KR20130022435A - Snp for diagnosing susceptibility to gastric cancer and method for diagnosing susceptibility to gastric cancer using the lox gene snp - Google Patents

Abstract

PURPOSE: LOX gene SNP for diagnosing gastric cancer susceptibility and a method for diagnosing gastric cancer susceptibility using the same are provided to quickly and easily diagnose gastric cancer susceptibility. CONSTITUTION: LOX gene SNP for diagnosing gastric cancer susceptibility contains 10-300 continuous polynucleotides containing 473th base of sequence number 1. A polynucleotide for diagnosing gastric cancer susceptibility has a sequence of sequence number 2. The risk of developing gastric cancer or differentiation of gastric cancer cells increases when the genotype of 473th base is G/A or A/A by substitution of 473th base.

Description

SNP for diagnosing susceptibility to gastric cancer and method for diagnosing susceptibility to gastric cancer using the LOX gene SNP}

The present invention relates to a SNP of the LOX gene for diagnosing gastric cancer susceptibility, the microarray and kit for detecting the LOX gene SNP, and a method for diagnosing gastric cancer sensitivity using the same.

Gastric cancer is particularly high in Asia and is the leading cause of cancer-related deaths (Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM.Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010; 127: 2893917). In Korea, 16.2% of cancer patients (20.3% of male cancer patients and 11.2% of female cancer patients) are estimated to be gastric cancer patients. The annual age-standardized incidence of gastric cancer is 61.2 / 100,000 for men and 23.9 / 100,000 for women (Jung, KW, Park S, Kong HJ, Won YJ, Boo YK, Shin HR, et al. Statistics in Korea: Incidence, Mortality and Survival in 2006-2007. J Korean Med Sci 2010; 25: 1113-21).

Chronic infection with H. pylori develops chronic gastritis, causing mucosal atrophy, intestinal metaplasia, dysplasia, and cancer (Yuasa Y. Control of gut differentiation and intestinal-type gastric carcinogenesis.Nat Rev Cancer 2003; 3: 592-600). However, only 3% of infected people develop gastric cancer, suggesting that other factors are involved in the development of gastric cancer (Yuasa Y., 2003). Thus, H. pylori infection is associated with mutations in pro- and anti-inflammatory cytokines such as interleukin (IL) and tumor necrosis factors (TNF). Genetic polymorphism of host factors, including host cancer, is likely to affect the risk of gastric cancer (Hamajima N, Naito M, Kondo T, Goto Y. Genetic factors involved in the development of Helicobacter pylori-related gastric cancer Cancer Sci 2006; 97: 1129-38).

Many common diseases, especially cancer, are not caused by one genetic variation in one gene, but involve complex interactions between various genes, along with environmental and lifestyle. Polymorphisms in the human genome potentially provide a wide range of genetic variations that affect normal physiology and cancer development. These polymorphic variants operate through various regulatory and metabolic pathways at different levels of the biological system (Lokotionov A. Common gene polymorphisms, cancer progression and prognosis. Cancer Lett 2004; 208: 1-33).

Genome-wide association studies (GWAS) use high-throughput genotyping technology to measure a large number of labeled polymorphisms at high concentrations across the entire genome. , New susceptible loci or genes for cancer have been identified (Carlson CS, Eberle MA, Kruglyak L, Nickerson DA.Mapping complex disease loci in whole-genome association studies. Nature 2004; 429: 446-52). To date, GWAS has been performed for more than 50 cancers and includes at least 15 different malignant tumors (Stadler ZK, Thom P, Robson ME, Weitzel JN, Kauff ND, Hurley KE, et al. Genome-wide association studies of cancer.J Clin Oncol 2010; 28: 4255-67). Recently, GWAS, which measures diffuse-type gastric cancer, has less than a single nucleotide polymorphism located in the first exon of the prostate stem cell antigen ( PSCA ) gene. It was found to be highly related to type gastric cancer (Sakamoto H, Yoshimura K, Saeki N, Katai H, Shimoda T, Matsuno Y, et al. Genetic variation in PSCA is associated with susceptibility to diffuse-type gastric cancer.Nat Genet 2008 40: 730-40). Although studies on the genetic susceptibility to gastric cancer are increasing, the fundamental exact genetic mechanism of sensitivity to gastric cancer has not yet been fully understood.

LOX is required for collagen maturation and becomes an elastomeric precursor of elastin in the biosynthesis of functional extracellular matrix (Kagan HM, Trackman PC.Properties and function of lysyl oxidase.Am J Respir Cell Mol Biol) 1991; 5: 206-10). The LOX gene inhibits the modification of the Ras oncogene in NIH 3T3 fibroblasts and has been termed the "Ras decision" gene (Contente S, Kenyon K, Rimoldi D, Friedman RM. Expression of gene rrg is associated with reversion of NIH 3T3 transformed by LTR-cH-ras.Science 1990; 249: 796-8, Kenyon K, Contente S, Trackman PC, Tang J, Kagan HM, Friedman RM. Lysyl oxidase and rrg messenger RNA. Science 1991; 253: 802. Both up- and down-regulation of LOX was detected in other cancer cell lines and primary tumors (Payne SL, Hendrix MJ, Kirschmann DA. Paradoxical roles for lysyl oxidases in cancer a prospect J Cell Biochem 2007; 101: 1338-54). LOX expression was significantly increased in colorectal and breast cancer tissues and was positively correlated with invasiveness and metastasis (Baker AM, Cox TR, Bird D, Lang G, Murray GI, Sun XF, et al. The role of lysyl oxidase in SRC-dependent proliferation and metastasis of colorectal cancer.J Natl Cancer Inst 2011; 103: 407-24., Nagaraja GM, Othman M, Fox BP, Alsaber R, Pellegrino CM, Ze ng Y , et. Gene expression signatures and biomarkers of noninvasive and invasive breast cancer cells: comprehensive profiles by representational difference analysis, microarrays and proteomics.Oncogene 2006; 25: 2328-38).

Interestingly, LOX is synthesized as a 50 kDa inert proenzyme (Pro-LOX) secreted into the extracellular environment, where it undergoes a proteolytic cleavage process and a functional 32 kDa enzyme (LOX) and 18 kDa propeptide (LOX-PP) (Bedell-Hogan D, Trackman P, Abrams W, Rosenbloom J, Kagan H. Oxidation, cross-linking, and insolubilization of recombinant tropoelastin by purified lysyl oxidase.J Biol Chem 1993; 268: 10345-50). LOX-PP inhibits the activity of phosphatidylinositol 3-kinase (PI3K), Akt, and MEK kinases (Jeay S, Pianetti S, Kagan), which maintain the tumor suppression function of LOX and induce the activity of nuclear factor-kB. HM, Sonenshein GE. Lysyl oxidase inhibits ras-mediated transformation by preventing activation of NF-kappa B. Mol Cell Biol 2003; 23: 2251-63). In addition, it has been reported that LOX is inactivated by methylation and allele loss in human gastric cancer (Kaneda A, Wakazono K, Tsukamoto T, Watanabe N, Yagi Y, Tatematsu M, et al. Lysyl oxidase is a tumor suppressor gene inactivated by methylation and loss of heterozygosity in human gastric cancers. Cancer Res. 2004; 64: 6410-5). In addition, monobasic polymorphism G473A / Arg158Gln (rs1800449) was found in a highly conserved region within LOX-PP. Gln variants encoded by the A allele also appear to have impaired tumor suppression ability as compared to the LOX-PP wild-type encoded by the G allele (Min C). , Yu Z, Kirsch KH, Zhao Y, Vora SR, Trackman PC, et al.A loss-of-function polymorphism in the propeptide domain of the LOX gene and breast cancer.Cancer Res 2009; 69: 6685-93). Accordingly, the present inventors hypothesized that other functions of LOX by polymorphism may be related to the development and / or progression of gastric cancer.

LOX for Koreans To elucidate the relationship between G473A polymorphism and gastric cancer susceptibility, we performed LOX by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on tissue samples collected from 458 gastric cancer patients and 282 healthy individuals. The genotype and allele frequency of the G473A polymorphism of the gene were analyzed. As a result, the G473A polymorphism of the LOX gene was found to be highly related to the susceptibility to the development and differentiation of gastric cancer in Koreans, thereby completing the present invention.

An object of the present invention is to provide a SNP of the LOX gene for diagnosing gastric cancer susceptibility.

Still another object of the present invention is to provide a microarray capable of detecting the SNP.

Still another object of the present invention is to provide a primer set capable of detecting the SNP.

Still another object of the present invention is to provide a kit capable of detecting the SNP of the LOX gene.

Still another object of the present invention is to provide a method of diagnosing susceptibility to gastric cancer using the SNP.

In order to achieve the above object, the present invention is a polynucleotide consisting of LOX gene SEQ ID NO: 1, gastric cancer susceptibility diagnostic polynucleotide consisting of 10 to 300 consecutive polynucleotides comprising the 473th base of SEQ ID NO: 1 To provide.

In one embodiment of the present invention, the gastric cancer susceptibility diagnostic polynucleotide may have a sequence of SEQ ID NO: 2.

In one embodiment of the present invention, When the 473th base G of SEQ ID NO: 1 is replaced with A and the 473th genotype is G / A or A / A, the risk of gastric cancer or differentiation of gastric cancer cells may be increased.

In addition, the present invention is characterized by comprising a polynucleotide capable of hybridizing allele-specific to a monobasic polymorphism (SNP) site of the polynucleotide of the present invention, a polypeptide encoded by it, or a cDNA thereof It provides a gastric cancer susceptibility diagnostic microarray.

The present invention also provides a primer set for detecting the SNP of the LOX gene for the diagnosis of gastric cancer susceptibility, characterized by amplifying the polynucleotide of the present invention.

In one embodiment of the present invention, the primer set may be composed of SEQ ID NO: 3 and SEQ ID NO: 4.

In addition, the present invention provides a kit for detecting SNP for gastric cancer susceptibility diagnosis comprising the primer set of the present invention.

In one embodiment of the present invention, the primer set is the primer set of claim 5, and may further comprise NotI restriction enzyme.

Further, according to the present invention,

Obtaining a nucleic acid sample from the test object;

Determining the genotype of the 473th base, the polymorphic site of the polynucleotide of SEQ ID NO: 1 from the nucleic acid sample; And

When the determined genotype is G / A or A / A, the present invention provides a method for diagnosing gastric cancer susceptibility comprising the step of determining that susceptibility to stomach cancer is high.

In one embodiment of the present invention, the step of determining the genotype, using a nucleic acid sample obtained from the test template as a template, a primer capable of amplifying a gene region comprising the 473th base of the polynucleotide of SEQ ID NO: 1 Performing a polymerase chain reaction (PCR) using; And identifying the PCR reaction by sequencing and restriction enzyme cleavage.

In one embodiment of the present invention, the primer is a primer set of claim 5, the restriction enzyme may be NotI.

In one embodiment of the present invention, the step of determining the genotype, hybridizing a nucleic acid sample obtained from the test object to the microarray of claim 3; And detecting the hybridization result.

The G / A and A / A genotypes of the LOX gene monobasic polymorphism (SNP) region of the present invention have been shown to be associated with the development of gastric cancer, particularly diffuse-type gastric cancer of Koreans. Therefore, as a method of determining the genotype of the SNP region of the LOX gene of the present invention, it is expected that the susceptibility to gastric cancer can be easily and quickly diagnosed. Seems to be.

1 is sequencing data showing three genotypes at the G473A site of the LOX gene. G / G homozygotes (top panel), A / A homozygotes (bottom panel), G / A heterozygotes (middle panel).
Figure 2 LOX by PCR-RFLP Genotype of G473A polymorphism is shown. Not1 restriction enzymes recognize the PCR product of the G-type allele and cleave it into two fragments. Therefore, if only 214 bp bands are visible, they are considered A / A homozygotes; if 214 bp bands and mixed bands (114 bp and 100 bp) are visible, they are heterozygotes; if only mixed bands are seen, they are considered G / G homozygotes. Can be. Lane 1: A / G, Lane 2: G / G, Lane 3: A / A. Lane M: 50-bp ladder.

The present invention relates to an SNP capable of diagnosing susceptibility to gastric cancer, wherein the SNP is located in the propeptide domain (LOX-PP) of the LOX gene (SEQ ID NO: 1). Specifically, the inventors hypothesized that a single nucleotide polymorphism (SNP) site located at the 473th base of the propeptide domain (LOX-PP) (SEQ ID NO: 1) of the LOX gene may be related to the development of gastric cancer in Koreans. In one embodiment of the present invention, a PCR product (SEQ ID NO: 2) containing this site was prepared to have a restriction enzyme site only at this position, and the relationship with gastric cancer was demonstrated through experiments (Table 1 and Table 2). .

The molecular mechanisms that explain the increased risk of gastric cancer development, in addition to infection with Helicobacter pylori, are not yet clear, but genetic factors, including polymorphisms in genes related to cancer development, may explain the change in personal sensitivity to gastric cancer. . LOX is an extracellular enzyme essential for normal biosynthesis of collagen and elastin (Kagan HM, Li W. Lysyl oxidase; Properties, specificity, and biological roles inside and outside of the cell.J Cell biochem 2003; 88: 660-72). Recently, the propeptide domain (LOX-PP) of LOX released from Pro-LOX has been used to modify Ras signaling, breast cancer, pancreatic cancer, lung cancer and prostate cancer cells; And to inhibit tumor formation by Her-2 / neu-driven breast cancer cells (Min C, Zhao Y, Romagnoli M, Trackman PC, Sonenshein GE, Kirsch KH. Lysyl oxidase propeptide sensitizes pancreatic and breast cancer cells to doxorubicin-induced apoptosis.J Cell Biochem, 2010; 111: 1160-8). LOX has tumor suppression in human gastric cancer cells, but recently active LOX enzymes have been known to enhance the invasion of MDA-MB-231 and MCF-7 breast cancer cells by ectopically expressing mature LOX enzymes. Erler JT, Bennewith KL, Nicolau M, Dornhofer N, Kong C, Le QT, et al. Lysyl oxidase is essential for hypoxia-induced metastasis.Nature 2006; 440: 1222-6). Thus, LOX and LOX-PP are expected to have different effects on the migration of cancer cells. In xenograft models, LOX-PP reduced the growth of pancreatic cancer cells and increased the cytotoxic effects of doxorubicin in pancreatic and breast cancer cells (Min C, Zhao Y, Romagnoli M, Trackman). PC, Sonenshein GE, Kirsch KH. Lysyl oxidase propeptide sensitizes pancreatic and breast cancer cells to doxorubicin-induced apoptosis.J Cell Biochem, 2010; 111: 1160-8).

Therefore, the present inventors investigated the potential association between LOX G473A polymorphism and gastric cancer in Korean to analyze whether the specific gene polymorphism of LOX-PP is related to the risk of gastric cancer. To this end, in one embodiment of the present invention, the incidence of genotype at the G473A region of the LOX gene in Korean gastric cancer patients was 54.4% for G / G, 34.3% for G / A, and 11.3% for A / A. (Table 1). Significant differences were observed in genotype and allele frequency of the G473A polymorphism of LOX-PP between gastric cancer patients and normal subjects ( P = 0.0294 and P = 0.0339, respectively; Table 1). These results indicate that the A allele of LOX may be a genetic vulnerability to Koreans' susceptibility to gastric cancer. Since there was no control for population stratification, the possibility of selection bias could not be completely ruled out, but because the control and cancer patients belonged to the same population, it is unlikely that population stratification would affect the results. see.

Molecular, histological and epidemiological studies show evidence that gastric adenocarcinoma is a heterogeneous disease with two major histological subtypes, intestinal- and diffuse-type (Solcia E, Klersy). C, Mastracci L, Alberizzi P, Candusso ME, Diegoli M, et al. A combined histologic and molecular approach identifies three groups of gastric cancer with different prognosis.Virchows Arch 2009; 455: 197-211, and Tajima Y, Yamazaki K, Makino R, Nishino N, Aoki S, Kato M, et al. Gastric and intestinal phenotypic marker expression in early differentiated-type tumors of the stomach: clinicopathologic significance and genetic background. Clin Cancer Res 2006; 12: 6469-79). Although both subtypes appear to be due to H. pylori -related chronic gastritis, epidemiologic and histopathological evidence shows that gastric adenocarcinoma is affected by genetic polymorphism, age of cancer development, and sex (Yuasa Y. Control). of gut differentiation and intestinal-type gastric carcinogenesis.Nat Rev Cancer 2003; 3: 592-600). In the present invention, when stratified according to gastric cancer tissue type, the incidence of A allele and AA homozygotes was higher among patients with subtype gastric cancer than in patients with gastrointestinal gastric cancer, and significant in genotype and allele frequency of these polymorphisms. There was no significant difference (P <0.05, Table 2). Interestingly, LOX appears to be a direct target of HIF-1, which is functionally required for hypoxia-induced metastasis (Schietke R, Warnecke C, Wacker I, Schodel J, Mole DR, Campean V, et al. The lysyl oxidases LOX and LOXL2 are necessary and sufficient to repress E-cadherin in hypoxia.J Biol Chem 2010; 285: 6658-69). It has also been reported that the induction of LOX is required to inhibit E-cadherin expression during hypoxia and to serve as a marker of epithelial-mesenchymal transition (Schietke R, et al., 2010). Therefore, the inventors found that the Korean LOX - PP It shows the possibility that polymorphism may affect the differentiation of gastric cancer. In addition, the above results may provide evidence that the polymorphic A allele has an impaired ability to reverse cancer cell invasion.

In conclusion, the present invention showed significant differences in genotype and allele frequency of LOX G473A polymorphism between gastric cancer patients and normal individuals ( P = 0.0294 and P = 0.0339, respectively; Table 1). ). LOX Gene polymorphisms can vary significantly when patients are classified according to histological subtypes of gastric cancer. Therefore, these results are LOX G473A polymorphism in the propeptide of the gene shows that it can be closely related to the sensitivity to gastric cancer development and differentiation.

Therefore, the present invention can provide a diagnostic polynucleotide for sensitivity to gastric cancer, that is, a diagnostic marker for gastric cancer, characterized in that it comprises the SNP site. The polynucleotide is not limited thereto, but is preferably 10 or more consecutive bases, and more preferably 10 to 300 consecutive bases. However, the length of the polynucleotide may be determined in various lengths in consideration of various factors such as the use type, the type of restriction enzyme used, the selection of PCR primers and the efficiency of the PCR reaction, and the length is not limited.

In addition, the present invention may provide a polynucleotide capable of allele specific hybridization with the polynucleotide of the present invention or its complementary polynucleotide, particularly the SNP site of the present invention. The allele-specific polynucleotide refers to a polynucleotide capable of hybridizing specifically to each allele. That is, it means hybridized to specifically distinguish the base of the polymorphic site in the sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In the present invention, the allele specific polynucleotide may be a primer. Here primers are the starting point for template-directed DNA synthesis under appropriate conditions (e.g. four different nucleoside triphosphates and polymerizers such as DNA, RNA polymerase or reverse transcriptase) in appropriate buffers and at appropriate temperatures. It refers to a single stranded polynucleotide that can act. Appropriate length of the primer may vary depending on the intended use. 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.

The primer hybridizes to the target DNA comprising the polymorphic site and initiates amplification of allele forms in which the primer exhibits complete homology. This primer can be used in pairs with a second primer that hybridizes to the other side. In one embodiment of the present invention, the primer may be a primer set of SEQ ID NO: 3 and SEQ ID NO: 4. The primer set may be manufactured according to the purpose of use. In the case of confirming genotyping using restriction enzymes, the number of restriction enzyme sites included in the PCR product using primers and the size of fragments cut by restriction enzymes are considered. shall.

In the present invention, the allele specific polynucleotide may be a probe. Probe (probe) in the present invention means a hybridization probe, it means an oligonucleotide capable of sequence-specific binding to the complementary strand of the nucleic acid. Such probes include peptide nucleic acids described in Nielsen et al., Science 254, 1497-1500 (1991). The probe of the present invention is an allele specific probe, in which a polymorphic site exists in a nucleic acid fragment derived from two members of the same species,

Hybridizes to derived DNA fragments, but not to fragments derived from other members. Hybridization conditions in this case show significant differences in hybridization strength between alleles and should be sufficiently stringent to hybridize to only one of the alleles. The probe of the present invention can be used for diagnostic methods for detecting alleles. The diagnostic method includes detection methods based on hybridization of nucleic acids such as Southern blotting, or the like, and may be provided in a form that is pre-coupled to a substrate of the microarray in a method using a microarray.

The present invention also encompasses the polynucleotides of the invention or their complementary polynucleotides, in particular polynucleotides capable of allele specific hybridization with the SNP sites of the invention, polypeptides encoded by them or cDNAs thereof. To provide a microarray for diagnosing gastric cancer susceptibility.

Microarrays according to the invention can be prepared by conventional methods known to those skilled in the art using polynucleotides, hybridized polypeptides or cDNAs thereof which hybridize the polynucleotides according to the invention with probes or them. have. For example, the polynucleotide may be immobilized on a substrate coated with an active group selected from the group consisting of amino-silane, poly-L-lysine, and aldehyde. In addition, the substrate may be selected from the group consisting of silicon wafer, glass, quartz, metal and plastic. As a method of immobilizing the polynucleotide on a substrate, a micropiping method using a piezoelectric method, a method using a pin-shaped spotter, or the like can be used.

In the microarray of the present invention, a primer, probe or antibody capable of measuring the SNP is used as a hybridizable array element and immobilized on a substrate. Preferred substrates may include suitable rigid or semi-rigid supports, such as membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. have. The hybridization array element is arranged and immobilized on the substrate, and such immobilization can be performed by a chemical bonding method or a covalent binding method such as UV. For example, the hybridization array element can be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and can also be bonded by UV at the polylysine coating surface. In addition, the hybridization array element can be coupled to the substrate via a linker (eg, ethylene glycol oligomer and diamine). On the other hand, when the sample to be applied to the microarray of the present invention is a nucleic acid can be labeled (labeled), it can be hybridized with the array element on the microarray. Hybridization conditions may vary, and detection and analysis of the degree of hybridization may vary depending on the labeling substance.

In addition, the present invention may provide a kit for detecting SNPs related to gastric cancer susceptibility diagnosis. The kit according to the present invention may further include a primer set used to separate and amplify DNA containing a corresponding SNP from a diagnostic subject in addition to the microarray of the present invention. The appropriate primer set is as described above, but is not limited thereto, and may be easily designed by those skilled in the art with reference to the sequences of SEQ ID NO: 3 and SEQ ID NO: 4 used in one embodiment of the present invention. In addition, the kit according to the present invention may further include reagents required for the polymerization reaction, such as dNTP, various polymerases and colorants. If the kit of the present invention is subjected to a PCR amplification process, the kit of the present invention may optionally contain reagents necessary for PCR amplification, such as buffers, DNA polymerases (eg, Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermal stability DNA polymerase obtained from Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase cofactors and dNTPs, when the kit of the present invention is applied to an immunoassay, The kit of may optionally comprise a secondary antibody and a substrate of the label. Further, the kit according to the present invention may be manufactured from a number of separate packaging or compartments containing the reagent components described above.

In addition, the present invention can provide a method for diagnosing susceptibility to gastric cancer using the SNP according to the present invention. The method comprises the steps of obtaining a nucleic acid sample from the test subject; Determining the genotype of the 473th base, the polymorphic site of the polynucleotide of SEQ ID NO: 1 from the nucleic acid sample; And determining that the susceptibility to gastric cancer is high when the determined genotype is G / A or A / A.

The method of separating nucleic acid from the test subject may be made through a method known in the art. For example, it can be done by directly purifying DNA from tissue or cells or by specifically amplifying and isolating specific regions using amplification methods such as PCR. In the present invention, nucleic acid or DNA is meant to include not only DNA but also cDNA synthesized from mRNA. The step of obtaining nucleic acid from the diagnostic target is, for example, PCR amplification, ligase chain reaction (LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), transcription amplification transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1173 (1989)) and self-sustaining sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1874 (1990)); Nucleic acid based sequence amplification (NASBA) and the like can be used.

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

< Example  1>

sample DNA Preparation of

<1-1> Subject

Gastric mucosal samples taken from 458 gastric cancer patients who underwent partial gastrectomy or total gastrectomy between 2000 and 2003 at the Catholic University College of Medicine were used in the examples of the present invention. All gastric cancer samples were identified pathologically as adenocarcinoma. Of the 458, 330 men (68.6%) and 128 women (31.4%) were on average 60 years (range 22-91 years) and had an initial diagnosis. Histologically, 250 samples were intestinal-type (54.6%) and 208 samples were diffuse-type (45.4%).

Samples used as controls were taken from healthy people who lived in the same area and had no history or family history of cancer or major diseases. The healthy control group consisted of 154 men and 128 women with an average age of 44 years. To exclude possible influences by ethnic groups, only Koreans were included. Informed consent was obtained from all participants and the study was approved by the Catholic University Institutional Review Board.

<1-2> DNA  extraction

Normal cells were obtained from a gastric mucosal site without cancer in a sample of gastric cancer patients. DNA extraction was performed using known modified single-step DNA extraction methods (Lee JY, Dong SM, Kim SY, Yoo NJ, Lee SH, Park WS.A simple, precise and economical microdissection technique for analysis of genomic DNA from archival tissue sections.Virchows Arch 1998; 433: 305-9). For the healthy control group, 2 mL whole blood was centrifuged to obtain leukocyte cell pellets of each blood sample from a buffy coat. Subsequently, genomic DNA was extracted from leukocytes using a Qiagen DNA Blood Mini Kit (Qiagen, Valencia, CA, USA) according to the producer's manual. DNA purity and concentration were measured with a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA).

< Example  2>

LOX  Gene G473A  Polymorphism test

<2-1> PCR - RFLP  ( Polymerase Chain Reaction - Restriction Fragment Length Polymorphism )

Genomic DNA amplification was performed using primers covering the R158Q polymorphic site in the propeptide (LOX-PP) region of the LOX gene. The primer sequences used are as follows:

Sense primer 5'-TTCCAAGCTGGCTACTCGAC-3 '(SEQ ID NO: 3); And

Antisense primer 5'-CAGGTCTGGGCCTTTCATAA-3 '(SEQ ID NO: 4)

PCR reactions were performed under the following standard conditions. 1 μl template DNA, 0.5uM of each primer, 0.2uM of each deoxynucleotide triphosphate, 1.5 mM MgCl ₂ , 0.4 unit of Ampli Taq gold polymerase, and 1 μl A 10 μl reaction mixture containing 10 × buffer (Perkin-Elmer, Foster City, CA, USA) was made, DNA denatured at 94 ° C. for 12 minutes, and denatured at 40 cycles (94 ° C. for 30 seconds). , Annealing at 57 ° C. for 30 seconds, and extension at 72 ° C. for 30 seconds. The final extension step was 5 min at 72 ° C. After amplification, PCR products were cleaved with 5 U of NotI (NEB, Ipswich, Mass., USA) at 37 ° C. for 4 hours. DNA fragments were then separated by electrophoresis on 2% agarose gel. To ensure the reliability of the RFLP results, PCR products were sequenced by fluorescent dideoxy chain termination using an ABI 3730XL Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's manual.

<2-2> Statistical Analysis

Two-sided Fisher's exact test was performed to assess the relationship between development of gastric cancer and genotype and allele frequency between normal healthy controls and gastric cancer patients, and between two histological types. The P -value <0.05 was considered statistically significant.

<2-3> LOX  Gene G473A  Polymorphism

LOX A 214 bp PCR fragment (SEQ ID NO: 2) containing a NotI polymorphic site was amplified. The A allele has no restriction site, whereas the G allele has one restriction site. After cleavage with NotI, three genotypes were identified: G / G homozygotes (114 / 100bp fragments); G / A heterozygote making 214bp, 114bp, and 100bp fragments; And A / A homozygotes (FIG. 2), which are 214bp fragments.

The genotype and allele frequency of G473A for LOX gene in Korean gastric cancer patients and controls are summarized in Table 1 and Table 2.

Genotype and Allele Distribution of LOX - PP (G473A) Gene Polymorphism in Gastric Cancer Patients and Healthy Controls
Stomach cancer
(N = 458) Control group
(N = 282) Crude OR
(95% CI) Adjusted ^a
OR (95% CI)
Number ratio(%) Number ratio(%) LOX-PP AA 52 11.3 16 5.7 1.472 (1.094-1.981) 1.583 (1.078-2.325) AG 157 34.3 100 35.5 1.047 (0.761-1.439) 1.089 (0.722-1.643) GG 249 54.4 166 58.9 1.00 1.00 G: A allele
Frequency ^b 655: 261
(71.5%: 28.5%) 432: 132
(76.6%: 23.4%) Trend test ^c 1.272 (1.009-1.602) 1.339 (0.993-1.806)

a. Age- and gender-specific adjustments

b. Two-sided Fisher's exact test: P = 0.0294 for genotype distribution and P = 0.0339 for allele frequency

c. Calculated in the logistic regression model using the number of A alleles in the genotypes as a continuous variable

Subgroup Analysis of LOX - PP (G473A) Genotyping Frequency in Gastric Cancer Patients and Healthy Controls
variable
LOX - PP Adjusted ^a OR (95% CI) Adjusted ^a OR (95% CI) Number of patients Number of controls GG versus GA GG versus AA AA AG GG AA AG GG age = 50 9 32 44 14 81 135 1.210 (0.710-2.062) 1.409 (0.895-2.218) > 50 43 125 205 2 19 31 0.963 (0.520-1.784) 1.775 (0.851-3.705) gender male 35 107 169 8 55 91 1.008 (0.585-1.737) 1.479 (0.879-2.489) female 17 50 80 8 45 75 1.163 (0.617-2.192) 1.671 (0.953-2.932) group
type Control group 16 100 166 Less than ^b 107 60 36 1.115 (0.688-1.807) 3.657 (1.614-8.283) Long ^c 142 97 16 0.872 (0.512 -1.486) 1.513 (0.532-4.305)

a. Adjustments to other covariates (years with continuous variables) shown in this table in a continuous regression model for age and gender; Adjust both age and gender in a continuous regression model for tissue type

b. Two-sided Fisher's exact test: P = 0.0001 for genotype distribution and P = 0.0021 for allele frequency

c. Fisher-way test (two-sided Fisher's exact test ): for genotype distributions P = 0.7607, P = 0.4771 for allele frequency of occurrence

The incidence of G / G, G / A, and A / A genotypes of G473A in normal healthy persons (n = 282) was 58.9%, 35.5%, and 5.7%, respectively. The genotyping frequency of all groups was consistent with that of the previously reported Caucasian population (Min C, Yu Z, Kirsch KH, Zhao Y, Vora SR, Trackman PC, et al. A loss-of-function polymorphism in the propeptide domain of the LOX gene and breast cancer.Cancer Res 2009; 69: 6685-93). The incidence of G and A alleles in the healthy control group was 76.6% and 23.4%, respectively. There was no statistical difference in genotype ( P = 0.9296) or allele frequency ( P = 0.8283) between males and females.

The incidence of G / G, G / A, and A / A, the genotype of G473A in gastric cancer patients, was 54.4%, 34.3%, and 11.3%, respectively. The incidence of G473A G and A alleles in gastric cancer patients was 71.5% and 28.5%, respectively. When comparing male and female patients, there was no statistical difference in genotype ( P = 0.9935) or allele frequency ( P = 0.9747).

However, we were able to observe statistically significant differences in genotype ( P = 0.0294) and allele incidence ( P = 0.0339) between gastric cancer patients and healthy control groups (Table 1). Risk of Diffuse Gastric Cancer in A Allele (G / A or A / A Genotype) Carriers When Genotypic Differences Are Compared Between Diffuse-Type Cancers and Controls and Intestinal-type Cancers and Controls It was statistically higher compared to this G / G genotype bearer ( P = 0.0001, Table 2). In addition, significant differences were found in allelic frequency between diffuse gastric cancer and healthy controls ( P = 0.0021). However, there was no significant difference in allele and genotype incidence between intestinal gastric cancer patients and controls ( P = 0.4771 and P = 0.77607, respectively). Putting these results together, the data is LOX G473A of the gene is associated with an increased risk of development and differentiation of gastric cancer.

So far I looked at the center of the preferred embodiment for the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> Industry Academic Cooperation Foundation of Catholic University <120> SNP for diagnosing susceptibility to gastric cancer and method          for diagnosing susceptibility to gastric cancer using the LOX          gene SNP <130> np11-1181 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1254 <212> DNA <213> LOX polynucleotides <400> 1 atgcgcttcg cctggaccgt gctcctgctc gggcctttgc agctctgcgc gctagtgcac 60 tgcgcccctc ccgccgccgg ccaacagcag cccccgcgcg agccgccggc ggctccgggc 120 gcctggcgcc agcagatcca atgggagaac aacgggcagg tgttcagctt gctgagcctg 180 ggctcacagt accagcctca gcgccgccgg gacccgggcg ccgccgtccc tggtgcagcc 240 aacgcctccg cccagcagcc ccgcactccg atcctgctga tccgcgacaa ccgcaccgcc 300 gcggcgcgaa cgcggacggc cggctcatct ggagtcaccg ctggccgccc caggcccacc 360 gcccgtcact ggttccaagc tggctactcg acatctagag cccgcgaagc tggcgcctcg 420 cgcgcggaga accagacagc gccgggagaa gttcctgcgc tcagtaacct gcggccgccc 480 agccgcgtgg acggcatggt gggcgacgac ccttacaacc cctacaagta ctctgacgac 540 aacccttatt acaactacta cgatacttat gaaaggccca gacctggggg caggtaccgg 600 cccggatacg gcactggcta cttccagtac ggtctcccag acctggtggc cgacccctac 660 tacatccagg cgtccacgta cgtgcagaag atgtccatgt acaacctgag atgcgcggcg 720 gaggaaaact gtctggccag tacagcatac agggcagatg tcagagatta tgatcacagg 780 gtgctgctca gatttcccca aagagtgaaa aaccaaggga catcagattt cttacccagc 840 cgaccaagat attcctggga atggcacagt tgtcatcaac attaccacag tatggatgag 900 tttagccact atgacctgct tgatgccaac acccagagga gagtggctga aggccacaaa 960 gcaagtttct gtcttgaaga cacatcctgt gactatggct accacaggcg atttgcatgt 1020 actgcacaca cacagggatt gagtcctggc tgttatgata cctatggtgc agacatagac 1080 tgccagtgga ttgatattac agatgtaaaa cctggaaact atatcctaaa ggtcagtgta 1140 aaccccagct acctggttcc tgaatctgac tataccaaca atgttgtgcg ctgtgacatt 1200 cgctacacag gacatcatgc gtatgcctca ggctgcacaa tttcaccgta ttag 1254 <210> 2 <211> 214 <212> DNA <213> LOX PCR product DNA sequence <400> 2 ttccaagctg gctactcgac atctagagcc cgcgaagctg gcgcctcgcg cgcggagaac 60 cagacagcgc cgggagaagt tcctgcgctc agtaacctgc ggccgcccag ccgcgtggac 120 ggcatggtgg gcgacgaccc ttacaacccc tacaagtact ctgacgacaa cccttattac 180 aactactacg atacttatga aaggcccaga cctg 214 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer <400> 3 ttccaagctg gctactcgac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> antisense primer <400> 4 caggtctggg cctttcataa 20

Claims (12)

In the polynucleotide consisting of SEQ ID NO: 1, the polynucleotide for gastric cancer susceptibility diagnostics comprising 10 to 300 consecutive polynucleotides comprising the 473th base of SEQ ID NO: 1. The method of claim 1,
The stomach cancer susceptibility diagnostic polynucleotide is a stomach cancer susceptibility diagnostic polynucleotide, characterized in that consisting of the sequence of SEQ ID NO: 2. The method of claim 1,
Gastric cancer susceptibility (poly) for diagnosing gastric cancer susceptibility (susceptibility) characterized in that the risk of gastric cancer or differentiation of gastric cancer cells increases when the 473th base G of SEQ ID NO: 1 is replaced with A and the 473th genotype is G / A or A / A. Nucleotides. Gastric cancer susceptibility diagnostic microorganism comprising a polynucleotide capable of hybridizing allele-specific to a single nucleotide polymorphism (SNP) site of the polynucleotide of claim 1, a polypeptide encoded by the same or a cDNA thereof Array. SNP detection primer set for gastric cancer susceptibility diagnosis, characterized in that for amplifying the polynucleotide of claim 1. The method of claim 5,
The primer set is SNP detection primer set for gastric cancer susceptibility diagnostic, characterized in that consisting of SEQ ID NO: 3 and SEQ ID NO: 4. SNP detection kit for gastric cancer susceptibility diagnostic comprising the primer set of claim 5. The method of claim 7, wherein
Said primer set is a primer set of claim 5, and further comprising a NotI restriction enzyme gastric cancer susceptibility diagnostic related SNP detection kit. Obtaining a nucleic acid sample from the test object;
Determining the genotype of the 473th base, the polymorphic site of the polynucleotide of SEQ ID NO: 1 from the nucleic acid sample; And
Gastric cancer susceptibility diagnostic method comprising the step of determining that the susceptibility to gastric cancer when the determined genotype is G / A or A / A. 10. The method of claim 9,
Determination of the genotype,
Performing a polymerase chain reaction (PCR) using a nucleic acid sample obtained from the test object as a template and using a primer capable of amplifying a gene region including the 473th base of the polynucleotide of SEQ ID NO: 1; And
And identifying the PCR reaction by sequencing and restriction enzyme cleavage. The method of claim 10,
The primer is a primer set of claim 5, wherein the restriction enzyme is characterized in that NotI. 10. The method of claim 9,
The determining of the genotype may include hybridizing a nucleic acid sample obtained from the test object to the microarray of claim 3; And detecting the hybridization result.

Images