KR20100036718A - Marker for the diagnosis of susceptibility to prostate cancer using retn gene and method for predicting and analyzing susceptibility to prostate cancer using the same - Google Patents

Abstract

PURPOSE: A marker for diagnosing prostate cancer susceptibility using RETN(resistin) gene is provided to diagnose risk of prostate cancer and predict susceptibility to prostate cancer. CONSTITUTION: A marker for diagnosing prostate cancer susceptibility contains polymorphic site of RETN gene. The polymorphic site of the RETN gene has 201th base of sequence number 1 in which C is substituted with G. A kit for diagnosing prostate cancer contains a primer for amplifying a polynucleotide containing 201th base of sequence number 1. The primer has sequences of sequence number 6 and 7. A method for predicting and determining prostate cancer susceptibility comprises: a step of collecting nucleic acid sample from a sample; a step of amplifying 201th base polymorphic site of sequence number 1 in the nucleic acid sample; and a step of determining amplified polymorphic site and analyzing risk of prostate cancer.

Description

Marker for the diagnosis of susceptibility to prostate cancer using RETN gene and method for predicting and analyzing susceptibility to prostate cancer using the same}

The present invention relates to a marker for diagnosing prostate cancer susceptibility using RETN gene and a method for predicting and determining prostate cancer susceptibility using the same. More specifically, a marker for diagnosing prostate cancer susceptibility based on polymorphism of RETN gene, prostate cancer susceptibility diagnostic kit using the same, micro A method of predicting and determining array and prostate cancer susceptibility.

Genetic differences between individuals are caused by differences in DNA sequences between individuals, and these differences are divided into mutations and polymorphisms. Polymorphisms lead to subtle phenotypic changes. If the frequency is more than 1% of the population, it is defined as a polymorphic genotype.

Among them, single nucleotide polymorphism (SNP) refers to a change in one nucleotide sequence consisting of A, T, C, and G on a genome. Two-thirds of these SNPs are known to be mutations between C and T in the nucleotide sequence, and SNP mutations are known to occur once every 1000 in the nucleotide sequence. In addition, SNPs account for about 90% of the mutations occurring in the human genome, and people with similar traits or the same family tree show the same or similar SNP patterns, so clinically susceptible to individual disease. It can be used as an index for predicting, and can be used as an index for predicting the effects and side effects on drugs. Furthermore, if it can be used in personalized medicine that uses diagnosis and treatment strategies appropriate to the genetic characteristics of an individual, and if the SNP pattern and disease record can be well integrated, medical statistics for the treatment of various diseases can be established. It is believed to be possible.

On the other hand, prostate cancer is one of the most incidence cancers in Western men, and the incidence of prostate cancer in East Asia including Korea has been increasing rapidly over the last decade. The cause of prostate cancer is not well known. Several risk factors associated with prostate cancer are known, but ethnicity, family history, and age are the major causes of the onset. Obesity has also been reported to be closely associated with the development and prevention of prostate cancer.

Resistin (RETN) is a recently discovered hormone believed to link obesity and diabetes (Steppan CM, Bailey ST, Bhat S, Brown EJ, Banerjee RR, Wright CM, Patel HR, Ahima RS and Lazar MA. The hormone resistin links obesity to diabetes.Nature 2001; 409: 307-312). This register is increased in mice obese by dietary or genetic causes. Residin-free rats have decreased blood glucose and weight gain, suggesting the role of resistin in hyperglycemia and obesity (Banerjee RR, Rangwala SM, Shapiro JS, Rich AS, Rhoades B, Qi Y , Wang J, Rajala MW, Pocai A, Scherer PE, Steppan CM, Ahima RS, Obici S, Rossetti L and Lazar MA.Regulation of fasted blood glucose by resistin.Science 2004; 303: 1195-1198).

However, the results of the role of resistin in obesity-related conditions in humans showed contradictory results. Some studies report increased levels of resistin expression in obesity, T2DM (Degawa-Yamauchi M, Bovenkerk JE, Juliar BE, Watson W, Kerr K, Jones R, Zhu Q and Considine RV. Serum resistin (FIZZ3) protein is increased in obese humans.J Clin Endocrinol Metabol 2003; 88: 5452-5455; Azuma K, Katsukawa F, Oguchi S, Murata M, Yamazaki H, Shimada A and Saruta T. Correlation between serum resistin level and adiposity in obese individuals.Obesity Res 2003 11: 997-1001), while other studies did not show any changes in resistin under these conditions (Lee JH, Chan JL, Yiannakouris N, Kontogianni M, Estrada E, Seip R, Orlova C and Mantzoros CS. resistin levels are not associated with obesity or insulin resistance in humans and are not regulate by fasting or leptin administration: cross-sectional and interventional studies in normal, insulin-resistant, and diabetic subjects.J Clin Endocrinol Metabol 2003; 88-4848-4856 Gerber M, Boettner A, Deidel B, Lammert A, Bar J, Schuster E, Thiery J, Kiess W and Kratzsch J. Serum resistin levels of obese and lean children and adolescents: biochemical analysis and clinical relevance. J Clin Endocrinol Metabol 2005; 90: 4503-4509). Similarly, genetic studies of the human registerin gene (RETN) in different populations have led to inconsistent results. For the -420C> G (rs1862513) polymorphism in the promoter, obesity, insulin resistance, and association with T2DM were consistently observed in several clusters (Engert, JC, Wohl MC, Williams SM, Lepage P, Loredo-Osti). JC, Faith J, Dore C, REnaud Y, Burtt NP, Villeneuve A, Hirschhorn JN, Altshuler D, Groop LC, Despres JP, Gaudet D and Hudson TJ. Diabetes 2002; 51: 1629-1634; Smith SR, Bai F, Charbonneau C, Janderova L and Argyropoulos G. Diabetes 2003; 52: 1611-1618; Osawa H, Yamada K, Onuma H, Murakami A, Ochi M, Kawata H, Nishimiya T, Niiya T, Shimizu I, Nishida W, Hashiramoto M , Kanatsuka A, Fujii Y, Ohashi J and Makino H. The G / G genotype of a resistin single-nucleotide polymorphism at -420 increases type 2 diabetes mellitus susceptibility by inducing promoter activity through specific binding Sp1 / 3.Am J Human Genet 2004 75: 678-686), for the + 62G> A (rs3745368) polymorphism in the 3'UTR, the results are consistent in association with the obesity-related phenotype. Tan MS, Chang SY, Chang DM, Tsai JC and Lee YJ.Association of resistin gene 3′-untranslated resion + 62G> A polymorphism with type 2 diabetes and hypertension in a Chinese population. J Clin Endocrinol Metabol 2003; 88: 1258-1263; Conneely KN, Silander K, Scott LJ, Mohlke KL, Lazaridis KN, Valle TT, Tuomilehto J, Bergman RN, Watanabe RM, Buchanan TA, Collins FS and Boehnke M. Variation in theresistin gene is associated with obesity and insulin-related phenotypes in Finnish subjects. Diabetologia 2004; 47: 1782-1788; Gouni-Berthold I, Giannakidou E, Faust M, Kratzsch J, Berthold HK and Krone W. Resistin gene 3'-untranslated region + 62G> A polymorphism is associated with hypertension but not diabetes mellitus type 2 in a German population. J Internat Med 2005; 258: 518-526). Effects on other SNPs have not been identified (Pizzuti A, Argiolas A, Di Paola R, Baratta R, Rauseo A, Bozzali M, Vigneri R, Dallapiccola B, Trischitta V and Frittitta L. An ATG repeat in the 3'-untranslated region of the human resistin gene is associated with a decreased risk of insulin resistance.J Clin Endocrinol Metabol 2002; 87: 2520-2524; Ma X, Warram JH, Trischitta V and Doria A. Genetic variants at the resistin locus and risk of type 2 diabetes in Caucasians.J Clin Endocrinol Metabol 2002; 87: 4407-4410; Livak KJ.Allelic discrimination using fluorogenic probes and the 5 'nuclease assay.Genet Anal 1999; 14: 143-149). Moreover, there have been no reports of evaluation of the role of RETN polymorphism in predicting prostate cancer.

In this regard, the present inventors attempted to uncover the polymorphism of the new function by scanning RETN in Korean population.Resistin polymorphism is associated with increased resistin blood concentration and overexpression of resistin mRNA in adipocytes. Assuming an association, while researching the potential role of RETN in susceptibility to prostate cancer, we found that a polymorphic region of the RETN gene is closely related to susceptibility to prostate cancer. The present invention was completed by developing a prostate cancer susceptibility diagnostic marker to be used.

Accordingly, it is an object of the present invention to provide a prostate cancer susceptibility diagnostic marker comprising a polymorphic site of the RETN gene.

Another object of the present invention is to provide a prostate cancer susceptibility diagnostic kit and a microarray for prostate cancer that can hybridize or amplify with a polymorphic site of the RETN gene.

On the other hand, another object of the present invention to provide a method for predicting and determining the prostate cancer susceptibility using the prostate cancer susceptibility diagnostic marker.

Still another object of the present invention is to provide a method for detecting polymorphism of the rs1862513 region through a sequencing reaction from a sample of a patient in order to provide information necessary for diagnosing prostate cancer.

In order to achieve the above object, the present invention provides a prostate cancer susceptibility diagnostic marker comprising a polymorphic site of the RETN gene.

In order to achieve another object of the present invention, the present invention provides a prostate cancer susceptibility diagnostic kit and a microarray for prostate cancer that can hybridize or amplify with a polymorphic site of the RETN gene.

In order to achieve another object of the present invention, the present invention provides a method for predicting and determining the prostate cancer susceptibility by using the prostate cancer susceptibility diagnostic marker.

In order to achieve another object of the present invention, the present invention provides a method for detecting the polymorphism of the rs1862513 region from the sample of the patient through sequencing in order to provide information necessary for the diagnosis of prostate cancer.

Hereinafter, the content of the present invention will be described in more detail.

The marker for diagnosing prostate cancer susceptibility of the present invention is characterized by including a polymorphic region of the RETN gene. More specifically, the polymorphic site of the RETN gene in the marker in the present invention is 20 to 100 in the polynucleotide consisting of SEQ ID NO: 1, 201 base C of SEQ ID NO: 1 is substituted by G and the 201 base It is characterized by consisting of a polynucleotide consisting of a continuous DNA sequence.

In one embodiment of the present invention, all nine potential functional polymorphic markers present in the RETN gene in the universal database resulting in changes in amino acids, 3'-UTR, splicing sites, promoters, etc. were selected, and patients with Korean prostate cancer The association with was investigated.

As a result, as shown in FIG. 1, four haplotypes were prepared with 9 SNPs in the Korean sample. The frequency of the minor alleles of each SNP was 0.315 (rs3760678), 0.289 (rs1862513), 0.063 (rs3219175), 0.243 (rs3219176), 0.477 (rs3219177), 0.153 (rs3745367), among 375 Koreans, respectively. 0.284 (rs3219178), 0.090 (rs3745268) and 0.090 (rs11270887) (see FIG. 1 and Table 3). These genotype distributions followed the Hardy-Weiberg equation (P> 0.05).

In another embodiment of the present invention, whether these polymorphisms are associated with prostate cancer was analyzed by logistic regression. As a result, as shown in Table 4, rs1862513 present in the promoter was found to be closely associated with the development of prostate cancer.

Thus, by combining these results, we analyzed the potential association between RETN gene polymorphism and prostate cancer risk. The rs1862513 polymorphism was clearly associated with an increased risk of prostate cancer. These results suggest that the RETN gene may be involved in the development of prostate cancer and that the rs1862513 polymorphism may be used as an effective marker for prostate cancer susceptibility.

Accordingly, the present invention provides a marker for diagnosing prostate cancer susceptibility comprising a polymorphic region of the RETN gene.

The term "diagnosis" as used herein includes all types of analyzes used to predict or derive a disease outbreak and to predict disease outbreaks. In addition, the term "susceptibility, sensitivity" in the present invention refers to the increase and decrease in the sensitivity to prostate cancer, that is, the risk of developing prostate cancer (prostate cancer). In the present invention, the term "polymorphism" refers to the occurrence of two or more alternative sequences or alternative alleles within a genetically determined population. Preferred polymorphic markers have two alleles which exhibit an incidence of at least 1%, more preferably at least 10% or 20% in the selected population.

On the other hand, the present invention provides a kit for diagnosing prostate cancer susceptibility comprising a primer capable of amplifying a polynucleotide comprising the 201 base of SEQ ID NO: 1.

The diagnostic kit may include not only the polynucleotide of the present invention but also reagents required for the polymerization reaction, for example, dNTP, polymerases of various kinds, and colorants.

The term “amplifiable primer” means template-directed DNA synthesis under appropriate conditions (eg, four different nucleoside triphosphates and polymerizers such as DNA, RNA polymerase or reverse transcriptase) in appropriate buffers and at appropriate temperatures. 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 15 to 30 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. The primer hybridizes to the target DNA comprising the polymorphic site and initiates amplification of an allelic form in which the primer shows complete homology. This primer is used in pairs with a second primer that hybridizes to the other side. By amplification the product is amplified from two primers, which means that certain allelic forms are present. Primers of the invention include polynucleotide fragments used in ligase chainreaction (LCR). In the present invention, the primer may be a primer having a nucleotide sequence represented by SEQ ID NOs: 6 and 7. In addition, the primer may further include a primer having a nucleotide sequence represented by SEQ ID NOs: 8 and 9 for genotyping according to the method of the present invention.

The present invention also includes a microarray for diagnosing prostate cancer comprising the polynucleotide of the present invention or a complementary polynucleotide thereof. More specifically, the present invention includes a polynucleotide consisting of 20 to 100 contiguous DNA sequences including the 201 base in which the 201st base C of SEQ ID NO: 1 is substituted with G in the polynucleotide consisting of SEQ ID NO: 1. It provides a microarray for diagnosing prostate cancer.

The microarray may include DNA or RNA polynucleotides. The microarray consists of conventional microarrays except that the polynucleotide of the present invention is included in the probe polynucleotide.

Methods for preparing microarrays by immobilizing probe polynucleotides on substrates are well known in the art. The term “probe polynucleotide” refers to a polynucleotide capable of hybridization, and refers to an oligonucleotide capable of sequence-specific binding to the complementary strand of a 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 polymorphic sites exist in nucleic acid fragments derived from two members of the same species, and hybridize to DNA fragments derived from one member, but not to fragments derived from other members. . In this case, hybridization conditions show significant differences in hybridization strength between alleles, and should be sufficiently stringent to hybridize to only one of the alleles. This can lead to good hybridization differences between different allelic forms. 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 blot and the like, and may be provided in a form that is pre-bound to the substrate of the DNA chip in a method using a DNA chip. The hybridization can usually be carried out under stringent conditions such as salt concentrations of 1 M or less and temperatures of 25 ° C. or higher. For example, conditions of 5 × SSPE (750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4) and 25-30 ° C. may be suitable for allele specific probe hybridization.

The immobilization of the probe polynucleotides associated with the prostate cancer of the present invention can also be readily prepared using this prior art. In addition, the hybridization of nucleic acids on microarrays and detection of hybridization results are well known in the art. The detection is accomplished by labeling a nucleic acid sample with a labeling substance capable of generating a detectable signal comprising, for example, a fluorescent substance such as Cy3 and Cy5, and then hybridizing onto a microarray and generating from the labeling substance. The hybridization result can be detected by detecting the signal.

The present invention further provides a method of predicting and determining prostate cancer susceptibility. More specifically,

a) obtaining a nucleic acid sample from a sample;

b) amplifying the polymorphic site of the 201 base of SEQ ID NO: 1 in the nucleic acid sample obtained in step a); And

c) determining the base of the amplified polymorphic site of step b) and analyzing the risk of prostate cancer development.

Obtaining nucleic acid from the sample of step a) of the method may be performed by a conventional DNA separation method. For example, the target nucleic acid can be amplified by PCR and purified. Ligase chain reaction (LCR) (Wu and Wallace, Genomics 4, 560 (1989), Landegren et al., Science 241, 1077 (1988)), 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)) and nucleic acid based sequence amplification (NASBA).

The base determination of step c) of the method comprises sequencing analysis, hybridization by microarray, allele specific PCR, dynamic allele-specific hybridization technique (DASH). , PCR extension analysis, Single Strand Conformation Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), Restriction Fragment Length Polymorphism (PCR-RFLP), Two-dimensional Gene Scanning (TDGS), Taq-Man or TOGATM. have.

These bases determine the risk of developing prostate cancer according to the genotype determined. The frequency of minor alleles in the rs1862513 polymorphic site of the present invention was higher in normal patients (frequency = 0.338) than in normal subjects (frequency = 0.224; P = 0.01, OR = 1.39 (95% CI: 1.09-1.54) ) (See Table 4).

Therefore, when determining the base of the polymorphic site amplified in step c) to predict and determine the prostate cancer susceptibility, if the genotype of the 201 base of SEQ ID NO: 1 determined in step c) is GG risk of prostate cancer Can be determined to increase.

In addition, the present invention provides a method for detecting polymorphism at the rs1862513 polymorphic site through sequencing from a sample of a patient in order to provide information necessary for diagnosing prostate cancer. The polymorphic site rs1862513 represents the polymorphism of the 201 base of SEQ ID NO: 1 in the polynucleotide consisting of SEQ ID NO: 1, and the patient's sample can be used without limitation as long as it can isolate genomic DNA for genotyping. Can be. Base sequence analysis can use the methods described above for base determination.

For reference, reference may be made to the above-mentioned nucleotide and protein operations (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982); Sambrook et al. ., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989); Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182. Academic Press. Inc., San Diego, CA (1990). )).

Accordingly, the present invention provides markers, kits, microarrays, and prostate cancer susceptibility prediction and determination methods for prostate cancer susceptibility. The prostate cancer susceptible markers, kits, microarrays and prostate cancer susceptibility prediction and determination methods can be used as an efficient tool to analyze the risk of developing prostate cancer.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Experimental Materials and Methods

1. Subject

185 Korean patients with prostate cancer were recruited from Chung-Ang University Hospital, Samsung Medical Center and Prostate Bank. All patients showed clinical symptoms and physical examination findings for prostate cancer. 190 healthy, racially matched subjects were selected as controls. A written explanation was obtained from each subject. The experiment was approved by the Institutional Review Board of Chung-Ang University Hospital. Clinical profiles for the subjects are shown in Table 1 below.

Clinical profile Experimental group Control Target number 185 190 age 69.0 ± 7.6 68.2 ± 8.7 Current smoker rate (%) 23.1 33.1 Height (cm) 161.9 ± 9.7 159.9 ± 8.5 Weight (kg) 61 ± 11.7 62.8 ± 10.6 BMI (kg / m ² ) 23.6 ± 3.2 24.5 ± 3.5

2. Genotyping

To confirm the genotype of the polymorphic region, the amplification primers and probes were selected from TaqMan (Gouni-Berthold I, Giannakidou E, Faust M, Kratzsch J, Berthold HK and Krone W. Resistin gene 3'-untranslated region + 62G> A polymorphism is associated with hypertension but not diabetes mellitus type 2 in a German population.J Internat Med 2005; 258: 518-526). The sequences of the primers and probes are shown in Table 2 below. Primer Express (Applied Biosystems) was used to design PCR primers and MGB TaqMan probes. One allele probe was labeled with FAM dye and the other allele probe was labeled with fluorescent VIC dye. PCR reactions included TaqMan University Master Mix (Applied Biosystems) without UNG, 900 nM PCR primers and 200 nM TaqMan MGB-probe. The reaction was performed in a format using 384-well plates with a volume of 5 ml total using 20 ng genomic DNA. The PCR reaction was heated at 50 ° C. for 2 minutes and 95 ° C. for 10 minutes, followed by 40 cycles of 95 ° C. for 15 seconds and 60 ° C. for 1 minute. TaqMan assay plates were transferred to Prism 7900HT instrument (Applied Biosystems) and each fluorescence intensity was measured. Fluorescence data files in each plate were analyzed using automation software (SDS 2.2).

rs3760678 Forward AAGTTGTATGTTATATGTAT SEQ ID NO: 2 Reverse AATTAACTGGGCGTGGTGGC SEQ ID NO: 3 Probe-1 (VIC) TTCTTTTTGAGGTGGAGTCTT SEQ ID NO: 4 Probe-2 (FAM) TTCTTTTTGAGATGGAGTCTT SEQ ID NO: 5 rs1862513 Forward AATCCGGCACACGAATTCC SEQ ID NO: 6 Reverse TGAATGTGGTATGTCATTCTC SEQ ID NO: 7 Probe-1 (VIC) AGGGCCTCCCTCTTCATGTCC SEQ ID NO: 8 Probe-2 (FAM) AGGGCCTCCGTCTTCATGTCC SEQ ID NO: 9 rs3219175 Forward TCCTGACCAGTCTCTGGACA SEQ ID NO: 10 Reverse GTCTCTGAGACCCTTGGGGA SEQ ID NO: 11 Probe-1 (VIC) CTGCTCAGGAGCTTCCTC SEQ ID NO: 12 Probe-2 (FAM) CTGCTCAGGGGCTTCCTC SEQ ID NO: 13 rs3219176 Forward TCCTGACCAGTCTCTGGACA SEQ ID NO: 14 Reverse GGAGCCGGCGACCTCCTGGA SEQ ID NO: 15 Probe-1 (VIC) ATGGCACCAGCGGGGAGGCTG SEQ ID NO: 16 Probe-2 (FAM) ATGGCACCAGTGGGGAGGCTG SEQ ID NO: 17 rs3219177 Forward CTCCTCCAGCCCTTACTGTC SEQ ID NO: 18 Reverse AGGTCTGTGCCAGGGATCAG SEQ ID NO: 19 Probe-1 (VIC) CCAAGGGTCTCAGAGACCTC SEQ ID NO: 20 Probe-2 (FAM) CCAAGGGTCTTAGAGACCTC SEQ ID NO: 21 rs3745367 Forward TCTCTGTCTCCTCCTCCTCC SEQ ID NO: 22 Reverse CCAATGCTGCTTATTGCCCT SEQ ID NO: 23 Probe-1 (VIC) CCTTATCCACAGCTCCAAAC SEQ ID NO: 24 Probe-2 (FAM) CCTTATCCACGGCTCCAAAC SEQ ID NO: 25 rs3219178 Forward GTATTTAGGGCAATAAGCAG SEQ ID NO: 26 Reverse GCGAAGCCTGCAGCCCGGAA SEQ ID NO: 27 Probe-1 (VIC) CCACCCTCACTCCCTGCCT SEQ ID NO: 28 Probe-2 (FAM) CCACCCTCACTGCCTGCCT SEQ ID NO: 29 rs3745368 Forward CCCACCCTCAGCCTCCCAGC SEQ ID NO: 30 Reverse GGAAGCAGTTGGAGACCCCA SEQ ID NO: 31 Probe-1 (VIC) GGGGTTGCGGAGGAGCTGG SEQ ID NO: 32 Probe-2 (FAM) GGGGTTGCGGGGGAGCTGG SEQ ID NO: 33 rs11270887 Forward GACACTGGTGTCCACCCTCA SEQ ID NO: 34 Reverse AAGGACCCAGCCACTTCCTG SEQ ID NO: 35 Probe-1 (VIC) CCTGGAGATGATGATGATGAT SEQ ID NO: 36 Probe-2 (FAM) AATAAACCTGGAGATGATGAT SEQ ID NO: 37

3. Statistical Analysis

Significant departures of genotype frequency from Hardy-Weinberg equilibrium in each single nucleotide polymorphism (SNP) were examined by χ2 analysis. The difference in genotype frequency between the patient and the control group was examined by the χ2 test and the calculation of the odds ratios (ORs) with 95% confidence intervals (CIs). Logistic regression models were used to analyze SNPs and haplotypes of three alternative models (co-dominant, dominant and recessive models). The haplotype of the RETN gene was analyzed by an EM algorithm (Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps.Bioinformatics 2005; 21: 2635; Stephens M, Smith NJ, Donnelly P. A. New statistical method for haplotype reconstruction from population data.Haploview version 2.05 based on Am J Hum Genet 2001; 68: 978-989). Linkage disequilibrium between locus | Lewontin's | Measured using the absolute value of D ' | (Hedrick PW. Gametic disequilibrium measures: proceed with caution. Genetics 1987; 117: 331-341). P -values less than 0.05 were considered significant.

Results and Discussion

The inventors have sequenced the RETN gene to find the genetic polymorphism of prostate cancer-related candidate genes and examined the association with Korean prostate cancer patients. Nine of the known RETN monobasic polymorphisms were selected (Xu JY, Sham PC, Xu A, Tso AWK, Wat NMS, Cheng KW, Fond CHY, Janus ED and Lam KSL.Resistin gene polymorphisms and progression of glycemia in southern Chinese: a 5-year prospective study). These are three in the promoter, four in the introns and two in the 3'UTR. The locations and allele frequencies of these nine polymorphic sites are shown in FIG. 1 and Table 3.

Nine monobasic polymorphisms were subjected to large-scale genotyping based on location, LDs, frequency, and haplotype tagging status. As a result, as shown in FIG. The frequency of the minor alleles of each SNP was 0.315 (rs3760678), 0.289 (rs1862513), 0.063 (rs3219175), 0.243 (rs3219176), 0.477 (rs3219177), 0.153 (rs3745367), among 375 Koreans, respectively. 0.284 (rs3219178), 0.090 (rs3745268) and 0.090 (rs11270887) (see FIG. 1 and Table 3). These genotype distributions followed Hardy-Weiberg equation (P> 0.05).

The association between RETN gene polymorphism and prostate cancer was analyzed by logistic regression. As a result, as shown in Table 4, rs1862513 present in the promoter was found to be closely associated with the development of prostate cancer.

The frequency of minor alleles in rs1862513 was higher in patients with prostate cancer (frequency = 0.338) than in normal subjects (frequency = 0.224; P = 0.01, OR = 1.39 (95% CI: 1.09-1.54)). Rs3745368 reported to be associated with an obesity-related phenotype (Tan MS, Chang SY, Chang DM, Tsai JC and Lee YJ.Association of resistin gene 3'-untranslated resion + 62G> A polymorphism with type 2 diabetes and hypertension in a Chinese population.J Clin Endocrinol Metabol 2003; 88: 1258-1263; Conneely KN, Silander K, Scott LJ, Mohlke KL, Lazaridis KN, Valle TT, Tuomilehto J, Bergman RN, Watanabe RM, Buchanan TA, Collins FS and Boehnke M Variation in the resistin gene is associated with obesity and insulin-related phenotypes in Finnish subjects.Diabetologia 2004; 47: 1782-1788.) No association with the prostate cancer phenotype was observed. No other SNPs showed any association between the prostate cancer phenotypes (see Table 4).

In conclusion, rs1862513 in RETN SNP shows a significant association between prostate cancer susceptibility in Korean population. These results suggest that RETN plays an important role in the development of prostate cancer.

As described above, the prostate cancer susceptibility diagnostic marker of the present invention has an effect of diagnosing the degree of susceptibility to prostate cancer. Therefore, the marker of the present invention and its application method can be used for the purpose of predicting and determining susceptibility to prostate cancer.

Figure 1 shows the gene map (A), haplotypes (LD), LD coefficient (LD coefficient, C) for the RETN polymorphism. (The coding exons in A are shown as test blocks and the 5 'and 3' UTRs as white blocks. Only those with frequencies greater than 0.02 are shown in B. Linkage disequilibrium coefficients (| D '| and r in C) ² ))

<110> Chungang University Industry-academic cooperation foundation <120> Marker for the diagnosis of susceptibility to prostate cancer          using RETN gene and method for predicting and analyzing          susceptibility to prostate cancer using the same <130> NP08-0112 <160> 37 <170> KopatentIn 1.71 <210> 1 <211> 701 <212> DNA <213> Homo sapiens <400> 1 cagctaacca aatccggcac acgaattcct gcaccgcagc tctttctttg aggcctcttg 60 gggtggggct tcctggcttg gctaataagt ccctgggccc ccaaccctcc ggtcccacat 120 ccggggccaa gaggaagccc ctgagcagac agtaagggct ggaggaggaa gggagccttc 180 ccacttccaa cagggcctcc ctcttcatgt ccagagactg gtcaggaggt ggtgccccag 240 ggataatgcc aggggctgtg gtctgaggaa caggtagaca agcagagttt tgcatgcaag 300 ggtggctgat gcaaacatga caaaattaat gcctcttgct aggcatggtg cggacaagca 360 cttgtagtcc cagctactaa ggaggctgac gtgagagaat tgcttgagcc cgggagttcg 420 aagctacagt gacttatgat cacagcactg cactccagtc tgggcaacag agcaagacca 480 cttctctaaa atagtaataa taattatgtc tctgggtgag aatgacatac cacattcata 540 cccaaatgcc catgagcaat agaactggta aataaaatca tggtttatgg ccggtggctc 600 acgcctgtaa tcccagcact ttgggaggcc aaggcgggcg gatcacttga ggtcaggagc 660 ttgagaccaa cctggccaac atgatgaaac cctgtctcca t 701 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3760678 forward <400> 2 aagttgtatg ttatatgtat 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3760678 reverse <400> 3 aattaactgg gcgtggtggc 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rs3760678 VIC <400> 4 ttctttttga ggtggagtct t 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rs3760678 FAM <400> 5 ttctttttga gatggagtct t 21 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> rs1862513 forward <400> 6 aatccggcac acgaattcc 19 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rs1862513 reverse <400> 7 tgaatgtggt atgtcattct c 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rs1862513 VIC <400> 8 agggcctccc tcttcatgtc c 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rs1862513 FAM <400> 9 agggcctccg tcttcatgtc c 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3219175 forward <400> 10 tcctgaccag tctctggaca 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3219175 reverse <400> 11 gtctctgaga cccttgggga 20 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> rs3219175 VIC <400> 12 ctgctcagga gcttcctc 18 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> rs3219175 FAM <400> 13 ctgctcaggg gcttcctc 18 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3219176 forward <400> 14 tcctgaccag tctctggaca 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3219176 reverse <400> 15 ggagccggcg acctcctgga 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rs3219176 VIC <400> 16 atggcaccag cggggaggct g 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rs3219176 FAM <400> 17 atggcaccag tggggaggct g 21 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3219177 forward <400> 18 ctcctccagc ccttactgtc 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3219177 reverse <400> 19 aggtctgtgc cagggatcag 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3219177 VIC <400> 20 ccaagggtct cagagacctc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3219177 FAM <400> 21 ccaagggtct tagagacctc 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3745367 forward <400> 22 tctctgtctc ctcctcctcc 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3745367 reverse <400> 23 ccaatgctgc ttattgccct 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3745367 VIC <400> 24 ccttatccac agctccaaac 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3745367 FAM <400> 25 ccttatccac ggctccaaac 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3219178 forward <400> 26 gtatttaggg caataagcag 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3219178 reverse <400> 27 gcgaagcctg cagcccggaa 20 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> rs3219178 VIC <400> 28 ccaccctcac tccctgcct 19 <210> 29 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> rs3219178 FAM <400> 29 ccaccctcac tgcctgcct 19 <210> 30 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3745368 forward <400> 30 cccaccctca gcctcccagc 20 <210> 31 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs3745368 reverse <400> 31 ggaagcagtt ggagacccca 20 <210> 32 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> rs3745368 VIC <400> 32 ggggttgcgg aggagctgg 19 <210> 33 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> rs3745368 FAM <400> 33 ggggttgcgg gggagctgg 19 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs11270887 forward <400> 34 gacactggtg tccaccctca 20 <210> 35 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> rs11270887 reverse <400> 35 aaggacccag ccacttcctg 20 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rs11270887 VIC <400> 36 cctggagatg atgatgatga t 21 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> rs11270887 FAM <400> 37 aataaacctg gagatgatga t 21  

Claims (9)

In a polynucleotide consisting of SEQ ID NO: 1, a prostate cancer consisting of a polynucleotide consisting of 20 to 100 contiguous DNA sequences comprising 201 base C of SEQ ID NO: 1 substituted with G and comprising the 201 base ) Susceptibility diagnostic markers. Prostate cancer diagnostic kit comprising a primer capable of amplifying a polynucleotide comprising the 201 base of SEQ ID NO: 1. The kit according to claim 2, wherein the primer is a primer having a nucleotide sequence represented by SEQ ID NOs: 6 and 7. The kit according to claim 3, wherein the primer further comprises a primer having a nucleotide sequence represented by SEQ ID NOs: 8 and 9. Prostate cancer diagnostic microarray comprising the polynucleotide of claim 1. a) obtaining a nucleic acid sample from a sample; b) amplifying the polymorphic site of the 201 base of SEQ ID NO: 1 in the nucleic acid sample obtained in step a); c) determining the base of the amplified polymorphic site of step b) and analyzing the risk of prostate cancer development. The method of claim 6, wherein the risk of developing prostate cancer is increased when the genotype of the 201 base of SEQ ID NO: 1 determined in step c) is GG. The method of claim 6, wherein the base of step c) is determined by sequencing analysis, hybridization by microarray, allele specific PCR, dynamic allele-specific technique hybridization (DASH), PCR extension analysis, Single Strand Conformation Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), Restriction Fragment Length Polymorphism (PCR-RFLP), Two-dimensional Gene Scanning (TDGS), Taq-Man and TOGATM And is selected from the group consisting of: A method for detecting polymorphism of the 201 base of SEQ ID NO: 1 in a polynucleotide consisting of SEQ ID NO: 1 through sequencing reactions from a patient's sample to provide information necessary for diagnosing prostate cancer.

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