WO2015167087A1 - Method for predicting risk of ankylosing spondylitis using dna copy number variants - Google Patents

Abstract

The present invention relates to a method for predicting the risk of ankylosing spondylitis using DNA copy number variants. It was verified that the risk of ankylosing spondylitis can be effectively predicted by using a primer for detecting DNA copy number variants, of the present invention, and the sensitivity and specificity to the prediction can be improved by grafting single nucleotide polymorphism (SNP) measurement results, and thus more fundamental approaches to the prevention and treatment of ankylosing spondylitis will be expected.

Description

Methods for predicting the risk of ankylosing spondylitis using DNA replication variation

The present invention relates to a method for predicting the risk of developing ankylosing spondylitis, and more particularly, to a method for predicting the risk of developing ankylosing spondylitis (AS) by identifying copy number variants (CNV) of a specific chromosomal location About

Ankylosing spondylitis (AS) is a form of acute spondyloarthropathies (SpA) that usually begins in the late teens to early 20s and typically shows vertebral stiffness in the 30s and 40s. The ankylosing spondylitis It is a chronic progressive systemic disease characterized by chronic inflammation of the spine and peripheral joints or eye, heart, and intestinal involvement. The prevalence of spondyloarthritis varies from country to country, but it is usually 1 to 2%, and its social and economic impact on patients and society is adversely affected by mobility limitation.

The cause of the disease is unclear, but it is presumed that genetic abnormalities, infections, and immune-inflammatory responses are likely to be involved in the disease, similar to systemic rheumatic diseases such as rheumatoid arthritis and systemic lupus erythematosus. Genetically, a histocompatibility antigens gene called "HLA-B27" is presumed to be involved in the onset, and recent studies suggest that ERAP1 (endoplasmic reticulum aminopeptidase 1) and IL23R (interleukin 23 receptor) genes are involved. Ankylosing spondylitis caused by ankylosing spondylitis begins slowly and, occasionally, arthritis is accompanied by intermittent knee and ankle. Symptoms are ambiguous and it is difficult to distinguish from general back pain or nonspecific joint pain by repeating aggravation and improvement. European studies have shown that it takes an average of 10 years to diagnose ankylosing spondylitis from the onset of symptoms of spondyloarthropathy. Therefore, the patient will seek a specialist with a certain degree of disease progression, at which time he often misses the initial treatment point.

Recently, as new knowledge about the progression and treatment of this disease has been accumulated, it has been found that early treatment is effective in inhibiting the treatment and progress of disease, and early diagnosis and prediction of the onset are important. Therefore, predicting the onset of ankylosing spondylitis has become a major subject and has been studied (Korean Patent Publication No. 10-2008-0072643). However, there is still a shortage of ankylosing spondylitis (AS) Is a form of severe spondyloarthroaphy (SpA), usually beginning in the late teens to early 20s and typical vertebral stiffness in the 30s and 40s. The chronic inflammation of the cephalad joints and vertebrae and peripheral joints It is a chronic progressive systemic disease characterized by eye, heart, and intestinal involvement. The prevalence of spondyloarthritis varies from country to country, but it is usually 1 to 2%, and its social and economic impact on patients and society is adversely affected by mobility limitation.

The cause of the disease is unclear, but it is presumed that genetic abnormalities, infections, and immune-inflammatory responses are likely to be involved in the disease, similar to systemic rheumatic diseases such as rheumatoid arthritis and systemic lupus erythematosus. Genetically, a histocompatibility antigens gene called "HLA-B27" is presumed to be involved in the onset, and recent studies suggest that ERAP1 (endoplasmic reticulum aminopeptidase 1) and IL23R (interleukin 23 receptor) genes are involved. Ankylosing spondylitis caused by ankylosing spondylitis begins slowly and, occasionally, arthritis is accompanied by intermittent knee and ankle. Symptoms are ambiguous and it is difficult to distinguish from general back pain or nonspecific joint pain by repeating aggravation and improvement. European studies have shown that it takes an average of 10 years to diagnose ankylosing spondylitis from the onset of symptoms of spondyloarthropathy. Therefore, the patient will seek a specialist with a certain degree of disease progression, at which time he often misses the initial treatment point.

Recently, as new knowledge about the progression and treatment of this disease has been accumulated, it has been found that early treatment is effective in inhibiting the treatment and progress of disease, and early diagnosis and prediction of the onset are important. Therefore, predicting the onset of ankylosing spondylitis has been the subject of major problems and studies have been made (Korean Patent Laid-open No. 10-2008-0072643).

DISCLOSURE OF THE INVENTION The present invention has been made in order to solve the above problems, and the present inventors have made efforts to predict the risk of ankylosing spondylitis, and as a result, confirmed that the incidence of ankylosing spondylitis and the DNA replication number of specific chromosomal loci are related , Thereby completing the present invention.

Accordingly, it is an object of the present invention to provide a marker composition for predicting the risk of developing ankylosing spondylitis using DNA replication water variation.

Another object of the present invention is to provide a method for predicting ankylosing spondylitis risk using DNA replication number variation.

It is still another object of the present invention to provide a composition for predicting the risk of developing ankylosing spondylitis comprising a primer for detecting DNA replication water variation.

Another object of the present invention is to provide a kit for predicting the risk of developing ankylosing spondylitis comprising a primer for detecting DNA replication water variation.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, there is a risk of ankylosing spondylitis including chromosome 1q32.2 position, 2q31.2 position, 6p21.32 position, 13q13.1 position and 16p13.3 gene marker combination A marker composition for prediction is provided.

In one embodiment of the invention, the marker composition may further comprise a combination of single nucleotide polymorphism (SNP) markers rslO865331 and rs27044.

(A) performing a PCR by treating a sample genomic DNA sample with a primer set for detecting DNA replication water variation, (b) sequencing the PCR result, and (c) And determining from the result whether the specimen has a DNA replication number variation. The present invention also provides a method for predicting an onset incidence of ankylosing spondylitis.

In one embodiment of the present invention, the primer set of step (a) comprises a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2. A primer set for detecting water variation; A primer set for detecting DNA replication number variation at 1q32.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4; A primer set for detecting the DNA replication number variation at the 2q31.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6; A primer set for detecting the DNA replication number variation at the 6p21.32 position on the chromosome, consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8; A primer set for detecting the DNA replication number variation at the 11q22.1 position on the chromosome, which is composed of the forward primer of SEQ ID NO: 9 and the reverse primer of SEQ ID NO: 10; A primer set for detecting the DNA replication number variation at the 13q13.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 11 and the reverse primer of SEQ ID NO: 12; A primer set for detecting the DNA replication number variation at the 14q24.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 13 and the reverse primer of SEQ ID NO: 14; A primer set for detecting the DNA replication number variation at the 16p13.3 position on the chromosome, consisting of the forward primer of SEQ ID NO: 15 and the reverse primer of SEQ ID NO: 16; A primer set for detecting the DNA replication number variation at the 22q11.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 17 and the reverse primer of SEQ ID NO: 18; A primer set for detecting the DNA replication number variation at the 1q32.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 21 and the reverse primer of SEQ ID NO: 22; A primer set for detecting the DNA replication number variation at the 13q13.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 23 and the reverse primer of SEQ ID NO: 24; And a primer set for detecting the DNA replication number variation at the 14q24.2 position on the chromosome, which is composed of the forward primer of SEQ ID NO: 25 and the reverse primer of SEQ ID NO: 26.

In another embodiment of the present invention, the PCR of step (a) may be genomic quantitative PCR or deletion-typing PCR.

In another embodiment of the present invention, when the DNA replication number is in any one or more selected from the group consisting of 1p34.2 position, 11q22.1 position, 14q24.2 position and 22q11.1 position on the chromosome, It can be predicted as low risk for ankylosing spondylitis.

In another embodiment of the present invention, the method comprises the steps of: cloning DNA in any one or more selected from the group consisting of 1q32.2 position, 2q31.2 position, 6p21.32 position, 13q13.1 position and 16p13.3 position on the chromosome If there is a mutation, it can be predicted as a high risk group of ankylosing spondylitis.

In another embodiment of the present invention, there is provided a method for detecting a single nucleotide polymorphism comprising: (d) performing a PCR (polymerase chain reaction) by treating a sample genomic DNA sample with a primer set for detecting a single nucleotide polymorphism; And (e) determining whether a single nucleotide polymorphism is present from the PCR result. The method may further comprise the step of determining the presence or absence of a single nucleotide polymorphism from the PCR result.

In another embodiment of the present invention, the primer set of step (d) comprises a primer set for detecting a single base polymorphism that specifically binds to a SNP marker (rs10865331) comprising the nucleotide sequence of SEQ ID NO: 29; And a single base polymorphism detecting primer set that specifically binds to the SNP marker (rs27044) consisting of the nucleotide sequence of SEQ ID NO: 30.

In another embodiment of the present invention, the method may be predicted to be a high-risk group of ankylosing spondylitis when a single base polymorphism is present on any one or more selected from the group consisting of rs10865331 and rs27044 on chromosomes.

The present invention A primer set for detecting the DNA replication number variation at the 1p34.2 position on the chromosome, consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2; A primer set for detecting DNA replication number variation at 1q32.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4; A primer set for detecting the DNA replication number variation at the 2q31.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6; A primer set for detecting the DNA replication number variation at the 6p21.32 position on the chromosome, consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8; A primer set for detecting the DNA replication number variation at the 11q22.1 position on the chromosome, which is composed of the forward primer of SEQ ID NO: 9 and the reverse primer of SEQ ID NO: 10; A primer set for detecting the DNA replication number variation at the 13q13.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 11 and the reverse primer of SEQ ID NO: 12; A primer set for detecting the DNA replication number variation at the 14q24.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 13 and the reverse primer of SEQ ID NO: 14; A primer set for detecting the DNA replication number variation at the 16p13.3 position on the chromosome, consisting of the forward primer of SEQ ID NO: 15 and the reverse primer of SEQ ID NO: 16; A primer set for detecting the DNA replication number variation at the 22q11.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 17 and the reverse primer of SEQ ID NO: 18; A primer set for detecting the DNA replication number variation at the 1q32.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 21 and the reverse primer of SEQ ID NO: 22; A primer set for detecting the DNA replication number variation at the 13q13.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 23 and the reverse primer of SEQ ID NO: 24; And a primer set for detecting the DNA replication number variation at the 14q24.2 position on the chromosome, the primer set consisting of the forward primer of SEQ ID NO: 25 and the reverse primer of SEQ ID NO: 26, the onset of ankylosing spondylitis A composition for predicting risk is provided.

In one embodiment of the present invention, the composition comprises a primer set for detecting a single base polymorphism that specifically binds to a SNP marker (rs10865331) comprising the nucleotide sequence of SEQ ID NO: 29; And a primer set for detecting a single base polymorphism that specifically binds to a SNP marker (rs27044) consisting of the nucleotide sequence of SEQ ID NO: 30.

The present invention can provide a kit for predicting the risk of developing ankylosing spondylitis comprising the above composition.

The method according to the present invention includes a step of measuring the DNA replication number and confirming the relationship between the DNA replication number and the incidence of ankylosing spondylitis. In addition, it was confirmed that the sensitivity and specificity of predicting the incidence of ankylosing spondylitis can be improved by combining the results of single nucleotide polymorphism (SNP) measurement with DNA replication number variation. As a result, It is anticipated that it will be possible to anticipate the onset early and effectively prevent and treat it.

FIG. 1 shows the ratio of the signal intensities in the array-CGH of the lq32.2 region to the log2 ratio.

FIG. 2 shows a result of color-signal intensity ratio in the array-CGH of the lq32.2 region.

FIG. 3 shows the result of genomic quantitative PCR for the DNA replication number variation in the lq32.2 region.

Figure 4 shows the number of replications of CNV regions (CNVRs) (CNVRs) in lq32.2 (a), 2q31.2 (b), 6p21.32 (c), 13q13.1 <2n, 2n,> 2n), respectively.

FIG. 5 shows (a) a schematic diagram for performing deletion typing PCR, (b) an image of electrophoresis gel of deletion typing PCR product, and (c) DNA sequence around the deletion site by sequencing.

FIG. 6 shows the results of the Odd ratio of AS incidence according to the number of replications (HOM, HET, 2n) in regions 1q32.2, 13q13.1 and 14q24.2 in Deletion typing PCR.

FIG. 7 shows the results of the Odd ratio value of the AS incidence rate of subjects who had at least one copy of mutation of CNVRs mutation increasing the incidence of AS.

FIG. 8 shows the results of the Odd ratio value of the AS incidence rate for subjects who had at least one copy of mutation in the number of copies of CNVRs that reduced the incidence of AS.

FIG. 9 shows the receiver-operating characteristic (ROC) curve of the AS incidence prediction set using five types of CNVRs that increase the incidence of AS.

FIG. 10 shows the ROC curve of the AS incidence prediction set using five types of CNVRs and five SNPs (rs27037, rs27434, rs10865331, rs27044, rs30187) that increase the incidence of AS.

FIG. 11 shows the ROC curve of a set of predicted AS incidence using five types of CNVRs and four SNPs (rs27434, rs10865331, rs27044, rs30187) that increase the incidence of AS.

Figure 12 shows the ROC curves of the predicted set of AS incidence using five types of CNVRs and two SNPs (rs10865331, rs27044) that increase the incidence of AS.

FIG. 13 shows the results of ROC curve analysis for only five individuals with HLA-B27 positive (501 patients in AS, 30 in control group) for the combination of five CNVRs and two SNPs markers.

The present invention provides a marker composition for predicting the risk of ankylosing spondylitis, comprising a genetic marker combination of 1q32.2 position, 2q31.2 position, 6p21.32 position, 13q13.1 position and 16p13.3 position on the chromosome.

As used herein, the term " markers for predicting onset risk " refers to polypeptides or nucleic acids (for example, mRNAs) that show a significant increase or decrease in gene expression level or protein expression level in individuals with a higher risk of developing ankylosing spondylitis Etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.) and the like.

In addition, a marker composition for predicting the risk of ankylosing spondylitis, which further comprises a combination of single nucleotide polymorphism (SNP) markers rs10865331 and rs27044 in order to increase the sensitivity and specificity of predicting the risk of developing ankylosing spondylitis in the marker composition .

As used herein, the term single nucleotide polymorphism (SNP) refers to a genetic change or variation in a DNA sequence (A, T, G, C) Or more of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of the nucleotide polymorphism Lt; / RTI &gt;

(A) performing a PCR by treating a sample genomic DNA sample with a primer set for detecting DNA replication water variation, (b) sequencing the PCR result, and (c) And determining whether the sample has a variation in the number of DNA replicas from the result.

In step (a), a primer set for detecting DNA replication variation is treated with a sample genomic DNA sample to predict the risk of developing ankylosing spondylitis.

The term "DNA copy number variation (CNV)" used in the present invention means that a specific nucleotide sequence of 1 kb or more is deleted (0 n or 1 n) or amplified (3 n or more) It is one of the structural variations of the gene, in addition to dislocation, amplification, and translocation. Generally means amplification or deletion of a DNA fragment having a size of 1 kb or more. According to the results of the Wellcome Trust Control Consortium (WTCCC), which recently conducted a CNV association analysis, only about 40% of all CNV genotypes are reported as CNVs that can be used for association analysis. In addition, unlike SNP analysis, the CNV study has a poor signal-to-noise ratio, so the WTCCC study used statistical methods to predict the genotype of CNV in order to predict the CNV genotype. In recent WTCCC studies, it has been recommended to use signal intensity as an alternative method to predict CNV genotypes.

The term " PCR (Polymerase Chain Reaction) " used in the present invention means that DNA synthesis reaction by two types of primers and DNA synthesizing enzymes located within a specific DNA region is repeated in vitro to amplify a specific DNA region by several hundred thousand times How. Generally, one cycle of the PCR process involves denaturing the double stranded DNA, annealing the two types of primers with the target single stranded DNA in between, separating the single stranded DNA by extending the primer And extrapolating a sequence complementary to the target region. In the PCR method, double strand separation occurs at heating (over 90) and primer binding (50-60) and DNA synthesis (70-76) are performed at relatively low temperatures.

As used herein, the term " primer " means an oligonucleotide complementary to a 5 'end sequence and a 3' end sequence adjacent to a target nucleic acid used in a polymerase chain reaction, quot; forward primer " and " reverse primer " refer to primers that bind to the 3 'and 5' ends of a certain region of a gene amplified by a polymerase chain reaction, respectively.

The primer set used in step (a) is for binding to a specific chromosome site having a DNA replication number variation, and preferably includes a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: , A primer set for detecting the DNA replication number variation at the 1p34.2 position on the chromosome; A primer set for detecting DNA replication number variation at 1q32.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4; A primer set for detecting the DNA replication number variation at the 2q31.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6; A primer set for detecting the DNA replication number variation at the 6p21.32 position on the chromosome, consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8; A primer set for detecting the DNA replication number variation at the 11q22.1 position on the chromosome, which is composed of the forward primer of SEQ ID NO: 9 and the reverse primer of SEQ ID NO: 10; A primer set for detecting the DNA replication number variation at the 13q13.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 11 and the reverse primer of SEQ ID NO: 12; A primer set for detecting the DNA replication number variation at the 14q24.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 13 and the reverse primer of SEQ ID NO: 14; A primer set for detecting the DNA replication number variation at the 16p13.3 position on the chromosome, consisting of the forward primer of SEQ ID NO: 15 and the reverse primer of SEQ ID NO: 16; A primer set for detecting the DNA replication number variation at the 22q11.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 17 and the reverse primer of SEQ ID NO: 18; A primer set for detecting the DNA replication number variation at the 1q32.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 21 and the reverse primer of SEQ ID NO: 22; A primer set for detecting the DNA replication number variation at the 13q13.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 23 and the reverse primer of SEQ ID NO: 24; And a primer set for detecting the DNA replication number variation at the 14q24.2 position on the chromosome, which is composed of the forward primer of SEQ ID NO: 25 and the reverse primer of SEQ ID NO: 26.

In the step (a), PCR is preferably genomic quantitative PCR or deletion type PCR, but is not limited thereto.

The term " genome quantitative polymerase chain reaction (qPCR) " used in the present invention is a method for measuring a trace amount of DNA, and is a method for measuring a fluorescence signal generated by a PCR reaction in each cycle of the reaction. The amount of test sequence introduced into the qPCR reaction will determine the intensity of the signal generated during cycling and by comparing this signal with the signal recorded from a known standard the DNA can be accurately quantified over the photoluminescent concentration.

The term " deletion-typing polymerase chain reaction (PCR) " used in the present invention is a method for detecting the exact size and deletion breakpoint of a deletion sequence, and PCR And then compared with the deletion sites identified by sequencing. In the absence of deletion, a PCR amplification of a predicted size is identified, whereas if there is a deletion, a PCR amplification that is reduced in length by that size is identified.

In step (b), the PCR result obtained in step (a) is sequenced to predict the risk of developing ankylosing spondylitis.

In step (c), the sample determines whether there is a variation in the number of DNA copies from the sequencing result obtained in step (b) to predict the risk of developing ankylosing spondylitis.

More specifically, in the case where the DNA replication number varies in any one or more selected from the group consisting of 1p34.2 position, 11q22.1 position, 14q24.2 position and 22q11.1 position on the chromosome, prediction of the low risk of ankylosing spondylitis have.

The DNA replication number may occur more than once, and the incidence of ankylosing spondylitis decreases as the number of CNVR deleted at 1p34.2 position, 11q22.1 position, 14q24.2 position and 22q11.1 position increases.

In addition, DNA replication numbers in any one or more of the 1q32.2, 2q31.2, 6p21.32, 13q13.1, and 16p13.3 positions on the chromosome can be predicted to be a high risk of developing ankylosing spondylitis.

The DNA replication number can occur at the same time, and the incidence of ankylosing spondylitis increases as the number of CNVR deleted at 1q32.2 position, 2q31.2 position, 6p21.32 position, 13q13.1 position and 16p13.3 position increases do.

(D) A primer set for detection of a single nucleotide polymorphism is subjected to a PCR (polymerase chain reaction) by treating a sample of a genomic DNA sample to increase the sensitivity and specificity of predicting the risk of developing ankylosing spondylitis ; And (e) determining whether there is a single nucleotide polymorphism from the performed PCR product.

In the present invention, when a single nucleotide polymorphism is present in at least one selected from the group consisting of rs10865331 and rs27044, it can be predicted as a high risk group of ankylosing spondylitis. In addition, the primer set of step (d) for measuring whether a single nucleotide polymorphism is a primer set for detecting a single base polymorphism that specifically binds to SNP marker (rs10865331) comprising the nucleotide sequence of SEQ ID NO: 29; And a single base polymorphism detecting primer set that specifically binds to the SNP marker (rs27044) consisting of the nucleotide sequence of SEQ ID NO: 30.

According to one embodiment of the present invention, 10-CNVRs associated with AS (5 CNVRs that increase the risk of AS outbreaks and 5 CNVRs that reduce the risk of AS inventions) were determined and verified by performing array-CGH analysis and qPCR , And then qPCR was performed to confirm the relationship with the number of replications of ankylosing spondylitis and CNVRs by independent verification (Examples 1 to 3). In addition, deletion-typing PCR was performed to confirm the exact size and boundary of CNVRs, and the association between AS homozygous deletion and AS heterozygosity was confirmed (Examples 4 to 5). In addition, it was confirmed that the risk of AS was significantly increased as the number of replications of five CNVRs increased in the same manner as in Example 3 (Example 6), and the number of replications of CNVRs (SNPs) associated with the risk of AS were found to be associated with a higher risk of onset, and based on these results, a method for predicting the risk of AS with excellent sensitivity and specificity was identified (Examples 7 to 8).

As another aspect of the present invention, A primer set for detecting the DNA replication number variation at the 1p34.2 position on the chromosome, consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2; A primer set for detecting DNA replication number variation at 1q32.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4; A primer set for detecting the DNA replication number variation at the 2q31.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6; A primer set for detecting the DNA replication number variation at the 6p21.32 position on the chromosome, consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8; A primer set for detecting the DNA replication number variation at the 11q22.1 position on the chromosome, which is composed of the forward primer of SEQ ID NO: 9 and the reverse primer of SEQ ID NO: 10; A primer set for detecting the DNA replication number variation at the 13q13.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 11 and the reverse primer of SEQ ID NO: 12; A primer set for detecting the DNA replication number variation at the 14q24.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 13 and the reverse primer of SEQ ID NO: 14; A primer set for detecting the DNA replication number variation at the 16p13.3 position on the chromosome, consisting of the forward primer of SEQ ID NO: 15 and the reverse primer of SEQ ID NO: 16; A primer set for detecting the DNA replication number variation at the 22q11.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 17 and the reverse primer of SEQ ID NO: 18; A primer set for detecting the DNA replication number variation at the 1q32.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 21 and the reverse primer of SEQ ID NO: 22; A primer set for detecting the DNA replication number variation at the 13q13.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 23 and the reverse primer of SEQ ID NO: 24; And a primer set for detecting the DNA replication number variation at the 14q24.2 position on the chromosome, the primer set consisting of the forward primer of SEQ ID NO: 25 and the reverse primer of SEQ ID NO: 26, the onset of ankylosing spondylitis A composition for predicting risk is provided.

In another aspect of the present invention, the present invention provides a kit for predicting the risk of developing ankylosing spondylitis comprising the above composition.

The kit of the present invention may further comprise at least one container containing a partitioned carrier means for containing a sample, a buffer for PCR reaction and a container containing a DNA polymerase, said carrier means comprising at least one container such as a bottle, And each container contains the independent components used in the method of the present invention, and those of ordinary skill in the art can readily dispense the necessary formulation in the container.

As a means for enhancing the sensitivity and specificity of predicting the risk of developing ankylosing spondylitis in the kit / composition, a primer set for detecting a single base polymorphism that specifically binds to SNP marker (rs10865331) comprising the nucleotide sequence of SEQ ID NO: 29 ; And a primer set for detecting a single base polymorphism that specifically binds to a SNP marker (rs27044) consisting of the nucleotide sequence of SEQ ID NO: 30.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

Example 1. Identification of CNVRs by array-CGH analysis

1-1. Study subjects and statistical analysis

All study subjects were composed of Koreans.

A total of 309 AS patients (278 men, 31 women, 22.28.3 years old) were recruited from the Rheumatism Hospital of Hanyang University Hospital for the analysis of Array-CGH (Comparative genomic hybridization) And used as a discovery group. A total of 309 healthy controls (214 males, 95 females, 35.313.6 years of age) were recruited for the comparative analysis. These patients were diagnosed with AS by the 1984 Modified New York Criteria. This study was conducted with the approval of the institutional review board (CUMC11U199) and was made in writing. The characteristics of the subjects in the discovery phase are shown in Table 1 below.

Table 1

Statistical analysis was performed using Stata software (version 10.0; Stata Corporation, College station, TX) and SPSS for Windows (version 11.5; SPSS, Chicago, IL). P value <0.05 was considered significant.

1-2. Experimental Method

Whole-genome array-CGH was performed using an Agilent 180K SurePrint G3 Human CNV Microarray. The array was used to screen genetic variation data and regions of DNA replication variants classified by the 1000 genome project. Genomic DNA isolated from patients and controls was labeled with Cy5-dCTP (PerkinElmer, Waltham, MA) and NA1085 DNA, the reference DNA, was labeled with Cy3-dCTP (PerkinElmer). The labeled DNA was hybridized on an Agilent 180K array with a hybridized buffer, human Cot-1 DNA (HybMasker, ConnectaGen, Seoul, Korea). Array slides were incubated at 65 for 24 hours, then the array slides were cleaned and scanned and analyzed using Feature Extraction Software (Agilent Technologies).

Probe mapping was performed through the UCSC genome browser (Human NCBI36 / hg18). The quality of the data was evaluated by the CGH-QCTM-Sep09 QC metric set, and all the data satisfied the above-mentioned quality evaluation.

1-3. Detection of copy number variants (CNVs)

Based on the log2 ratios, the CNVs were detected using the ADM2 algorithm in Genomic Workbench 7.0 software under a default setting with a slight modification (minimum log2 ratio = 3, minimum number of consecutive probes in a CNV = 4).

A total of 629,186 CNVs were identified from 618 samples. The median of the number of CNVs identified for each genome was 999 (range 488-1515), the median size of CNVs was 2.0 Kb (101bp to 5.9 Mp), and the general characteristics of the identified CNVs are shown in Table 2 below .

Table 2

1-4. Identification of Copy number variants regions (CNVRs) associated with AS risk

After CNV extraction, CNV regions (CNVR) were identified by overlapping the CNVs from 618 individuals using CNVRuler software.

A total of 3,706 CNVRs were identified, and 1,357 CNVRs with a frequency of more than 5% were analyzed for their association with the risk of AS. Relevance analysis was carried out through logistic regression analysis to prevent the experiment results from being different according to age and gender. We used the false discovery rate (FDR) method to solve the problem of multiple comparisons. In this study, CNVRs with P <0.05 and FDR <0.2 were considered significant.

Through the discovery stage of the AS-associated risk-associated CNVR, 227 CNVRs out of 1,357 CNVRs were found to meet P <0.05, FDR <0.2 criteria, ie, a significant association with AS outbreak was recognized.

Example 2. Validation of CNVRs Associated with AS-Onset Risk Using Genomic qPCR (Validation)

As a result of observing the array-CGH signal intensities of the 227 AS-associated risk CNVRs found in Example 1, quantitative PCR (qPCR) verification was performed on 79 CNVRs with relatively clear signal intensity. Quantitative PCR (qPCR) Primer was designed for each CNVR locus as the first step of quantitative PCR (qPCR) verification. The primer set of 57 loci among 79 loci was r ² = 0.99, amplification efficiency (90% ~ 110%). Therefore, quantitative PCR (qPCR) verification was performed on 57 loci. Quantitative PCR (qPCR) was performed by randomly selecting 22 samples from the discovery group.

Through these procedures, the 10 most reliable chromosomal regions from which consistent results were obtained, in which the results of array-CGH and qPCR coincide by more than 80%, were selected for independent replication, Respectively.

Table 3

Nine of the 10 CNVRs tested with qPCR were in deletion form and one in gain form. Subjects with CNVRs in the 1q32.2 (HHAT), 2q31.2 (PRKRA), 6p21.32 (HLA-DPB1), 13q13.1 (EEF1DP3) and 16p13.3 areas had significantly higher incidence of AS than the control group The subjects with CNVRs in the 1p34.2 (BMP8A), 6p24.3 (BMP6), 11q22.1 (CNTN5), 14q24.2 (RGS6) and 22q11.1 (IL17RA) . Results of the verification of the CNVR Array-CGH profile and qPCR in the 1q32.2 region are shown in FIGS.

As shown in FIGS. 1 to 3, the ratio of the signal intensity of 1q32.2 region was confirmed by log2 ratio value and color (negative value and green), and the decrease of copy number was verified through qPCR.

Example 3. Independent replication of the risk of AS-associated CNVRs using genomic qPCR (independent replication)

3-1. Study subjects and statistical analysis

All subjects were composed of Korean people.

Two independent assays were carried out on the ten types of AS - associated risk - associated CNVR. Step 1 independent verification was performed on 491 AS patients and 671 normal controls. Two - step independent verification was performed on 134 AS patients and 274 normal control subjects. All the subjects in the first and second independent verification groups were recruited from Hanyang University Rheumatism Hospital and Eulji University Hospital. This study was conducted with the approval of the institutional review board (CUMC11U199) and was made in writing. The characteristics of the study subjects in the replication step are shown in Table 4 below.

Table 4

Statistical analysis was performed using Stata software (version 10.0; Stata Corporation, College station, TX) and SPSS for Windows (version 11.5; SPSS, Chicago, IL). P value <0.05 was considered significant.

3-2. Preparation of primer sets for genomic qPCR

Genomic quantitative PCR (qPCR) for ten AS-associated risk-associated CNVR isolates was performed by designing a set of amplification primers specific for each locus and using this to identify CNVRs that were found to be associated with AS at the discovery stage To perform independent replication of

Information on the primers used in this experiment is shown in Table 5 below.

Table 5

Quantitative PCR was performed using the Viia7 system (Life Technologies, Carlsbad, Calif.) and NA10851 sample was used as a calibrator. The number of replications for each subject is defined as 2- ^CT , where Ct is the difference in threshold cycle for the values obtained from the reference gene (HS6ST3) and the calibrator DNA (individual / calibrator) in the sample to be identified, Rounded to the nearest integer. In the qPCR, the Ct value of the control group was defined to be less than 0.3.

3-3. Independent verification results of CNVRs related to AS

The independent validation group qPCR results over the two steps of 10 CNVRs are shown in Table 6.

Table 6

The first-step independent validation results (491 experimental and 617 control), 9 of the 10 CNVRs showed consistently significant results, and the direction of the odd ratio was the same as the discovery step (5 CNVR increased risk Five CNVRs were identified as risk reduction, five risk-increasing and four protective CNVRs. On the other hand, one (6p24.3) CNVRs did not show significance in discovery group in independent validation group.

In the two-step independent validation (134 experimental and 274 control groups), the four CNVRs were consistent with the discovery group, and the direction of the odd ratio was the same as the discovery step and independent verification (See Table 3). That is, the CNVRs in the regions 1q32.2, 2q31.2, 6p21.32, and 16p13.3 were consistently significant in all three sets.

In addition, in this independent verification, the reference DNA of qPCR as well as NA10851 was used as a reference for a person having the CNVR locus of 2n as a reference.

As shown in Fig. 4, 1q32.2 (A), 2q31.2 (B), 6p21.32 (C), 13q13.1 (D), 16p13.3 ) Individuals with deletion duplication number of region CNVRs showed higher AS incidence than individuals with 2n. The odd ratio increased with decreasing number of copies, and showed a significant dependence on the number of copies (r ² = 0.944 ~ 0.999).

Individuals with copy number variants of CNVRs in the 1p34.2, 11q22.1, 14q24.2, and 22q11.1 regions that were found to reduce incidence at the discovery stage showed lower AS incidence than individuals with 2n. However, the odd ratio did not show a significant dependence on the number of duplicates.

(CNVR: 1q32.2, 2q31.2, 6p21.32, 2q31.2, 2q31.2, 2q31.2, 2q31.2, 2q31.2, 2q31.2, 2q31.2, 2q31.2, 2q31.2, 2q31.2, 2q31.2, 2q31.2, 2q31.2, 2q31.2, 2q31.2, 2q31.2, 2q31.2, 2q31.2, and 2q31.22, respectively) 13q13.1, 16p13.3; low AS-related association CNVR: 1p34.2, 11q22.1, 14q24.2, 22q11.1) were all significant with AS, and individual discovery, one-step independent verification, Significantly higher than step-independent verification (Table 6 combined column).

Example 4. Size and Boundary of CNVRs Using deletion-typing PCR

4-1. Experimental Method

To confirm the exact size and boundaries of deletion for the eight deletion CNVRs among the nine validated CNVRs, we performed deletion-typing PCR. The deletion-typing primer for the first step was designed, and it was confirmed that three CNVRs (1q32.2, 13q13.1 and 14q24.2) among 8 CNVRs could be confirmed by PCR. Then, deletion-typing PCR was performed using primer pairs for these three CNVRs. Amplicons of deleted alleles were sequenced using PCR-direct sequencing.

A set of primers to detect deletion sites were designed within the flanking sequence of the putative deletion site. The experimental strategy is shown in FIG. 5 (a), and information on the primers is shown in Table 7 below.

Table 7

5 (a), when the PCR containing the predicted deletion region is performed, the PCR amplified in the expected size appears when there is no deletion, whereas when the PCR amplification is performed in the presence of deletion, The principle is that water will appear. In Fig. 5 (a), a white box inside indicates a predicted deletion region, an outer white box indicates an actual deletion region, a blue arrow indicates a primer set for searching deletion, and a black vertical bar indicates microhomology sequences.

4-2. Check the size and boundaries of CNVRs

5 (b) and 5 (c), in the 1q32.2 region, the length of the deleted region confirmed by deletion typing PCR was ~ 2.4 Kb, and the length of the deleted region confirmed by sequencing was 2,412 bp , And the mechanism of CNVRs was nonhomologous end joining (NHEJ) by 2bp microhomology (AT).

In the 13q13.1 region, the length of the deletion region confirmed by deletion typing PCR was ~ 6.2 Kb, the length of the deleted region confirmed by sequencing was 6.161 bp, which was almost the same, and the mechanism of CNVRs was 4bp microhomology ( GGCT).

In the 14q24.2 region, the length of the deletion region confirmed by deletion typing PCR was ~ 5.1 Kb, and the length of the deleted region confirmed by sequencing was almost the same as 5.129 bp. The mechanism of CNVRs was 7bp microhomology (AAAAGTA ). In FIG. 5 (b), HOM represents homozygous deletion and HET represents heterozygous deletion. In FIG. 5 (c), the yellow bar represents a microhomology sequence and the red box represents deleted sequences.

Example 5. Comparison of AS incidence according to deletion and AS incidence between homozygous deletion and heterozygosity deletion

The results of deletion-typing PCR and PCR-direct sequencing of three CNVRs (1q32.2, 13q13.1 and 14q24.2) in 849 AS patients and 922 normal controls are shown in Table 8 Respectively.

Table 8

The incidence of AS between 1q32.2, 13q13.1, 14q24.2 and 2n were compared using deletion-typing PCR and 1q32.2, 13q13.1, 14q24.2 region The incidence of AS between homozygous deletion and heterozygous deletion was also compared among the subjects with CNVRs.

As shown in FIG. 6, subjects with deletion CNVRs in the 1q32.2 region showed higher AS incidence (OR = 1.43, 95% CI 1.17-1.75, P = 4.9x10 ^-4 ) in the case of homozygous deletion, it showed higher incidence compared to the heterozygous deletion (OR = 2.21, P = 3.1x10 -5 in HOM versus OR = 1.29, P = 0.020 in HET).

Deletion of the 13q13.1 region The subjects with CNVRs showed a higher incidence of AS than those with 2n (OR = 1.26, 95% CI 1.03-1.54, P = 0.023). In the case of homozygous deletion, comparison showed a high incidence (OR = 1.59, P = 9.3x10 -4 in HOM versus OR = 1.09, P = 0.473 in HET).

Subjects with a deletion of the 14q24.2 region had a lower incidence of AS (OR = 0.62, 95% CI 0.41-0.95, P = 0.027) than the subjects with 2n, and the odds ratio was not correlated with the dose.

Example 6. Confirmation of the Correlation between Simultaneous Replication Number of CNVRs and AS Incidence

We compared AS incidence among subjects with at least one replication of CNVRs mutations that increase AS outbreaks, or those with at least one replication mutation of CNVRs that reduce AS outbreaks and those that did not. There were 5 CNVRs (1q32.2, 2q31.2, 6p21.32, 13q13.1, 16p13.3) and 3 types of CNVRs (1p34.2, 11q22, and 16p13.3) for the risk of developing AS. 1, 14q24.2).

As shown in FIG. 7 and Table 9, subjects having four or more copies of the variation in the number of CNVRs that increased the risk of developing AS were approximately 18 times more likely to be infected than those without the copy number variation of CNVRs (OR = 17.98, 95% CI 6.02-53.70, P = 2.3x10 ^-7 ). Odd ratio increased with increasing number of CNVRs (r ² = 0.999).

Table 9

Also, as shown in FIG. 8 and Table 10, subjects with two or more copies of the number of copies of CNVRs that reduced the risk of developing AS had a 5.2-fold lower incidence than those without copies of the copy number of CNVRs (OR = 0.19, 95% CI 0.12-0.32, P = 4.0x10 &lt; ^-10 &gt;). Odd ratio decreased as the number of CNVRs decreased (r ² = 0.9849).

Table 10

This result implies that it is possible to more accurately predict the onset of ankylosing spondylitis by using the result of the determination of the number of copies of specific CNVRs.

Example 7. Confirmation of the relationship between single nucleotide polymorphism (SNP) and AS incidence

7-1. Experimental Method

The subjects of SNP (single nucleotide polymorphism) analysis were 701 patients (339 patients, 362 control subjects) who had DNA of ankylosing spondylitis patients and control group who underwent 5 types of CNVRs test to increase the incidence of ankylosing spondylitis.

We have selected 5 SNP markers (rs27037, rs27434, rs10865331, rs27044, rs30187) that increase the incidence of ankylosing spondylitis in Koreans. We performed qPCR reaction using primers and probes specific for the five SNP markers. The genotype of each SNP marker was analyzed based on the fluorescence signal generated by PCR reaction in each cycle. SNP marker-specific primers and probes used in this analysis were purchased from Life Technologies, and the information on the used markers is shown in Table 11 below.

Table 11

7-2. Independent verification of single nucleotide polymorphisms (SNPs) associated with AS

The genotype of each of the five SNP markers was used to evaluate the risk of developing ankylosing spondylitis. The genotype results are shown in Table 12 below.

Table 12

As shown in Table 12, the SNPs of four SNPs (rs27434, rs10865331, rs27044, and rs30187) among the five SNPs used in the present example were confirmed to have a significant relationship between genotype and AS incidence.

Example 8. Verification of Accuracy of AS Prediction Using CNVRs and Single Base Polymorphism (SNP).

Receiver-Operating Characteristic (ROC) curve analysis was performed based on the five CNVRs identified in Example 6 related to AS and the five SNPs identified in Example 7. In this study, we aimed to identify the risk prediction model of AS with excellent sensitivity and specificity.

ROC analysis was performed using five sets of CNVRs, five sets of CNVRs and five sets of SNPs, five sets of CNVRs and four sets of SNPs (rs27434, rs10865331, rs27044 , rs30187), and five sets of CNVRs and two sets of SNPs (rs10865331, rs27044) satisfying P <0.01.

Table 13

As shown in Table 14 and Figs. 9 to 12, superior prediction results were confirmed when SNPs (Area value; 0.652, 0.667, 0.684) were used together (Area value; 0.632) than when only CNVRs were used. In particular, when SNPs were independently verified, two SNPs identified as P <0.01 and five different CNVRs (ROC-Set3) were the best.

Table 14

In addition, ROC curve analysis of only the HLA-B27-positive individuals (501 patients in the AS and 30 in the control group) was performed on the set 3 marker combinations (5 kinds of CNVRs + 2 kinds of SNPs) (Area = 0.707), which is better than the result (Area = 0.684) in the entire analysis target, as shown in FIG.

This result implies that the incidence of ankylosing spondylitis can be more accurately predicted by combining the results of SNVs and the number of copies of CNVRs compared to the method of predicting only the number of copies of CNVRs.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

The present invention relates to a method for predicting an onset risk of ankylosing spondylitis using DNA copy number variants, and, unlike the conventional method using the HLA-B27 antigen gene, the DNA replication number and single nucleotide polymorphism SNP) can be used to predict the onset of ankylosing spondylitis more accurately. Thus, using the method or composition of the present invention, it can be used for early prevention and diagnosis of ankylosing spondylitis as well as for applications based on it.

<210> 1

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> 1p34.2 forward primer

<400> 1

gagtcagcac agaagtccta tc 22

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> 1p34.2 reverse primer

<400> 2

cccttctcct cacacctaaa c 21

<210> 3

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 1q32.2 forward primer

<400> 3

acctgtgaag gatggattgg caga 24

<210> 4

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 1q32.2 reverse primer

<400> 4

tggcctttga gcactgactg acta 24

<210> 5

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 2q31.2 forward primer

<400> 5

taaaggcctg ttagtgctgt ccct 24

<210> 6

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 2q31.2 reverse primer

<400> 6

gcgacagtct ttcttactgg tgct 24

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 6p21.32 forward primer

<400> 7

attgcttggc tccattgctg aagg 24

<210> 8

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 6p21.32 reverse primer

<400> 8

tttcagtgag ctcaggaacc ctgt 24

<210> 9

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 11q22.1 forward primer

<400> 9

tctcttcctg gtctctccac ttca 24

<210> 10

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 11q22.1 reverse primer

<400> 10

aatgaagggc tgctgtatcg tggt 24

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 13q13.1 forward primer

<400> 11

ccaagcctgg acatcaatga gcta 24

<210> 12

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 13q13.1 reverse primer

<400> 12

atgccatagg ctttccaaga tgcc 24

<210> 13

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 14q24.2 forward primer

<400> 13

tgtgaaaggc tctctcattg ccct 24

<210> 14

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 14q24.2 reverse primer

<400> 14

atctgctctg catgggacag aagt 24

<210> 15

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 16p13.3 forward primer

<400> 15

tcaacatcca gcatcccaca ctca 24

<210> 16

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 16p13.3 reverse primer

<400> 16

acttgatggg aggagaaacc caca 24

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> 22q11.1 forward primer

<400> 17

ccctgtgagc tggtttctat t 21

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> 22q11.1 reverse primer

<400> 18

tgaccggtgt atttggtgtc 20

<210> 19

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 6p24.3 forward primer

<400> 19

tgagtgaagt tccctgaacg agca 24

<210> 20

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 6p24.3 reverse primer

<400> 20

atatggcctg aaagacctcg cact 24

<210> 21

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

&Lt; 223 > 1q32.2 forward primer (2)

<400> 21

agtgctttaa gggtgcgttg ttgg 24

<210> 22

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> 1q32.2 reverse primer (2)

<400> 22

actttcacag cacctccaca gact 24

<210> 23

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

&Lt; 223 > 13q13.1 forward primer (2)

<400> 23

aggcattccc acatgcacaa acag 24

<210> 24

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

&Lt; 223 > 13q13.1 reverse primer (2)

<400> 24

agttcactgc agtcaccctt ggaa 24

<210> 25

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

&Lt; 223 > 14q24.2 forward primer (2)

<400> 25

aacccacaag cagaggaagg agaa 24

<210> 26

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

&Lt; 223 > 14q24.2 reverse primer (2)

<400> 26

atgtgtgtgg tgctgacatt gctg 24

<210> 27

<211> 51

<212> DNA

<213> SNP marker_rs27037

<400> 27

attattatta ttacaattgt tagggttgtt ttttctttta gaaattcaaa g 51

<210> 28

<211> 51

<212> DNA

<213> SNP marker_rs27434

<400> 28

agtgtgaatg agcttatacc tggtgggcca gttcatgggc cacagtcatt g 51

<210> 29

<211> 51

<212> DNA

<213> SNP marker_rs10865331

<400> 29

actgactttg gtgccgtatc taccaggtgg tcaagctgat gccattgcaa g 51

<210> 30

<211> 51

<212> DNA

<213> SNP marker_rs27044

<400> 30

tgcacacagg cgaggagtag tagttgactc cgcagcattc gctctgagac t 51

<210> 31

<211> 51

<212> DNA

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Claims (13)

1. A marker composition for predicting the onset risk of ankylosing spondylitis, comprising a genetic marker combination as set forth in Table 15 below at positions 1q32.2, 2q31.2, 6p21.32, 13q13.1 and 16p13.3 on chromosomes.

   [Table 15]

   
2. The method according to claim 1,

   A marker composition, further comprising a combination of single nucleotide polymorphism (SNP) markers rslO865331 and rs27044, set forth in Table 16 below.

   [Table 16]

   
3. A method for predicting the risk of developing Ankylosing spondylitis, comprising the steps of:

   (a) performing a PCR (polymerase chain reaction) by treating a sample genomic DNA sample with a primer set for detecting DNA copy number varation;

   (b) sequencing the performed PCR product; And

   (c) determining whether the sample has a DNA replication number variation from the sequencing result.
4. The method of claim 3,

   The primer set of step (a) comprises a primer set for detecting DNA replication number variation at 1p34.2 position on a chromosome, which is composed of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2;

   A primer set for detecting DNA replication number variation at 1q32.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4;

   A primer set for detecting the DNA replication number variation at the 2q31.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6;

   A primer set for detecting the DNA replication number variation at the 6p21.32 position on the chromosome, consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8;

   A primer set for detecting the DNA replication number variation at the 11q22.1 position on the chromosome, which is composed of the forward primer of SEQ ID NO: 9 and the reverse primer of SEQ ID NO: 10;

   A primer set for detecting the DNA replication number variation at the 13q13.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 11 and the reverse primer of SEQ ID NO: 12;

   A primer set for detecting the DNA replication number variation at the 14q24.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 13 and the reverse primer of SEQ ID NO: 14;

   A primer set for detecting the DNA replication number variation at the 16p13.3 position on the chromosome, consisting of the forward primer of SEQ ID NO: 15 and the reverse primer of SEQ ID NO: 16;

   A primer set for detecting the DNA replication number variation at the 22q11.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 17 and the reverse primer of SEQ ID NO: 18;

   A primer set for detecting the DNA replication number variation at the 1q32.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 21 and the reverse primer of SEQ ID NO: 22;

   A primer set for detecting the DNA replication number variation at the 13q13.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 23 and the reverse primer of SEQ ID NO: 24; And

   A primer set for detecting the DNA replication number variation at the 14q24.2 position on the chromosome, which is composed of the forward primer of SEQ ID NO: 25 and the reverse primer of SEQ ID NO: 26.
5. The method of claim 3,

   Wherein the PCR in step (a) is genomic quantitative PCR or deletion-typing PCR.
6. The method of claim 3,

   This method can be used to predict a low risk of ankylosing spondylitis when DNA replication numbers are present in any one or more selected from the group consisting of 1p34.2 position, 11q22.1 position, 14q24.2 position and 22q11.1 position on the chromosome &Lt; / RTI &gt;
7. The method of claim 3,

   The method comprises the steps of: when a DNA replication number is present in at least one selected from the group consisting of 1q32.2 position, 2q31.2 position, 6p21.32 position, 13q13.1 position and 16p13.3 position on chromosome, a high risk of ankylosing spondylitis . &Lt; / RTI &gt;
8. The method of claim 3,

   Further comprising the steps of:

   (d) performing a PCR (polymerase chain reaction) by treating a sample genomic DNA sample with a primer set for detecting a single nucleotide polymorphism (SNP); And

   (e) determining whether there is a single nucleotide polymorphism from the performed PCR product.
9. 9. The method of claim 8,

   The primer set of step (d)

   A primer set for detecting a single base polymorphism that specifically binds to a SNP marker (rs10865331) consisting of the nucleotide sequence of SEQ ID NO: 29; And

   A primer set for detecting a single base polymorphism that specifically binds to a SNP marker (rs27044) consisting of the nucleotide sequence of SEQ ID NO: 30.
10. 9. The method of claim 8,

    Wherein said method is predicting a high-risk group of ankylosing spondylitis when there is a single base polymorphism in any one or more selected from the group consisting of rs10865331 and rs27044 on chromosomes.
11. A primer set for detecting the DNA replication number variation at the 1p34.2 position on the chromosome, consisting of a forward primer of SEQ ID NO: 1 and a reverse primer of SEQ ID NO: 2;

    A primer set for detecting DNA replication number variation at 1q32.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 3 and the reverse primer of SEQ ID NO: 4;

    A primer set for detecting the DNA replication number variation at the 2q31.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 5 and the reverse primer of SEQ ID NO: 6;

    A primer set for detecting the DNA replication number variation at the 6p21.32 position on the chromosome, consisting of the forward primer of SEQ ID NO: 7 and the reverse primer of SEQ ID NO: 8;

    A primer set for detecting the DNA replication number variation at the 11q22.1 position on the chromosome, which is composed of the forward primer of SEQ ID NO: 9 and the reverse primer of SEQ ID NO: 10;

    A primer set for detecting the DNA replication number variation at the 13q13.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 11 and the reverse primer of SEQ ID NO: 12;

    A primer set for detecting the DNA replication number variation at the 14q24.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 13 and the reverse primer of SEQ ID NO: 14;

    A primer set for detecting the DNA replication number variation at the 16p13.3 position on the chromosome, consisting of the forward primer of SEQ ID NO: 15 and the reverse primer of SEQ ID NO: 16;

    A primer set for detecting the DNA replication number variation at the 22q11.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 17 and the reverse primer of SEQ ID NO: 18;

    A primer set for detecting the DNA replication number variation at the 1q32.2 position on the chromosome, consisting of the forward primer of SEQ ID NO: 21 and the reverse primer of SEQ ID NO: 22;

    A primer set for detecting the DNA replication number variation at the 13q13.1 position on the chromosome, consisting of the forward primer of SEQ ID NO: 23 and the reverse primer of SEQ ID NO: 24; And

    A primer set for detecting DNA replication number variation at the 14q24.2 position on the chromosome, which is composed of the forward primer of SEQ ID NO: 25 and the reverse primer of SEQ ID NO: 26, the risk of developing ankylosing spondylitis Composition for prediction.
12. 12. The method of claim 11,

    Said composition comprising a primer set for detecting a single base polymorphism that specifically binds to a SNP marker (rs10865331) comprising the nucleotide sequence of SEQ ID NO: 29; And

    A primer set for detecting a single base polymorphism that specifically binds to a SNP marker (rs27044) consisting of the nucleotide sequence of SEQ ID NO: 30; and at least one primer set selected from the group consisting of a primer set for detecting a single base polymorphism.
13. A kit for predicting the risk of developing ankylosing spondylitis comprising the composition of claim 12.

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