip carrying complementary probes.

[0056] (4) Detection of differential methylation - sulfite sequencing: Another method for detecting nucleic acids containing methylated CpG includes the steps of contacting a sample containing nucleic acid with a reagent that modifies unmethylated cytosine, and amplifying the CpG-containing nucleic acid in the sample using CpG-specific oligonucleotide primers. Here, the oligonucleotide primers can distinguish between modified methylated and unmethylated nucleic acids to detect methylated nucleic acids. The amplification step is optional, preferably but not required. The method relies on a PCR reaction to distinguish between modified (e.g., chemically modified) methylated and unmethylated DNA.

[0057] (5) Bisulfite sequencing method: Another method for detecting nucleic acids containing methylated CpG comprises the following steps: contacting a sample containing nucleic acids with a reagent that modifies unmethylated cytosine, and amplifying the nucleic acids containing CpG in the sample using methylation-independent oligonucleotide primers. Here, the oligonucleotide primers may amplify methylated nucleic acids without distinguishing between modified methylated and unmethylated nucleic acids. The amplified products may be sequenced by the Sanger method using sequencing primers, or by a next generation sequencing method, with reference to bisulfite sequencing for detecting methylated nucleic acids.

[0058] (6) Next generation sequencing methods can be performed by sequencing by synthesis or sequencing by ligation. Instead of creating bacterial clones, this method spatially separates individual DNA fragments and amplifies them in situ (clonal amplification) and sequences them. This is sometimes called massively parallel sequencing because hundreds of thousands of fragments are read simultaneously.

[0059] Basically, it is a sequencing-by-synthesis method that splices single-nucleotide or dinucleotide sequences together to obtain a signal, such as pyrosequencing, ion torrent, and Solexa.

[0060] NGS instruments based on sequencing by synthesis include Roche's 454 platform, Illumina's HiSeq platform, LifeTechnology's Ion PGM platform, and Pacific BioSciences' PacBio platform. 454 and Ion PGM use emersion PCR as a clonal amplification method, while HiSeq uses bridge amplification. The sequencing by synthesis method reads the sequence by detecting phosphates, hydrogen ions, or pre-added fluorescence generated when DNA is synthesized by attaching one nucleotide after another. For sequence detection, 454 uses a pyrophosphate sequencing method using phosphates, while Ion PGM uses hydrogen ion detection. HiSeq and PacBio detect fluorescence to decode the sequence.

[0061] Sequencing by ligation is a sequencing technology that uses DNA ligase to identify nucleotides at specific positions in a DNA sequence. Unlike most sequencing technologies that use polymerases, this technology does not use polymerases, but instead uses the characteristic of DNA ligases that they do not ligate unmatched sequences. The SOLiD system is an example. In this technology, two bases are read at a time, with gaps, and are independently repeated five times with primer resets so that each base is ultimately read twice in duplicate to improve accuracy.

[0062] In the case of sequencing by ligation, dinucleotide primers corresponding to a given sequence from a set of 16 dinucleotide primer combinations are sequentially ligated, and these ligated combinations are finally analyzed to complete the sequence of the DNA.

[0063] Here, the next generation sequencing method may be performed by sequencing by synthesis or sequencing by ligation.The methylated DNA-specific binding protein is not limited to MBD2bt, and the antibody is not limited to the 5'-methyl-cytosine antibody.

[0064] For the primers used in the present invention, the treatment with a reagent such as sulfite according to the above step (a) results in the cytosine at the 5'-CpG'-3 site remaining cytosine if methylated and converting to uracil if unmethylated. Therefore, primers corresponding to the site where the 5'-CpG-3' sequence exists can be prepared for the sequence converted after treatment with a reagent such as sulfite.

[0065] The primers can be made "substantially" complementary to each strand of the locus to be amplified. This means that the primers have sufficient complementarity to hybridize to the corresponding nucleic acid strands under polymerization reaction conditions.

[0066] On the other hand, the present invention also provides a composition for diagnosing lung cancer, which composition includes (a) one or more reagents for differentially modifying methylated PCDHGA12 gene and CDO1 gene, unmethylated PCDHGA12 gene and CDO1 gene; and (b) primers for specifically amplifying methylated PCDHGA12 gene and methylated CDO1 gene.

[0067] The constructs included in the composition according to the present invention overlap with the previously described constructs, so their descriptions apply equally.

[0068] In another aspect, the present invention relates to a kit for detecting target DNA methylation comprising the composition.

[0069] In one example, the kit may include a compartmentalized carrier device for accommodating a sample, a container containing reagents, and a container containing primers for each of the methylated PCDHGA12 gene and the methylated CDO1 gene. In some cases, the kit may also include a container containing a probe for detecting each of the amplified products of the methylated PCDHGA12 gene and the methylated CDO1 gene.

[0070] The carrier device is suitable for holding one or more containers, such as bottles or tubes, each container containing a separate component for use in the method of the present invention. According to the description of the present invention, a person skilled in the art can easily dispense the desired formulation from the container.

[0071] The present invention will now be described in more detail with reference to the following examples. These examples are intended only to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention should not be construed as being limited by these examples.

[0072] Example 1. Validation of primers and probes for detecting PCDHGA12 and CDO1 methylation genes

[0073] Primer sets for multiplex polymerase chain reaction (multiplex PCR) for simultaneous detection of methylation were constructed for PCDHGA12 and CDO1 genes. Methylation-specific oligonucleotide primers and probe sets (Table 1) were designed using MethPrimer (http: / / www.urogene.org / cgi-bin / methprimer / met hprimer.cgi) for PCDHGA12 and CDO1 gene sequences converted with sulfite. PCDHGA12 gene was designed based on exon 1 region sequence, and CDO1 gene was designed based on promoter region sequence. Oligonucleotide primers and probes specific for control gene COL2A1 were also designed (Table 1).

[0074] Table 1. Sequences of primers and probes for PCDHGA12 and CDO1 methylation gene amplification

[0075]

[0076] To confirm the methylation-specific detection of the designed primers and probes for multiplex PCR of PCDHGA12 and CDO1 genes, a quantitative methylation-specific real-time polymerase chain reaction (qMSP) method was performed using methylated and unmethylated control DNA (EpiTect PCR Control DNA Set (Qiagen, cat. no. 59695)). The COL2A1 gene was used as an internal control gene.

[0077] A total of 25 μl of PCR reaction solution (sulfite-converted template human DNA, 20 ng / 5 μl; Qplex MasterMix (CellSafe, Korea), 10 μl; methylation-specific PCR primers (IDT, USA), 1 μl each (1 to 10 pmoles / μl); methylation-specific TaqMan probes (IDT, USA), 1 μl each (2.5 to 5 pmoles / μl); DW 1 μl) were prepared and subjected to PCR reaction with sulfite-converted template human DNA (Table 2). The PCR conditions were performed at 95°C for 5 minutes, followed by 5 cycles of 95°C for 15 seconds and 72°C for 45 seconds, the qMSP step was performed at 95°C for 15 seconds and 58°C for 45 seconds, for a total of 35 cycles, and the amplification of the PCR product of qMSP was checked in real time using a CFX96 (Bio-Rad) instrument ( Figure 1 ).

[0078] Table 2. qMSP reaction solution

[0079]

[0080] like Figure 1As shown, in methylated DNA, PCDHGA12 and CDO1 genes and the control gene COL2A1 were amplified together, while in unmethylated DNA, PCDHGA12 and CDO1 genes were not amplified, and only the control gene COL2A1 was amplified, confirming that the methylation-specific primers PCDHGA12 and CDO1 genes worked properly.

[0081] Example 2. Confirmation that the combination of CDO1 and PCDHGA12 gene methylation in bronchial lavage fluid DNA can improve lung cancer detection performance

[0082] To determine whether the combination of CDO1 gene and PCDHGA12 gene methylation (Korean Patent No. 10-1106727), an existing methylation biomarker for early lung cancer diagnosis (Table 3), can improve the detection of lung cancer, the methylation of PCDHGA12 and CDO1 genes was analyzed in bronchial lavage fluid DNA collected by bronchoscopy from 111 patients, including 38 patients with benign lung disease (Konyang University Hospital) and 73 patients with cancer (Konyang University Hospital). Figure 2 ). The methylation of PCDHGA12 and CDO1 genes in these samples was measured using the qMSP method described in Example 2.

[0083] The PCR instrument was CFX96 (Bio-Rad), and sulfite conversion was performed using the EZ Methylation Gold Kit (Cat. No. D5006, Zymo Research, USA).

[0084] The methylation level of each gene was expressed by the 35-dCt (PCDHGA12 or CDO1 Ct-COL2A1Ct) (Chung W et al., Cancer Epidemiol Biomarkers Prev., 2011; 20(7): 1483-1491) method. The higher the 35-dCt value, the higher the methylation level. In order to evaluate the performance of each gene in lung cancer diagnosis, the MedCalc program (MedCalc software, Ballgier) was used to perform receiver operating characteristic (ROC) analysis to determine the optimal cutoff value for positive results and calculate sensitivity and specificity.

[0085] When methylation of the CDO1 gene was measured using sulfite-treated and converted bronchial lavage DNA, the methylation positive cutoff was calculated to be 27.5, and only 1 of 38 patients with benign lung disease was methylated positive, with a specificity of 97.4%; in lung cancer patients, 59 of 73 patients were methylated positive, with a sensitivity of 80.8%. When methylation of the PCDHGA12 gene was combined, 3 of 38 patients with benign lung disease were methylated positive, with a specificity of 92.1%, indicating no significant decrease (P = 0.615, Fisher's exact test). In terms of sensitivity, 68 of 73 lung cancer patients were methylated positive, and an additional 9 lung cancer patients were positive compared to CDO1 alone, for a total of 68 (68 / 73), indicating a significant increase in sensitivity to 93.2% (P < 0.047, Fisher's exact test) (Table 3). Therefore, the combination of CDO1 gene methylation and PCDHGA12 gene methylation significantly increased the ability to detect lung cancer.

[0086] Table 3. Sensitivity and specificity of combined detection of PCDHGA12 and CDO1 methylation

[0087]

[0088] Industrial availability

[0089] The present invention is useful because by detecting methylation with high detection sensitivity using primers that specifically amplify multiple methylated lung cancer marker genes, detection of lung cancer can be improved compared to detection methods using a single marker gene, thereby enabling accurate and rapid diagnosis of lung cancer.

[0090] Although certain aspects of the present invention have been described in detail above, it is obvious to those skilled in the art that these specific descriptions are only preferred examples and are not intended to limit the scope of the present invention. Therefore, the essential scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for providing information for lung cancer diagnosis, the method comprising the following steps: (a) treating a sample with one or more agents that differentially modify methylated PCDHGA12 (protocadherin gamma subfamily A, 12) gene and CDO1 (cysteine ​​dioxygenase type 1) gene, and unmethylated PCDHGA12 gene and CDO1 gene; as well as (b) Primers for specifically amplifying the methylated PCDHGA12 gene and the CDO1 gene are processed.

2. The method according to claim 1, wherein: The reagent is sulfite, bisulfite, disulfite or a combination thereof.

3. The method according to claim 1, wherein: The treatment with the reagent converts at least one cytosine base into a base other than uracil or cytosine.

4. The method according to claim 1, wherein: The primers for specifically amplifying the methylated CDO1 gene may include a primer pair of SEQ ID Nos: 4 and 5.

5. The method according to claim 1, wherein: The primers for specifically amplifying the methylated PCDHGA12 gene may include a primer pair of SEQ ID Nos: 1 and 2.

6. The method according to claim 1, wherein: The method may further comprise the following step (c): treating probes capable of complementary hybridization with the methylated PCDHGA12 gene and the methylated CDO1 gene specifically amplified by the primers in the above step (b), respectively.

7. The method according to claim 6, wherein: The probe capable of complementarily hybridizing to the amplified methylated CDO1 gene may include, for example, the sequence of SEQ ID NO:

6.

8. The method according to claim 6, wherein: The probe capable of complementarily hybridizing with the amplified methylated PCDHGA12 gene may include, for example, the sequence of SEQ ID NO:

3.

9. The method according to claim 1, wherein: The methylation detection can be performed by a method selected from the group consisting of: PCR, methylation-specific PCR, real-time methylation-specific PCR, PCR using a methylated DNA-specific binding protein, PCR using a methylated DNA-specific binding antibody, quantitative PCR, gene chip, sequencing, synthase sequencing, and sequencing by ligation.

10. The method according to claim 6, wherein: The methylation is detected by detecting a substance that binds to the probe and exhibits fluorescence.

11. A composition for diagnosing lung cancer, comprising one or more reagents for differentially modifying methylated PCDHGA12 (protocadherin gamma subfamily A, 12) gene and CDO1 (cysteine ​​dioxygenase type 1) gene, unmethylated PCDHGA12 gene and CDO1 gene; and primers for specifically amplifying the methylated PCDHGA12 gene and the CDO1 gene.

12. The composition according to claim 11, wherein The reagent is sulfite, bisulfite, disulfite or a combination thereof.

13. The composition according to claim 11, wherein The treatment with the reagent converts at least one cytosine base into a base other than uracil or cytosine.

14. The composition according to claim 11, wherein The primers for specifically amplifying the methylated CDO1 gene may include a primer pair of SEQ ID Nos: 4 and 5.

15. The composition according to claim 11, wherein The primers for specifically amplifying the methylated CDO1 gene may include a primer pair of SEQ ID Nos: 1 and 2.

16. The composition according to claim 11, wherein The composition further comprises the following step (c): treating probes capable of complementary hybridization with the methylated PCDHGA12 gene and the methylated CDO1 gene specifically amplified by the primers, respectively.

17. The composition according to claim 16, wherein The probe capable of complementarily hybridizing with the amplified methylated CDO1 gene may include the sequence of SEQ ID NO:

6.

18. The composition according to claim 16, wherein The probe capable of complementarily hybridizing with the amplified methylated PCDHGA12 gene may include the sequence of SEQ ID NO:

3.

19. A kit for diagnosing lung cancer, comprising the composition according to any one of claims 11 to 18.

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