KR101106014B1 - Polynucleotides derived from chromosome 1 comprising single nucleotide polymorphism, microarrays and diagnostic kits comprising the same, and analytic methods using the same - Google Patents

Abstract

The present invention provides polynucleotides or their complementary polynucleotides comprising a single-nucleotide polymorphism (SNP) derived from genes on chromosome 1, in particular ZMPSTE24, GPR37L1, LMX1A, SRGAP2, FAM5C, EDEM3 genes. In addition, the polynucleotide or its complementary polynucleotide may be used as a marker for clinical diagnosis of intestinal type of gastric cancer.

ZMPSTE24, GPR37L1, LMX1A, SRGAP2, FAM5C, EDEM3, Enteric Gastric Cancer

Description

Polynucleotides derived from chromosome 1 comprising single nucleotide polymorphism, microarrays and diagnostic kits comprising the same, and analytic methods using the same}

The present invention relates to polynucleotides or their complementary polynucleotides comprising monobasic polymorphisms derived from genes on chromosome 1, in particular, ZMPSTE24, GPR37L1, LMX1A, SRGAP2, FAM5C, EDEM3 genes; Amplification primers or enteric gastric cancer diagnostic probes for diagnosing intestinal type of gastric cancer comprising the polynucleotide or its complementary polynucleotides; A microarray comprising the probe; And an analysis method for providing information necessary for diagnosing enteric gastric cancer using the polynucleotide or its complementary polynucleotide.

The human genome consists of 22 pairs of homologous chromosomes and two sex chromosomes. But every human being has a different appearance and character. This is because each person has the same number of chromosomes, but all of the phenotypes of the genes expressed from that chromosome are different. The diversity of these gene expressions in individuals is due to the genetic diversity of the genome carrying the gene. Much research has been done on this genetic diversity, and the structure of the genetic genome has recently been revealed.

Genetic diversity in the genome is represented by transposable elements, short tandem repeats (STRs), microsatellites, sequence tagged sites (STS), variable number tandem repeats (VNTRs), and single nucleotide polymorphisms (SNPs). Known. Dual SNPs are monobasic, appearing at a frequency of about one thousand nucleotides on the human genome and taking the form of single nucleotide variations between individuals of the same species. The meaning in the genetics represented by these monobasic polymorphisms makes a big difference in each individual depending on its location. For example, when a monobasic polymorph is present at a location that encodes a protein, it may affect the structure of the protein, altering protein function and associated with disease. In another example, if a monobasic polymorph is present on another gene that does not encode a protein, ie, on a promoter or intron, the expression level of the protein may differ for each, resulting in a decrease in the overall activity of the protein. In addition, a protein may be abnormally expressed through alterative splicing.

Monobasic polymorphs can be used as indicators of specific phenotypes, such as disease. To do this, first of all, a large number of monobasic polymorphisms present in the human genome are identified and the single monopolymorphism is found in a number of human genome samples. An assay will be conducted to determine if there is a genetic variation marker present in more than 1% of the population. Assayed SNPs will be used in association studies to find the cause due to genetic differences such as specific diseases or drug responses. For example, if a single base polymorphism is found to be significantly different in the comparison between the patient and control group, it will be selected as a very useful single base polymorphism and subjected to clinical trials. It is a genetic indicator of economically useful value and will be available for actual clinical use.

Conventional techniques for the discovery of such monobasic polymorphisms include Restriction Fragment Length Polymorphism (RFLP) using restriction enzymes, TaqMan ^™ probe method using Allele-specific hybridization, and melting temperature ( dynamic allele-specific hybridization (DASH) using melting temperature (Tm) and pyrosequencing using polymerization. Recently, microarray technology has discovered a single base polymorphism by integrating and planting probes using a principle of single base extension on a small substrate, followed by hybridization and extension with target DNAs.

The incidence and mortality rate of gastric cancer is decreasing, but it is still the second most common malignancy in the world. In Korea, the incidence of cancer in men and women is ranked first in gastric cancer and second in mortality. Helicobacter pylori (H. pylori) infection, which is closely related to gastric cancer, is the most common chronic infection in humans, affecting more than 50% of the world's population. The prevalence of H. pylori infection in Korea is high at 46.6%, 69.4% in adults over 16 years old, and 78.5% in 40s. Gastric cancer is classified into Intestinal type of gastric cancer and Diffuse type of gastric cancer by Lauren classification. Enteric gastric cancer is associated with H. pylori infection in the order of chronic gastritis, atrophic gastritis, intestinal metaplasia, displasia, and intestinal type of gastric cancer. do. Diffuse gastric cancer, on the other hand, can be induced from normal mucous membranes regardless of H. pylori infection. A high H. pylori infection rate in Korea means a high risk for intestinal gastric cancer.

Early gastric cancer has a 5-year survival rate of 95% to 100%, but 80% of cases have no symptoms. This implies the need for early diagnosis, along with the importance of early diagnosis. Since only 1 to 2 out of 100 H. pylori infections develop gastric cancer, it is not appropriate to determine H. pylori infection as a risk group for gastric cancer. Predicting the risk of developing gastrointestinal cancer among patients with chronic gastritis at all stages of gastric cancer and identifying the factors involved in the development of gastric cancer is important for public health and is an essential requirement for tests to prevent stomach cancer. Finding monobasic polymorphisms that may be specific for enteric gastric cancer can help diagnose early gastric cancer, furthermore, to identify pathophysiological pathways and to develop therapeutic methods.

The present inventors conducted a study to find a single nucleotide polymorphism specific to Korean patients with enteric gastric cancer. Specific monobasic polymorphic sites were found to complete the present invention.

Accordingly, an object of the present invention is to provide a polynucleotide or a complementary polynucleotide thereof comprising a monobasic polymorphism derived from ZMPSTE24, GPR37L1, LMX1A, SRGAP2, FAM5C, EDEM3 genes as a monobasic polymorphism associated with enteric gastric cancer.

In addition, an object of the present invention is to provide an amplification primer or a gastric cancer diagnostic probe for diagnosing enteric gastric cancer consisting of the polynucleotide or a complementary polynucleotide thereof.

Another object of the present invention is to provide a microarray including the probe.

It is also an object of the present invention to provide a method for diagnosing enteric gastric cancer using the polynucleotide or its complementary polynucleotide.

According to one aspect of the invention, in the polynucleotide consisting of the DNA sequence of SEQ ID NO: 1, the 501th base (polymorphic site) is A, a polynucleotide consisting of 10 or more consecutive DNA sequences comprising the 501th base ( a); In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, the polynucleotide (b) consisting of ten or more consecutive DNA sequences comprising the 101 st base (polymorphic site) G and comprising the 101 st base; In a polynucleotide consisting of a DNA sequence of SEQ ID NO: 3, a polynucleotide (c) consisting of ten or more consecutive DNA sequences comprising the 401 th base (polymorphic site) G and comprising the 401 th base; A polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, wherein the 501th base (polymorphic site) is G and the polynucleotide consists of 10 or more consecutive DNA sequences comprising said 501th base; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 5, the polynucleotide (e) consisting of ten or more contiguous DNA sequences comprising the 301 th base (polymorphic site) is C and the 301 th base; A polynucleotide consisting of the DNA sequence of SEQ ID NO: 6, wherein the 501th base (polymorphic site) is G, and the polynucleotide (f) is composed of 10 or more consecutive DNA sequences comprising the 501th base; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 7, a polynucleotide (g) consisting of at least 10 contiguous DNA sequences comprising a 262 th base (polymorphic site) G and comprising the 262 th base; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 8, a polynucleotide (h) consisting of 10 or more contiguous DNA sequences including the 2187th base (polymorphic site) G and comprising the 2187th base; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 9, a polynucleotide (i) consisting of 10 or more consecutive DNA sequences comprising the 301th base (polymorphic site) G and comprising the 301th base; And a polynucleotide consisting of the DNA sequence of SEQ ID NO: 10, wherein the 201 base (polymorphic site) is A, and the polynucleotide (j) is composed of 10 or more consecutive DNA sequences comprising the 201 base. One or more selected polynucleotides or complementary polynucleotides thereof are provided.

In addition, according to another aspect of the present invention, an amplification primer for diagnosing enteric gastric cancer, comprising the polynucleotide or a complementary polynucleotide thereof, is provided.

In addition, according to another aspect of the present invention, there is provided a enteric gastric cancer diagnostic probe and a microarray comprising the polynucleotide or a complementary polynucleotide thereof.

In addition, according to another aspect of the present invention, there is provided a kit for diagnosing enteric gastric cancer comprising the polynucleotide or a complementary polynucleotide thereof.

In addition, according to another aspect of the invention, to provide information necessary for the diagnosis of enteric gastric cancer, (i) obtaining a nucleic acid sample from a sample; And (ii) analyzing the nucleotide sequence of the polymorphic site of at least one polynucleotide selected from polynucleotides of SEQ ID NOs: 1 to 10 or complementary polynucleotides thereof from the nucleic acid sample, provided that the polymorphic site is 501 base for DNA sequence, 101 base for DNA sequence of SEQ ID NO: 2, 401 base for DNA sequence of SEQ ID NO: 3, polymorphic site is 501 base for DNA sequence of SEQ ID NO: 4, polymorphism The site is the 301th base for the DNA sequence of SEQ ID NO: 5, the polymorphic site is the 501st base for the DNA sequence of SEQ ID NO: 6, the polymorphic site is the 262th base for the DNA sequence of SEQ ID NO: 7, and the polymorphic site is 2187 base for the DNA sequence of SEQ ID NO: 8, the polymorphic site is 301 base for the DNA sequence of SEQ ID NO: 9, DNA sequence of SEQ ID NO: 10 If there is provided a second method 201, which means the base respectively), detecting a single nucleotide polymorphism in a sample comprising a.

One or more polynucleotides selected from the group consisting of polynucleotides (a) to (j) according to the present invention or complementary polynucleotides thereof may be usefully used for the diagnosis of enteric gastric cancer.

In addition, a primer or probe consisting of the polynucleotide or its complementary polynucleotide, and a kit comprising the polynucleotide or the complementary polynucleotide thereof may be usefully used for the diagnosis of enteric gastric cancer.

In addition, the analysis method of the present invention can be usefully used to provide information necessary for diagnosing gastric cancer.

The present invention provides polynucleotides or complementary polynucleotides selected from the group consisting of polynucleotides (a) to (j) derived from the sequences of SEQ ID NOs 1 to 10 newly found to be associated with enteric gastric cancer. That is, the present invention provides a polynucleotide consisting of the DNA sequence of SEQ ID NO: 1, wherein the 501st base (polymorphic site) is A, and is composed of 10 or more consecutive DNA sequences comprising the 501th base (a); In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 2, a polynucleotide (b) consisting of ten or more consecutive DNA sequences comprising the 101 st base (polymorphic site) G and comprising the 101 st base; In a polynucleotide consisting of a DNA sequence of SEQ ID NO: 3, a polynucleotide (c) consisting of ten or more consecutive DNA sequences comprising the 401 th base (polymorphic site) G and comprising the 401 th base; A polynucleotide consisting of the DNA sequence of SEQ ID NO: 4, wherein the 501th base (polymorphic site) is G and the polynucleotide consists of 10 or more consecutive DNA sequences comprising said 501th base; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 5, the polynucleotide (e) consisting of ten or more contiguous DNA sequences comprising the 301 th base (polymorphic site) is C and the 301 th base; A polynucleotide consisting of the DNA sequence of SEQ ID NO: 6, wherein the 501th base (polymorphic site) is G, and the polynucleotide (f) is composed of 10 or more consecutive DNA sequences comprising the 501th base; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 7, a polynucleotide (g) consisting of at least 10 contiguous DNA sequences comprising a 262 th base (polymorphic site) G and comprising the 262 th base; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 8, a polynucleotide (h) consisting of 10 or more contiguous DNA sequences including the 2187th base (polymorphic site) G and comprising the 2187th base; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 9, a polynucleotide (i) consisting of 10 or more consecutive DNA sequences comprising the 301th base (polymorphic site) G and comprising the 301th base; And a polynucleotide consisting of the DNA sequence of SEQ ID NO: 10, wherein the 201 base (polymorphic site) is A, and the polynucleotide (j) is composed of 10 or more consecutive DNA sequences comprising the 201 base One or more polynucleotides selected from or complementary polynucleotides thereof are provided.

The DNA sequences of SEQ ID NOs: 1 to 10 are monobasic polymorphisms showing statistically significant difference (significance level p value <0.05) in patients with chronic gastritis and intestinal gastric cancer, and they are located on chromosome 1, ZMPSTE24, GPR37L1. , LMX1A, SRGAP2, FAM5C, EDEM3 genes. The ZMPSTE24 gene encodes zinc metalloproteinase, and mutations in this gene have been reported to be associated with mandibuloacral dysplasia and restrictive dermopathy. GPR37L1 (G protein-coupled receptor 37 like 1) gene has yet to be identified. The LMX1A (LIM homeobox transcription factor 1, alpha) gene is known to stimulate insulin transcription by attaching to an insulin promoter. The SRGAP2 (SLIT-ROBO Rho GTPase activating protein 2) gene encodes an ARHGAP family protein and is responsible for melanoma, germ cell tumors, chondrosarcoma and retinoblastoma. It has been reported to be expressed. FAM5C (family with sequence similarity 5, member C) genes have been reported to be associated with myocardial infarction, and EDEM3 (ER degradation enhancer, mannosidase alpha-like 3) genes are known to be glycoprotein-induced ERADs (glycoproteins). It has been reported to enhance endoplasmic reticulum-associated degradation and mannose trimming.

The present inventors selected ZMPSTE24, GPR37L1, LMX1A, SRGAP2, FAM5C, and EDEM3 genes located on chromosome 1 as candidate gene groups related to enteric gastric cancer, and developed various diagnostic indicators at the molecular level in the diagnosis of enteric gastric cancer. The study was conducted. We performed genotyping and statistical analysis on 24 Korean patients with chronic gastritis and 21 patients with gastrointestinal gastric cancer. Surprisingly, only a single polymorphic polymorphic polymorphism was found in patients with chronic gastritis and intestinal gastric cancer. It was found that this represents a significant difference. In particular, the monobasic polymorphisms of SEQ ID NOS: 1 to 10 showed a risk allele at each polymorphic region as a result of Hardy-Weinberg Equilibrium and logistic regression analysis.

The present invention includes a polynucleotide hybridizing with at least one polynucleotide selected from the group consisting of the polynucleotides (a) to (j) or a complementary polynucleotide thereof, and the length of the polynucleotide or the complementary polynucleotide thereof is 10 To 100 nucleotides, preferably 20 to 60 nucleotides, more preferably 40 to 60 nucleotides.

At least one polynucleotide selected from the group consisting of the polynucleotides (a) to (j) according to the present invention is a polymorphic sequence. A polymorphic sequence refers to a sequence comprising a polymorphic site representing a monobasic polymorphism in a nucleotide sequence. A polymorphic site refers to a site where a monobasic polymorphism occurs in the polymorphic sequence. The polynucleotide can be DNA or RNA.

The invention also includes an allele specific primer for amplification for enteric gastric cancer, comprising at least one polynucleotide selected from the group consisting of the polynucleotides (a) to (j) or complementary polynucleotides thereof. “Primer” herein refers to the synthesis of template-directed DNA synthesis under appropriate conditions (eg, four different nucleoside triphosphates (ATP, GTP, CTP, TTP) and DNA polymerase) in a suitable buffer and at a suitable temperature. It refers to a single stranded oligonucleotide that can act as a starting point. The appropriate length of the primer may vary depending on the purpose of use, but is usually 10 to 100, preferably 10 to 80 nucleotides. Short primer molecules generally require lower temperatures to form stable hybrids with the template. The primer sequence need not be completely complementary to the template, but should be sufficiently complementary to hybridize with the template. It is preferred that the primer 3'-terminal alignment with the single nucleotide polymorphism of SEQ ID NO: 1 to 10. The primer hybridizes to the target DNA comprising the polymorphic site and initiates amplification in allelic form of DNA where the primer exhibits complete homology. This primer is used in pairs with a second primer that hybridizes to the other side. The product is amplified from two primers by amplification, and only the amplified product is specifically stained and visualized. This means that certain allelic forms exist. If the allele-specific polynucleotides used herein undergo different denaturation, Infinium ^™ Assay I can be used by other methods as well.

The present invention includes a probe for diagnosing enteric gastric cancer and a microarray for diagnosing enteric gastric cancer comprising the polynucleotide (a) to (j) selected from the group consisting of one or more polynucleotides or complementary polynucleotides thereof. The present invention also includes a kit for diagnosing enteric gastric cancer, comprising a polynucleotide selected from the group consisting of polynucleotides (a) to (j) or complementary polynucleotides thereof.

The present invention provides a nucleic acid sample from a sample in order to provide information necessary for the diagnosis of enteric gastric cancer; And (ii) analyzing the nucleotide sequence of the polymorphic site of at least one polynucleotide selected from polynucleotides of SEQ ID NOs: 1 to 10 or complementary polynucleotides thereof from the nucleic acid sample, provided that the polymorphic site is 501 base for DNA sequence, 101 base for DNA sequence of SEQ ID NO: 2, 401 base for DNA sequence of SEQ ID NO: 3, polymorphic site is 501 base for DNA sequence of SEQ ID NO: 4, polymorphism The site is the 301th base for the DNA sequence of SEQ ID NO: 5, the polymorphic site is the 501st base for the DNA sequence of SEQ ID NO: 6, the polymorphic site is the 262th base for the DNA sequence of SEQ ID NO: 7, and the polymorphic site is 2187 base for the DNA sequence of SEQ ID NO: 8, the polymorphic site is 301 base for the DNA sequence of SEQ ID NO: 9, DNA sequence of SEQ ID NO: 10 In the case, the sample containing the 201st base means, respectively) include a method of detecting a single nucleotide polymorphism.

The sample includes blood, tissue, cells, and the like, and a nucleic acid sample can be obtained according to a conventional method. For example, in the case of blood, a nucleic acid sample can be obtained as follows. Transfer blood to a centrifuge tube and add low concentrations of salt component buffer (10 mM Tris-HCl [pH 7.6], 10 mM KCl, 10 mM MgCl ₂ , and 2 mM EDTA). Then, after adding Nonidet P-40, the resulting solution was mixed well and centrifuged at about 2,200 rpm for 10 minutes at room temperature. The resulting mass is resuspended in a high concentration of salt component buffer (10 mM Tris-HCl [pH 7.6], 10 mM KCl, 10 mM MgCl ₂ , 0.4M NaCl, and 2 mM EDTA). Genomic DNA is mixed with 50ul of 10% SDS and reacted at about 55 ° C overnight. After the reaction, each genomic DNA is transferred to a 1.5 ml centrifuge tube and 0.3 ml of 6 M NaCl is added. After mixing the genomic DNA well, centrifuge for 10 minutes at about 12,000rpm. After centrifugation, the upper layer containing genomic DNA is collected, transferred to a new test tube, and the same amount of 100% ethanol is added at room temperature. Mix the genomic DNA well until it is settled, and wash the genomic DNA with 70% ethanol when settling. At the end of this procedure, open the test tube lid and allow the solution to dry. The nucleic acid sample can be obtained by resuspending genomic DNA by adding TE buffer solution (pH 8.0).

Here, the step of analyzing the nucleotide sequence of the polymorphic site is a sequence comprising a polymorphic site as a polynucleotide or a complementary polynucleotide sequence thereof selected from the group consisting of polynucleotides of SEQ ID NO: 1 to 10, wherein the polymorphic site is 501th base for the DNA sequence of SEQ ID NO: 1, 101 base for the DNA sequence of SEQ ID NO: 2, 401 base for the DNA sequence of SEQ ID NO: 3, and 501th base for the DNA sequence of SEQ ID NO: 4 Base, the polymorphic site is the 301th base for the DNA sequence of SEQ ID NO: 5, the polymorphic site is the 501th base for the DNA sequence of SEQ ID NO: 6, the polymorphic site is the 262th base for the DNA sequence of SEQ ID NO: 7, The polymorphic site is 2187th base for the DNA sequence of SEQ ID NO: 8, and the polymorphic site is No. 301 for the DNA sequence of SEQ ID NO: 9 The second base, in the case of the DNA sequence of SEQ ID NO: 10 means 201 base each) can be carried out using a primer or a probe as a template. The primer or probe is as described above.

In addition, the step of analyzing the nucleotide sequence of the polymorphic site is a step of hybridizing the nucleic acid sample in a microarray to which a polynucleotide selected from the group consisting of polynucleotides (a) to (j) or a complementary polynucleotide thereof is immobilized. Wherein the polynucleotides (a) to (j) are as defined above); And analyzing the obtained hybridization results, wherein the lengths of the DNA sequences of the polynucleotides or complementary nucleotides immobilized on the microarrays may be 10 to 100 nucleotides, respectively.

The results obtained according to the method for detecting a monobasic polymorphism in a sample of the present invention may provide information necessary for diagnosing gastrointestinal gastric cancer. If one or more G, C, G, G, G, G, A (risk alleles) are present, it can be determined that they belong to a high risk group of gastric cancer. The more the nucleic acid sequence having the risk allele is detected in one sample, the higher the probability of belonging to the risk group can be determined.

In addition, analyzing the nucleotide sequence of the polymorphic site may include genotype analysis of the polymorphic site of the DNA sequence selected from the group consisting of the DNA sequence of SEQ ID NO: 1 to 10. That is, the genotypes of the polymorphic regions of SEQ ID NOs: 1 to 10 are A / A and A / G (SEQ ID NO: 1), G / G and A / G (SEQ ID NO: 2), G / G (SEQ ID NO: 3), G / G (SEQ ID NO: 4), C / C and A / C (SEQ ID NO: 5), G / G and A / G (SEQ ID NO: 6), G / G and A / G (SEQ ID NO: 7), G / G and If one or more A / G (SEQ ID NO: 8), G / G (SEQ ID NO: 9), and A / A (SEQ ID NO: 10) are present, the risk of developing gastrointestinal cancer may be high. The more the nucleic acid sequence having the risk genotype is detected in one sample, the higher the probability of belonging to the risk group can be determined.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example

1. Selection of subjects

This study was performed with 21 patients who were clinically diagnosed as enteric gastric cancer and 24 patients who were diagnosed with chronic gastritis from Korea Ajou University Hospital. All 45 patients were infected with Helicobacter pylori. The size of the clusters used is shown in Table 1 below.

group Normal Patient group total Survey 24 21 45

2. Preparation of Genomic DNA

Blood was collected from enteric gastric cancer patients and chronic gastritis patients to prepare genomic DNA (gDNA). 5 ml of total venous blood was obtained from each volunteer's anterior venous vein and transferred to a Vacutainer tube containing 15% EDTA. To extract genomic DNA, the blood is transferred to a centrifuge tube and a low concentration of salt buffer solution (10 mM Tris-HCl [pH 7.6], 10 mM KCl, 10 mM MgCl ₂ , and 2 mM EDTA) is added. 5 ml was added. And Nonidet P-40 was added. The resulting solution was mixed well and centrifuged at 2,200 rpm for 10 minutes at room temperature. The obtained mass was resuspended in a high concentration salt buffer solution (10 mM Tris-HCl [pH 7.6], 10 mM KCl, 10 mM MgCl ₂ , 0.4M NaCl, and 2 mM EDTA). The genomic DNA was mixed with 50ul of 10% SDS and reacted overnight at 55 ° C.

After the reaction, each genomic DNA was transferred to a 1.5 ml centrifuge tube and 0.3 ml of 6 M NaCl was added. After mixing the genomic DNA well, it was centrifuged for 10 minutes at 12,000rpm. After centrifugation, the upper layer containing genomic DNA was collected and transferred to a new test tube, and the same amount of 100% ethanol was added at room temperature. Mix well until genomic DNA precipitates and wash genomic DNA with 70% ethanol when precipitated. At the end of this process, open the test tube lid and let the solution dry. Then, TE buffer solution (pH 8.0) was added to resuspend the genomic DNA.

Prepared genomic DNA using the ^{Quant-iT TM PicoGreen dsDNA reagent (} Molecular Probes, Invitrogen) for the Infinium Assay II is adjusted to a concentration of 750ng / 15uL.

3. Genotyping of the Whole Genome-Infinium II Assay

Whole-genome amplification (WGA) Genomic DNA (750ng / 15uL) was dissolved at room temperature (22 ° C) and transferred to each well of 0.8mL micotiter plate. 15 uL of 0.1N NaOH was added and reacted at room temperature for 10 minutes. After the reaction, add 270uL of MP1 solution and 300uL of AMM solution in turn, and mix well. The reaction was centrifuged at 280g for 20 hours in a 37 ℃ oven.

Fragmentation and Purification of Amplified Genomic DNA After the whole genome DNA amplification step, the cells were centrifuged at 50 g for 1 minute and transferred to three extra wells at 150 μL. As a result, four wells become identical genomic DNA. 50 μL of FRG solution was added to each well, followed by vortex for 1 minute at 1600 rpm. After centrifugation at 50g at room temperature, the reaction was carried out at 37 ℃ for 1 hour. Genomic DNA was centrifuged at 50g, 100uL of PA-1 solution was added and vortexed at 1600rpm, centrifuged at 50g at room temperature, and left at 37 ° C for 5 minutes. After centrifugation at 50g for 1 minute at room temperature, 300uL of 100% isopropanol was added and reacted at 4 ° C for 30 minutes. After centrifugation at 3000g for 20 minutes at 4 ℃ to confirm the genomic DNA clumps, the top layer was removed by inverting the microtiter plate. After drying the genomic DNA for 1 hour at room temperature, 42uL of RA1 solution was added, and reacted for 1 hour at 48 ℃ oven.

Preparation and Sentrix ^R BeadChip (Sentrix ^R ) BeadChip was prepared while resuspending amplified genomic DNA in RA1 solution. The BeadChip was removed and placed in a hybridization chamber containing PB2 solution until genomic DNA was added.

After resuspension of hybridized amplified genomic DNA (48 ° C., 1 hour reaction), vortex for 1 minute at 1800 rpm, centrifuge at 280 g, and denature amplified genomic DNA for 20 minutes in a 95 ° C. heat block. , Centrifuged at 280 g. Genomic DNA divided into four wells was pooled into one well and centrifuged at 280g. After removing the BeadChip Chamber from the hybridization chamber (Hybridization Chamber), the genomic DNA was added, the BeadChip Chamber was put back into the hybridization chamber, and reacted by shaking at 48 ° C. at an appropriate speed for 18 hours.

After 18 hours of hybridization with single base extension (SBE) , the BeadChip was taken out, washed sequentially with the WB1 solution and the PB1 solution, and then flowed 150 μL of RA1 solution. 450uL of the XC1 solution, 450uL of the XC2 solution, and 200uL of the TEM solution were sequentially reacted for 10 minutes, 5 minutes, and 15 minutes, respectively, and 450uL of 95% formamide / 1 mM EDTA was flowed. 450 uL of XC3 solution was flowed and genomic DNA stained with LTM and ATM solution according to Illuminas protocol. After disassembling the BeadChip chamber, the BeadChip is washed sequentially with PB1 solution, XC4 solution, and dried using vacuum desiccator. The final step was to scan the Illumina BeadArray reader and save an image file for each BeadChip.

Genotyping Analysis of genotypes from image files was performed using the Bead Studio Version 3.2 software to genotype each single polymorph.

4. Statistical Analysis

The Hardy-Weinberg equilibrium test was performed to determine the genetic distribution of the alleles that make up the monobasic polymorphisms. In this case, the criteria of HWE p value> 0.05 and Minor Allele Frequency (MAF)> 0.1, which are the criteria for polybasic polymorphism, were applied. Using the frequency of incidence between intestinal gastric cancer patients and chronic gastritis patients, the significance level of each polybasic polymorphism was set to <0.05 and analyzed using SAS software version 9.1.3. Correlation analysis was performed to compare significant differences between alleles in the incidence of intestinal gastric cancer and in patients with chronic gastritis. The significance level was <0.05. Odds Ratio was calculated by logistic regression model and dominant, recessive and co-dominant models were applied. In analyzing the significance of each model, the majority allele is A1 and the minor allele is A2. The dominant model compares the sum of the frequency of A1A1 with the frequency of A1A2 and A2A2. The Recessive trait model compares the sum of A1A1 and A1A2 with the frequency of A2A2, while the Co-Dominant trait model compares the respective frequencies for A1A1, A1A2 and A2A2.

5. Results

(1) Single base polymorphism associated with enteric gastric cancer

Table 2 shows six sets showing the highest score among the results of screening SNP sets strongly associated with gastric cancer of 510,000 monobasic polymorphisms.

Monobasic Polymorphic Set Accuracy Sensitivity Specificity rs12117796 rs16841280 rs2333715 rs3820380 rs10158553 97.78 100 95.83 rs12117796 rs16841280 rs971081 rs3820380 rs10158553 97.78 100 95.83 rs12117796 rs16841280 rs6691829 rs3820380 rs10158553 97.78 100 95.83 rs12117796 rs16841280 rs3860304 rs3820380 rs10158553 97.78 100 95.83 rs12117796 rs16841280 rs1171386 rs3820380 rs10158553 97.78 100 95.83 rs12117796 rs16841280 rs3795593 rs3820380 rs10158553 97.78 100 95.83

Table 2 shows the single nucleotide polymorphisms associated with intestinal gastric cancer using the forward selection method after extracting only the top 500 single nucleotide polymorphisms by χ ² value to find a single nucleotide polymorphism set strongly associated with long-term gastric cancer among 510,000 single nucleotide polymorphisms. The top six of the set extraction results. The steps of the forward selection method are as follows. First, not selecting a set to obtain a single recognition rate for the n sets of the single nucleotide polymorphisms for the n number of single nucleotide polymorphisms having highest recognition rate s _l and contained in the following, the set selected in the previous step s _i and s _j For each monobasic polymorphism s _j , we construct s _i ∪ { s _j } to find the recognition rate and select the set with the highest recognition rate as s _{i + 1} . If i is smaller than n , the second step is repeated, and if i is n , a set having the highest recognition rate among s _k (1 ≦ _k ≦ n) is output as a result. A decision tree was used as a recognizer for this experiment. In addition, the LOOCV method was used in evaluating the recognition rate because the number of data was too small to use separate data for training and evaluation of the recognizer. The LOOCV method trains the recognizer with the data except for each data P _i from the given data, and uses the trained recognizer for the remaining data P _i to determine whether the patient is present and to evaluate the correctness and wrongness of the results for all data. This means that the recognition rate is calculated by using the number correctly recognized by the recognizer and the wrong number. In Table 2, the meaning of each column is as follows.

A monobasic polymorphic set is a monobasic polymorphic set extracted by the forward selection method.

Accuracy refers to the ratio correctly determined for all samples.

Sensitivity refers to the rate at which patients are correctly identified as patients.

Specificity refers to the ratio of correctly determined normal to normal.

(2) Monobasic polymorphisms showing significance in ZMPSTE24, GPR37L1, LMX1A, SRGAP2, FAM5C, EDEM3 genes

Table 3 below shows the characteristics of the polymorphic sequences of SEQ ID NOs: 1 to 10, which are statistically significant as a result of analyzing the association between the patients with chronic gastritis and the patients with bowel gastric cancer.

SEQ ID NO: Reference SNP ID Allele location gene SNP Role Amino acid changes One rs12117796 G> A 40509484 ZMPSTE24 intron No change 2 rs16841280 A> G 163458326 LMX1A intron No change 3 rs3795593 G> A 200358669 GPR37L1 5'UTR No change 4 rs3820380 G> A 200363438 GPR37L1 intron No change 5 rs10158553 A> C 204687870 SRGAP2 intron No change 6 rs1171386 A> G 188578948 FAM5C intron No change 7 rs3860304 A> G 188501670 FAM5C intron No change 8 rs6691829 A> G 188456354 FAM5C intron No change 9 rs971081 G> A 182983080 EDEM3 intron No change 10 rs2333715 A> G 182962655 EDEM3 intron No change

Sequence number is a sequence number which represents each monobasic polymorphism.

Reference SNP ID refers to the name of the standard single nucleotide polymorphism (Reference SNP ID; rs), which is used to distinguish each single nucleotide polymorphism from the database of the National Institute of Biotechnology Information (NCBI) under the National Institute of Health. Name given.

Alleles represent major and minor alleles in each monobasic polymorph.

Position refers to the position of the standard base sequence on the chromosome (NCBI Genome build 36.3).

The SNP role indicates where the single base polymorphisms are located on the gene. Thus, it can be seen how each monobasic polymorphism works in gene expression and function.

A change in amino acid is a description of whether the monobasic polymorphs are changing the amino acids constituting the protein when the gene is expressed to produce a protein.

(3) Genotype and Risk Allele Analysis

Table 4 shows the allele frequency of the DNA sequences of ten monobasic polymorphisms (SEQ ID NOS: 1 to 10) belonging to the intestinal gastric cancer associated monobasic polymorphism set extracted from Table 2, and the chronic gastritis and typhoid gastric cancer patient groups for each genotype, respectively. Table 5 shows the results of the Hardy-Weinberg Equilibrium Test based on the genotype frequency of Table 4 and the analysis of gastric cancer (ie, Case-control association study). As a result, the following association analysis can identify monobasic polymorphisms and their genotypes that may affect intestinal gastric cancer.

In Table 4, the meaning of each column is as follows.

Sequence number is a sequence number which represents each monobasic polymorphism.

Reference SNP ID refers to the name of the standard single nucleotide polymorphism (Reference SNP ID; rs), which is used to distinguish each single nucleotide polymorphism from the database of the National Institute of Biotechnology Information (NCBI) under the National Institute of Health. Name given.

The allele frequency column indicates the two nucleotide bases that make up each monobasic polymorphism and indicates the frequency of each allele observed in the population. Here, an allele means a nucleotide sequence of nucleotides actually present at a single nucleotide polymorphism site, and a single nucleotide polymorphism may have two alleles due to its characteristics. According to the high and low frequency of these alleles, they are divided into large alleles and small alleles. In particular, the minor allele frequency (MAF) is used as a genetic reference for dividing a single nucleotide polymorphism and a point mutation based on 0.1. This criterion represents the stability of the polymorphism itself (that is, the site where a monobasic polymorphism occurs is constant), thus representing its potential use as a genetic marker.

Genotype consists of alleles that form each monobasic polymorphism, and shows what alleles are in the locus where the monobasic polymorphic site exists.

The genotype frequency column is made up of alleles that form a monobasic polymorphism and indicates what alleles are in the locus where the monobasic polymorphic site exists. Here, the genotype consisting of only large alleles is represented by the genotype consisting of the large major homotype, the large allele and the small allele is heterotyped, and the genotype consisting of only the small alleles is represented by the small minor homotype. It is called. The numbers represent the number of people of each genotype divided into patients with chronic gastritis and those with enteric gastric cancer. This can be called an observation.

Table 5 compares the frequency differences between the two alleles that form a single polymorphism and the three types of genotypes resulting from chronic gastritis patients and gastrointestinal cancer patients, and which alleles or genotypes are associated with the disease. This is the result of analysis. This association analysis was carried out in four categories: the allele model, the co-dominant genotype, the dominant genotype, and the recessive model. Meaning of each column in Table 5 is as follows.

The sequence number is a number representing each monobasic polymorphism.

Reference SNP ID refers to the name of the standard single nucleotide polymorphism (Reference SNP ID; rs), which is used to distinguish each single nucleotide polymorphism from the database of the National Institute of Biotechnology Information (NCBI) under the National Institute of Health. Name given.

HWE represents the state of the Hardy-Weinberg Equilibrium. Based on chi-value = 3.84 (p-value = 0.05, df = 1) in the chi-square (df = 1) test, it is judged by Hardy-Weinberg equilibrium HWE (Hardy-Weinberg Equilibrium) if it is greater than 3.84, If less than 3.84, it was judged as Hardy-Weinberg Disequilibrium. This indicates that the genotype and distribution of genotypes of the individual monobasic polymorphisms observed in both patients with chronic gastritis and intestinal gastric cancer are common.

The odds ratio (OR) represents the ratio of odds of risk allele among the group of chronic gastritis patients to odds of risk allele among patients with gastric cancer. 95% CI represents the 95% confidence interval of the odds ratio, indicating (lower confidence interval, upper confidence interval). Confidence intervals with a value of 1 cannot be considered significant for association with disease. If the odds ratio is greater than 1 and does not include 1 in the 95% confidence interval, minor alleles have a higher frequency in the disease group. If the odds ratio is less than 1, the majority allele has a higher frequency in the disease group. Have

In the correlation analysis on the allele type, the Logit_p value represents the statistical significance for the odds ratio. This means that the difference between the patients with chronic gastritis and the patients with gastrointestinal cancer is as significant as the odds ratio. The criterion for determining that there is a significant difference is when the Logit_p value indicates a value of 0.05 or less. These values were obtained through logistic regression analysis.

In the correlation analysis of allele types, Exact p value also means the significance of the difference between the patients with chronic gastritis and gastrointestinal gastric cancer like Logit_p value. This also recognizes a significant difference when the p value is less than 0.05. The exact p value is obtained using a statistical method called Fisher's exact test, which can be performed when the expected frequency required for the comparison of patients with chronic gastritis and those with enteric gastric cancer is less than or equal to 5 This is a statistical technique developed for the purpose. Therefore, the data shown in Table 5 is a very suitable assay.

The risk allele was a small allele if the odds ratio was greater than 1 when the p-value was significant and a majority allele if the odds ratio was less than 1.

In analyzing the significance of the disease according to the four analysis models, if the majority allele is A1 and the minor allele is A2, the dominant model compares the sum of the frequency of A1A1 with the frequency of A1A2 and A2A2. The recessive trait model compares the sum of A1A1 and A1A2 with the frequency of A2A2, and the co-dominant trait model compares the respective frequencies for A1A1, A1A2 and A2A2.

Correlation analysis of alleles can be used to determine how relevant alleles are to enteric gastric cancer by the ratio of the frequency with which alleles are present in patients with chronic gastritis and gastrointestinal gastric cancer. Since there is no significance when 1 is included in the 95% confidence interval, as shown in Table 5, the nucleotide sequences of the polymorphic sites of SEQ ID NOs: 1 to 10 are A, G, G, G, C, and G, respectively. If more than one G, G, G, or A (risk allele) exists, you may be more likely to develop gastric cancer

In addition, SEQ ID Nos. 1 to 10 show significance in alleles, co-occurrence and dominant models, and SEQ ID Nos. 1 to 2 and SEQ ID NOs: 5 to 8 show odds ratios> 1 and genotypes to minor alleles. Those who have a higher risk of developing gastrointestinal cancer than those who have Major Allele, and those who have at least one genotype with a small number of alleles are more likely to have The risk of catching was high. On the other hand, SEQ ID NOs: 3 to 4 and SEQ ID NOs: 9 to 10 have an odds ratio of <1, and those with genotype Minor Allele are more likely to have gastrointestinal gastric cancer than those with Major Allele. The risk of getting higher was also higher, and people with two alleles of the genotype had a higher risk of developing gastric cancer than those with any one of the minor alleles.

From the above results, the polymorphic regions of SEQ ID NOs: 1 to 10 are genotypes A / A and A / G (SEQ ID NO: 1), G / G and A / G (SEQ ID NO: 2), and G / G (SEQ ID NO: 3), respectively. , G / G (SEQ ID NO: 4), C / C and A / C (SEQ ID NO: 5), G / G and A / G (SEQ ID NO: 6), G / G and A / G (SEQ ID NO: 7), G One or more of / G and A / G (SEQ ID NO: 8), G / G (SEQ ID NO: 9), and A / A (SEQ ID NO: 10) may be considered to be at high risk for gastric cancer. In the case of SEQ ID NO: 1, A, SEQ ID 2 is G, SEQ ID 3 is G, SEQ ID 4 is G, SEQ ID 5 is C, SEQ ID 6 is G, SEQ ID 7 is G, SEQ ID 8 is G , SEQ ID NO: 9, G, SEQ ID NO: 10, A, respectively, appeared to be significantly higher in the gastrointestinal gastric cancer group than in the chronic gastritis group, indicating that each allele is susceptible to gastrointestinal gastric cancer. Can be. The more a nucleic acid sequence having the risk allele is detected in one sample, the higher the probability of belonging to a risk group can be determined. In contrast, in the case of SEQ ID NO: 1, G, SEQ ID NO: 2 is A, SEQ ID NO: 3 is A, SEQ ID NO: 4 is A, SEQ ID NO: 5 is A, SEQ ID NO: 6 is A, SEQ ID NO: 7 is A, and SEQ ID NO: 8 is A, SEQ ID NO: 9 and A, SEQ ID NO: 10, respectively, were significantly higher in the chronic gastritis group than in the gastrointestinal gastric cancer group, so that those with alleles were resistant to enteric gastric cancer. Can be predicted. Therefore, in the present invention, the single nucleotide polymorphism of SEQ ID NOs: 1 to 10 can be effectively used for diagnosis related to enteric gastric cancer. Specifically, it may be used as a target site as a primer or probe for diagnosis of enteric gastric cancer.

In view of the results of such an analysis, each allele may exist in the form of a homozygote or heterozygote in each individual. Mendel's genetic law and Hardy-Weinberg law show that the composition of the alleles that make up a group is maintained at a constant frequency and, if statistically significant, can give a biological function meaning. The monobasic polymorphism of the present invention exhibits a statistically significant difference between entero gastric cancer patients and chronic gastritis patients, indicating that these monobasic polymorphisms can be used for the diagnosis of enteric gastric cancer.

<110> College of Medicine Pochon CHA University Industry-Academic Cooperation Foundation <120> Polynucleotides derived from chromosome 1 comprising single          nucleotide polymorphism, microarrays and diagnostic kits          configuring the same, and analytic methods using the same <130> PN0243 <150> KR10-2008-0122469 <151> 2008-12-04 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 701 <212> DNA <213> Homo sapiens <400> 1 tcttttcccc ctgatttttc cctccctcac atctcctatt cctcttctct ttccatactc 60 tcaagtgctt cacaacacaa acactacaac gcagtcaaaa tggtctgttc agggccaggc 120 acagtggctt atgcctgtaa tcctggcact ttgggaagcc aaggtgggag gattgcttaa 180 gcccaagagt ttaagaccag cctgggtaac acagtgagac tctgtctcta caaataataa 240 tttttaaaaa acccaaatga aaaaacccaa agtggtctgt tcagtgcacc cagaaattat 300 catctatcag ctcttcagct ccactgatga ggtttatttt attttagaga tagggcctcc 360 ctgtgttgcc caggctggag tacagtggct atttcacagg catgatcata gtgcactaca 420 gctttgagct cctgggctca agcagtcctc ctgccttggc ctcctgagta gctgggacta 480 caggtgtgtg acaccatgcc yaagctgagg tttattttag tctgaattac ataatgctca 540 ttgtttcatt ttccattgat gggatcaaga tcagaaaatg ggcgctagtt ggatttacag 600 gtatctattg tatggcaaat taaagtaaga tgaaaaagag cagagaaaat gctctaaaaa 660 tgcttaagga agtgctattt caaccacatt gtacaattat g 701 <210> 2 <211> 201 <212> DNA <213> Homo sapiens <400> 2 taggttcaaa tgaataaggt ttgagtgtgt ttgtcctaat ttgggaagta acttaagtgg 60 atcgaatacc atctctcttt ggagtagtcc aattctgcta yattaattac tagtcttttg 120 aaggataaat tatctgagaa aattaaattg tttctaggca agggttggaa gacaaagggt 180 taattatctc tccaaattga a 201 <210> 3 <211> 801 <212> DNA <213> Homo sapiens <400> 3 ctagcaccta gtaagtgttc agtaagtgac agtgatggtt attgctgagt tttgaatgga 60 ggagctgcct tagaatcagg agacctgcgc cccagttccc attctgcccc acctccctgt 120 gtcaccctag gcaggccaca tttcctccct agtttcaggg gcctgaagca gatgccctct 180 tagggcccca cagcctgaca tgctgtaggg cctgagaagc ggcgtcgtgg aggacgagat 240 gtgtgagggc agcaaagagt gctatgtgtc cagcagaggg ccctgcccgg cctgtggccg 300 gaggctggga gggagggcag gcgagtgatg ccagacgcct gactggaggc ggatccagcc 360 ggccagctgc ctctctggag cccagctctt gggccccctg yactcacctg ctcttcctgg 420 gctggctgtc tcctgctcat ccagccatgc ggtggctgtg gcccctggct gtctctcttg 480 ctgtgatttt ggctgtgggg ctaagcaggg tctctggggg tgcccccctg cacctgggca 540 ggcacagagc cgagacccag gagcagcaga gccgatccaa gaggggcacc gaggatgagg 600 aggccaaggg cgtgcagcag tatgtgcctg aggagtgggc ggagtacccc cggcccattc 660 accctgctgg cctgcagcca accaagccct tggtggccac cagccctaac cccggcaagg 720 atgggggcac cccagacagt gggcaggaac tgaggggcaa tctgacagga gcaccagggc 780 agaggctaca gatccagaac c 801 <210> 4 <211> 701 <212> DNA <213> Homo sapiens <400> 4 gttgattggg aatgtgatga cacgtaaagt gcccgaccca taggaaatgc tcattaaatg 60 gagttgttac cattattata attccttcct tctttccttc cttccttcct tccttccttc 120 tttccttcct tccttccttc cttgtttcca cagggttttg ctctgtcacc caggctggag 180 tgcagtgtcg tgatcttggc tcactgcagc ctcagcctcc taggctcaag ccatcctcct 240 acctaagttt cccgagtagt tgggaccaca ggcgtgcctc accacgacag gctatttttt 300 tgtattttta gtagagacag ggttccatca tgttgcccag gctggccttg aactcctggg 360 ctcaagtgat ctgcctgcct cggcctctca aagtgctggg attacaggtg tgagtcaccg 420 cacccggccc attattataa tttctatgct tatcactcaa ttcatggtct aactgagcca 480 gaagccaggt gtccttcctt rtccccatag attttgacca agtgcttctc tcttccctca 540 catcctgccc acaggtctcc tctctgggag tcacgacttt cagcctctgt gccctgggca 600 ttgaccgctt ccacgtggcc accagcaccc tgcccaaggt gaggcccatc gagcggtgcc 660 aatccatcct ggccaagttg gctgtcatct gggtgggctc c 701 <210> 5 <211> 601 <212> DNA <213> Homo sapiens <400> 5 ggtctcagcc tactggatga tgagctccag ggagctgtcc ttgtgcatgt gacttagagc 60 tttggttttc ccccttgcat gagtggtgaa aagctgtaga atagcattcc cttccttcta 120 aagtcaggca gaacctccgc cctcacttct caaacatcaa accactgcag gctctggtcc 180 agtgcccggg gcacagagac ccctacagcc ctggcccctg agtcccagcc cagtgggtca 240 gagtcctgga ggggtgtggc tcacggggac aggtgccttc ctccagtttc ctccaagata 300 ktctcccaac ctacccacct gtttcttgtg cactcaacat ttattagcag cctctggaaa 360 aaccacaaga ttagtcaggg aacattacta ccgcagactg tgtgggaggc tagaagcacc 420 tgaggaacaa gagtcagcaa ccttgggttg agagggacct tctaaccgct ggattcacac 480 ccacctggaa ggctcatggc ctctcctctc ccttctttgg gaagtgagag aagtgccagc 540 cccgggagct aaggcagtaa ggaggtgagt gagggtggta gaaggggaga tagagcacta 600 a 601 <210> 6 <211> 1001 <212> DNA <213> Homo sapiens <400> 6 agacacctcc ctttatgatg tcaatctaag ggctgagcta agggaaataa atgtgattat 60 tgtaagtaac aatacttctg atctcagtta tataaactac atgtacattt ttaggaaaaa 120 aatgaataaa tatagatgtt taaacatatg aagtatatct caaatgataa atgtttgtat 180 ttagctgtgg cctagaataa atacagaatt ttcttatggg ccaaagacag aaaacggtgt 240 taagatcaag tgatgaaaag ctttgaatac aagtttaagg aggtagactt cattcagtag 300 gattcgaaga actatggtgg ctctttaaac agaactgcgg catagccatt aaatgtatct 360 aggaaaaata tctaccatca atcaaagtac aggttgaagt taagatgaac aggcgatgta 420 tcaattattg atttattaaa ctgagggaaa ctgctgcatg tataatgagg aagaaccacc 480 cagggttaga ttttgggaac rgaggaagag tgaattaaaa agttgacaga aaaataaaat 540 gagttaggtt tgaagaggta aatatttaaa attctgtggc agatatactt ggaagaaggt 600 ggctgtttaa ggctagagtt tgagagagaa gttaagacta cagatgaata aatgcagaaa 660 ctttgcacag taaagtgaaa acaaaaccaa attagcctga ttttgaatct tgattctgct 720 atgtactatg gtgagctact tgggaaagtc agttatcctt tccaaaactc agcttactta 780 actgtaaaat tggaaaataa taagcattct gcacaggtat tataaaggaa ggatgacata 840 taaattaagt gcatatttga atgccttgta tatagtacat gctcaacaat catttttact 900 tttattatta tcattacaat aataaaaacc attactagta ttattatatt taatctctaa 960 tttgttagag gtgttagatt aaagaaaatt ggtaacatag a 1001 <210> 7 <211> 1371 <212> DNA <213> Homo sapiens <400> 7 ccaaacaaaa gggttgctga aagaatgaaa acagcaagtt tgacaattat gtgctagatg 60 agtatccgat aatatgtttg tcggaggatc acaagttctt aatgtctaac ttcaatgaca 120 tggatttttg tcttttggac attgaaaagt tcagcttaat tgtaatacac tttacgtttt 180 acgtttgaga accaattgct actacctaaa tcctacatag atttcagtgt ttttcacaca 240 gtgtattaac acacaatgtt tyaatatatc ctatgcaggg aaattctgaa tttattacat 300 atatatacat acctgtatat gcatacacat gctttatctg aattttaaag gtgcagatga 360 aaactagtgc tttgtcatat ttccatttgg atattctatt ctatggtaaa aatttctgag 420 taaatgcaat ttctttcttt ttatttattt atttatttat ttttgagacg gagtctcgct 480 ctgtcgccca ggctggagtg cagtggcggg aacctccgct cactgcaagc tccgcctccc 540 gggttcacgc cattcttctg ccacagcctc cggagtagct ggaactactg gcgcccgcca 600 ccacgcccgg ctaatttata tatatatata tatatatatt tagtagagac ggggtttcac 660 cgtgttagcc aggatagtct cgatctcctg ccctcatgat ccgcccacct cggccttcca 720 tagtgctggg attacaggcg tgagccaccg tgcccggccc tgagtaaaat acagtttcat 780 tttatgcccc tcacttgttt gttttccata aattaatttt attttggacc atatgagtgt 840 tatgctaaac tttgttagaa agtcaataaa atctggagaa actgttctgc aaaagctaaa 900 tatctgacat tcagtaacta cgtgtttaaa gtgaccaata tcccaggcac tttttgaggt 960 acttgagaaa tatctgtgac caaaaataaa accccttgcc cttttggcat atatcactcc 1020 tttgttttac tatattcata agctagacct ttccacctgc agataagtta aaaaaaaaga 1080 ttttaagtgg aaaattgaat aatttgaaat attattaatg caggagagga gtcactcaca 1140 atttttgtgg acaagcggaa gttgagcaaa cgagctgaag gaagtgattc caccaccaat 1200 agctcttcgg tcactctgga gacgctacat cagctagccg cttcttattt cattgacagg 1260 gacagcaccc ttcggagact tcaccacatt caaattgcat ccactgccat aaaggtaaga 1320 atgagtttac gctaaaatta tcaccttcca ttgtttgttt cctctatatc c 1371 <210> 8 <211> 7297 <212> DNA <213> Homo sapiens <400> 8 aaaatcattt ttaattgatg tgtattttat tgtaatttga agaattataa ttgtatacat 60 ttatgaggta acactggatg ttattatata tgtatataat gaagaatgag taattcaaac 120 taattaacat atctaccacc tcaaatactt atttttttat ggtgagaata tttgaaatat 180 attagaaata tattctgttg gcaattttga aacatacatt ttattaacaa gtcaacatgc 240 tatgctatag atctcaaaac ctatttttcc ggtctaactg aaactttgca acatttgatg 300 atcacatcct catacctccc cccaccctcc ttgtcccctc tattcaccaa tgtctgcttc 360 cattattttg gttgttttta atgctctatt ttgatcaaat aaatcttaag ggagcctaca 420 tttactaact cttcctaaat ctatttaccc tttcattgtg taatttgtgt tatttccaac 480 aacaacaaca aaagcaaaat attttgttga agccatcatt gtgttttctc ttgtctggaa 540 catgttttgt caccttcaca cagtactagg ataattcctt tgggaccaac tccattcagg 600 actgccagtg ctctcattat ttacaacggc aatgcaaata ggggattcag gcacaactaa 660 gagccaagaa taaagcagac ctagcagtaa agaatcacgg agcaagcata tttaactata 720 aatgaaaaaa attggacaga taaaactgga attaataatt aatccttcaa tgcaattcac 780 agacatttac ccataagaaa caacagcaaa caggaaacca tgacctcctc aaaaagacag 840 aacaaaaatt gagtgactga cccaaacaag atgctgattt gtgagctctc tgaccaagaa 900 ttcaaaacag cagttttcag gaaactcagt gatctctaag ataacacaga aaagcaattc 960 tgaaatttat tagagaaatt taacaaagat attaaaataa taaaaaatca aacagaaatc 1020 atggaataga gaaatacatt tcctgaactg aacaattcat tagaggtctc aacagtagaa 1080 tggaccaagc agagaaaaga atcagtgagc taaaagactg gctatttgaa aatacatagt 1140 cagggccagg aatggtggct cacgcctgta atcccagcac tttgggaggc cgaggtgggt 1200 ggatcacgag gtcaagagat cgagaccatc ctggccaaca tggtgaaacc ctgtctctac 1260 taaaaataca aaaattagct ggatgtggtg gcggatgtct gtagtcccag ctactcagga 1320 agctgaggca ggagaatcgc ttgaacctgg gaggtggagg tttcagtgag ccaagatctc 1380 accactgcac tccagcctgg gtgacagagt gagactccaa aaaaaaaatt aaaataataa 1440 taataataat aaattttaaa aaaagggaaa tacacagtca gagggaaaca aacaagcaaa 1500 caaatgaaaa acatgaaaag gaacaaagac cacctataag atacagaaaa ttacctcaaa 1560 agaccaaatt gaagaattat tggtattcaa aagtgagctg agcaagatca aggagtggaa 1620 attttagtca aataaataat gaaaaactgt gcaaagcttg agaaatattc aggtatagaa 1680 cgatctggga atactagtca gattcaatcc cgataagact acctccaagg catataataa 1740 ccaaactccc taagatcaat gaaaaagaga gaatcctaaa agcagcaaga gaaaagaagc 1800 aaaaaggatg agttcatgtc ctttacaggg acatgggtga agctggaaac catcattctc 1860 agctaactat cgtaagatca gaaaaccaaa cactgcatgt tctcaatcat atgtgggagt 1920 tgaacaatga gatcacacgg acacagggag aggaacatca cacatcaggg cctgtcaggg 1980 gttgggggct aggggagggt taacattagg agaaatacct aatgttggtg acgggttgat 2040 gggtgtagca aaccaccatg gcccgtgtat acctatgtaa caaaactgca cgttctgcac 2100 atgtaacgca gaacttaaag tataattttt tttaaaaaag aagcaaataa cataaaagac 2160 ataaaaggag cttcaatttg tctggcraca aacttctcaa tggcattttc aatgtgttca 2220 aagataaaaa aaattgcaac tcaaaaatat tgtattaggc aaaatcatcc ttaaaacatg 2280 aaggagagat aaactatttc ccaaataaac aaaaatggag agaatttacc accaccagac 2340 tcatcttaca agaaatgata aaggaagctc ttcagtctga aagtaaaaac atacaaatat 2400 gccaaaaaaa agttttcaag gcataaatcc cactggtaaa attaagtaca aggacaaact 2460 cagaatactc tgtcacttta attttggtgt gcaattctct tgtaacctca tatgaagccc 2520 aaaagtaaaa tctgtcaaaa acagtaatag ctatagcaac tttctaagag atagtataaa 2580 aatctgtaaa cagaggagtt cgagaccagc ctggccaaca tggtgaaacc ctgtctctac 2640 tacaaacaca aaaattagtc gggcatggtg gcagatgcct gtaatcccaa ctacttgggt 2700 ggctgaagca agagaatcgc ttgaacccgg gaggcagagg ttgcagtgag ctgagatcgc 2760 accactacac tccagcctag gcgacagagt gagactccat ctcaaacaaa caaacaacaa 2820 aaaaagtaaa ctggggcaac taaaagtaaa catgtgggtg agtggagtta aagtgtaaaa 2880 ttttttgtgt gctttttgtc tttgttgttt ctgttcttag tttgttatcc aaggtaagtt 2940 gtcttctctt tagcataact tactatatct ataagatgat ttttgtaagc ctcataactt 3000 gagataaatt catggaaaat gaaaagcaac aaattaaaac acactaccaa tgaaaatcac 3060 ttaatcacaa agaaagacag taagaagaga agaagagact aaaacgaacc agaaaacaag 3120 ccacaaaatg gcagtagtgt gcacttattt atcaataata acactgaatg taaatgatct 3180 cacttcccca attataacgt gtatagtggc tgaatgaata aagaagtaag acccaactat 3240 atgttgcctt caaaaaatcc acttaatgta gaaagataca catagactga gcgtgaaggg 3300 gtggaaaaag atactcgatg caactggaaa acaaaggagc aggagtagct atacctatat 3360 cagacaaaat agattaaaaa tctaagattt aaaaagacat aggaggtcaa tataaaatga 3420 taaaggggtc aatacagcaa gataatataa caattatcaa tagttttatg cccaacaaca 3480 gagtttccta taagtatata aagcacacat tagtagagct aaaaatatat ataagactat 3540 aataaaataa cagcagggga atttaacacc ctcgtatcag taatgaacat atcatccaga 3600 cagaaaatca agaaagaaag agcacagtta aactacgcac tagatctaat gggccctaga 3660 tgacattaac aaaacatttc acccaactgc tgcagaatat acattctttc cttcagtgca 3720 tggaacattc ttcagaatag gtcatatctt aagcaacaaa aaagtctgaa taaattaaaa 3780 aataaaaatc ttatcaagta acttttctga ccacaatgga ataaaaacca caaattgaaa 3840 acaagaggaa ccttggaaaa tacacaaaca catagaaata aaacatccta ctggacagct 3900 aatgagtcaa ttaaaaaaat aagaaggcaa ttaaaaaaaa caaatgaaaa caaaaataca 3960 acaaaccaaa acttgagata caataacatc tatatcaagc gggatgttta tagtaataaa 4020 tgcctaaatt aaaagagtat aaagacttca aataacctaa caatgaacct caacgaacga 4080 gaaaagcaag aataaaccaa accccaaatt aatagaagaa ataataaaaa tcacagcaga 4140 tatgaataaa gttgagacaa aaaaatacat aagatcaaca aaaggaaaag ctgcatgttt 4200 gaaaagatat taacaaaccc ttagccagat taagaataaa ggaagaagag gcaaataaat 4260 aaaataagaa atgaaaaagg gaaataaatg aaatttatac tagaaaaata cataagatca 4320 atgatagaaa aagttgggtt tttgaaaaga taatcaaaac tgacaaattc tttgtctaag 4380 aaaaaaaaga cctaattaaa taaaatcaga aatgaaaaag gagacataac aactgagaac 4440 ccagaagcac aaagaatcgt tagagactat tgtgaacaac tgcatgccaa caaattggaa 4500 aacttggaag aaatgaataa attcctggac acacacaact taccaagatt gaacaatgaa 4560 gaaataaaaa aaaaattaac aagccaaaaa tcaatatggc tatcaaagag ataattcaaa 4620 gtcttccatc aaaatgaagc tcaggatcat tggcttcact gctaactcta ccaaaattta 4680 aagaagaacc aatatcagtt ctacccaaat tctccaaaaa tttggaagga aggtaatact 4740 ccaaactcat tatatgaggt cagcattacc ctgatactaa aaccagacaa aaacatgaca 4800 agaaaccaat aagaacacta cagatcaata tcactgatga gcagagatgc aaagataatc 4860 aacaaaacac tagcaaacca aattcaacaa cacattaaca tgatcaagtg aaattcatcc 4920 tagggataca aagatggttc aacacatgca aatcaacaaa cgtgatgcat cacattagca 4980 gaatcaagaa aataaacata tcattatttc aatagatgcc aaaaaagcac ttgataaaat 5040 tgaacaacag ttcatgataa aaactctcat caaaatgggt atagagagaa tacacttcaa 5100 aattataaag gccatgtatg acaaacccac agttaacact gtgctgaatg agaaaaataa 5160 ttttttctta tgagaaaggc cttttcttta aagactggaa caagataaga atgcttactt 5220 tcaccacttt tattcaacat gacatggaaa gtcctctcca gaacaagtag ggaaaagaac 5280 taaataaaaa gcataaaaac tggaaagaag aagtcaaatt agcgttgttt gtagacaaca 5340 caatttcata cctagaaaaa acctcaataa aaacaatgtt aaaattgata aaaacattca 5400 gtaaagttgc gggatacaaa ataaactaca gaaatcaaca aataatctga aaaaacaaga 5460 aagcaacctc atttactatg gctacaactg tataaaatac ctcaaagcaa tcaaaccaaa 5520 taatggaaag atctatataa gaaaaactat gaaattctta tgcaaaaagt acaagaaaac 5580 acacaaataa tgtaaagata tttcatatgt atggattgga atatttaatg ttgttaaaat 5640 gacaatacta ctaaagcaat ttacggattc agcacaatcc ccatcaaaac agtggcattc 5700 ttcacagaaa tagaaaaaaa aaattataaa atatgtgcag gacatcaaaa gatacccaaa 5760 taactaaagt aatcatgagc aaaaaagaac aaagctagaa tcatcacacc acctgacttc 5820 aaatttacta caaagctatc acaatcaaaa cagcatagta ctgggataaa agcagacaca 5880 tagaccaatg gaatagaata gagaaccata tatatatcca cacatttaca gccaactcaa 5940 ctttgacaaa gactacacat acgttgggga aaagaaaatc ttttaaataa atattgctgg 6000 aaaaaatgga taattttatg cagaaaaaat ctagacccct atctctcacc atacacaaaa 6060 atcaaataaa aataaattat agacttaaat ctaaaactta aaaatataaa actaaaaacg 6120 aaattattta ggaaacacta caggacattg gtctgggcaa agattttttt gtttgtaaaa 6180 cctcaacatt acagacaact aaagcaaaaa atagacaaat ggaattatat caagctaaaa 6240 agcctctgta cagtaaacaa tcagcaaagt gaagagataa cccacagaat gggagaaaat 6300 atttacaaac cattcatctg tccagggatt aataaccaga atacataagg agctcaaaca 6360 actaaataac caaaacaaaa tactaaaaac caaaactaat aacccaatta aagactgggc 6420 agaatatctg aatacatatt tctcataaga agacataaag atggcaaaca gatatatgaa 6480 ataatgctca acatcactaa ttatcagaga aatgcaagtc aaaactacaa tttaaaatgg 6540 gttgtatcaa aagacaggca ataacaggtg ctggtgagga tgcagagaaa ggggaaccct 6600 tctacactgt tggtggaaat atagattagt agagccacta aggagaactc tatggagttt 6660 gctcaaacac ctaaagtaga agtaccatat tattaatctc actactgaat atatatacaa 6720 aataaaggaa atcagtatat tgaatatata gctgcactcc cttatttatt gcagcactat 6780 tcccaataac caaatacgga ttcaatttaa ctgcccatca gtggatgaat agattaagag 6840 aatgtgttat atatacacaa tggaatatta ttcagccata aaatagaatg aaatcttgtc 6900 atctgcaaca acatggatgg aacaagaggc ctttatatta agtgaaataa gtcaatcaca 6960 gaaagacaaa tattctaacc cacatgttcc cactcatatg tggaagctaa aaaaagtaga 7020 actcatgaag atagagagta gaacggtggt ttccagaggc caggaagggt agtgggggct 7080 gggagaaaat gcagggaaaa taaaagaata aaaatgtatt tattaagtgt gcttaaaaat 7140 cgcaaagata gtaaatttta tatgtatgtt ttaaccaagt aaaaataaaa caatcataac 7200 aaaactcaca aaacactata aaattaaaat ttagataact tcattgtaga aataagtgaa 7260 gtgaaaataa gcactaatga gaatcatgtt gaaaaag 7297 <210> 9 <211> 601 <212> DNA <213> Homo sapiens <400> 9 atatttccag tgggaccata cgtgtccact gtaatatgta ttttaaaatg ttcaactatt 60 ttacaatgtg agtgactctt gctgtgtact tcttctatat tcaatctgtg tgaggtctgt 120 aacccaagat ttagtctaga atttacactt cctcttttca agttctcaac taacatgaca 180 cagttctaaa atattaaaga gaatacagtt gccttgtatt tgttatttga agtcagttgg 240 aaattttttt ccggtcccta cactcagaaa cacttttagc tttctgtagg gtaaaagtgt 300 rtaggttttg gagttagaag ggcttgagtt caaatcctgt ctcagctatt tacaggttgg 360 accctaaaca ggttatttaa cccatctgag cagtgtcttt ctatgtaaaa tgagaataat 420 tgcaaagagt tattgtagac tatagatttt tttttaaaga acaggaacca atttctttaa 480 tttttaatac attcaccttt tttatatgtc ttgtgcctgg tacataaatg cttagcaaat 540 ttttttgaaa gaactgaatt gaattcttgt gaagatgaag taaaataatg tttttttttt 600 t 601 <210> 10 <211> 673 <212> DNA <213> Homo sapiens <400> 10 gtatctttta taacaatttt gttctctgga cagtttgaac tagtaagtac aagaataggg 60 aagtagacca gccttagaag aaattagggt tagtttagaa tttgtgaaaa accagtctat 120 attatatatt aactagttca ccatagagaa aaaacggatt gttatggtat acatttgaaa 180 ggaaattaaa atttcattta ygggttttaa gagtttggaa atatagagtg tatcaccttg 240 ctggcaggct aacaacacaa gggaaatttt ttcaaaggtg tatttcttta ttaagtggaa 300 gacaaccaaa taggccacga gcggtagctc atgcctgtaa tcccagcact ttgtgaggcc 360 aaggcgagtg gatcacttga ggccagaagt ttgagaccag cctcaccaac atggcgtaac 420 cccatctcta ctaaaaatac aaaaatcagc caggcatggt tgcgtgtgcc tgtaatccca 480 gctacctggg aggctgaggc acgaatctct tgaacttggg aggtggaggt tgcagtgagc 540 tgagatagcg ccacagcact ccagcctgtg tgacagagca agacctttac tattacaata 600 tgaatctgga ttaactagac tcaaactgag cagacactga ttaaaattag aatatttttc 660 agcaatccaa ata 673  

Claims (11)

delete delete delete delete delete A polynucleotide consisting of the DNA sequence of SEQ ID NO: 6, wherein the 501th base (polymorphic site) is G, and the polynucleotide (f) is composed of 10 or more consecutive DNA sequences comprising the 501th base; In a polynucleotide consisting of the DNA sequence of SEQ ID NO: 7, a polynucleotide (g) consisting of at least 10 contiguous DNA sequences comprising a 262 th base (polymorphic site) G and comprising the 262 th base; And In the polynucleotide consisting of the DNA sequence of SEQ ID NO: 8, the polynucleotide consisting of 10 or more contiguous DNA sequences including the 2187th base (polymorphic site) G and the 2187th base (h) Enteric gastric cancer diagnostic kit comprising at least one polynucleotide selected from the group consisting of or complementary polynucleotides thereof. In order to provide information necessary for diagnosing enteric gastric cancer, (i) obtaining a nucleic acid sample from a sample; And (ii) analyzing the nucleotide sequence of the polymorphic site of at least one polynucleotide selected from the polynucleotides of SEQ ID NOS: 6-8 or its complementary polynucleotides from the nucleic acid sample, provided that the polymorphic site is Means a 501st base for a DNA sequence, a 262th base for a DNA sequence of SEQ ID NO: 7, and a 2187th base for a DNA sequence of SEQ ID NO: 8, respectively). 8. The method of claim 7, wherein analyzing the base sequence of the polymorphic site comprises a polynucleotide selected from the group consisting of polynucleotides of SEQ ID NOs: 6-8 or a complementary polynucleotide sequence thereof comprising the polymorphic site; A polymorphic site means a 501st base for the DNA sequence of SEQ ID NO: 6, a 262th base for the DNA sequence of SEQ ID NO: 7, and a 2187th base for the DNA sequence of SEQ ID NO: 8) respectively; A method for detecting a monobasic polymorph in a sample, characterized in that carried out using. 8. The method of claim 7, wherein analyzing the base sequence of the polymorphic site comprises hybridizing the nucleic acid sample to a polynucleotide selected from the group consisting of polynucleotides (f) to (h) or a microarray to which a complementary polynucleotide thereof is immobilized. A step wherein the polynucleotides (f) to (h) are as defined in claim 6; And analyzing the obtained hybridization result. 10. The method of claim 9, wherein the length of the DNA sequence of the polynucleotide immobilized on the microarray or its complementary polynucleotide is 10 to 100 nucleotides each. The method according to claim 7, wherein the step of analyzing the nucleotide sequence of the polymorphic site is a polymorphic site of the DNA sequence selected from the group consisting of the DNA sequence of SEQ ID NO: 6 to 8, wherein the polymorphic site is the DNA sequence of SEQ ID NO: 6 Single base in the sample, characterized in that the genotype analysis of the 501st base, the 262th base for the DNA sequence of SEQ ID NO: 7, and the 2187th base for the DNA sequence of SEQ ID NO: 8, respectively) How to detect polymorphisms.