KR102090901B1 - Composition to predict sports injuries - Google Patents

Abstract

The present invention predicts in advance whether genetic sensitivity or resistance to sports injuries using a single-base mutation polymorphism present in a specific gene as a genetic marker, and personalized exercise programs for preventing elite prospects or preventing sports injuries A method for preventing sports injuries using a useful genetic marker for predicting sports injuries for application to the GOL constituting the AA genotype constituting the rs # 970547 polymorphism (SNP) of the COL12A1 gene and the rs # 679620 SNP of the MMP3 gene A genotype and a composition for predicting sports injury comprising any one or more of the CC genotypes constituting the rs # 1800955 SNP of the DRD4 gene.

Description

Composition to predict sports injuries}

The present invention relates to a composition for predicting sports injuries, and more specifically, whether genetic sensitivity or resistance to sports injuries is predicted in advance using a single-base mutation polymorphism present in a specific gene as a genetic marker, It relates to a composition for predicting sports injury for application to a personalized exercise program to prevent elite prospects or sports injury.

Research to search for useful genetic markers that can predict the sensitivity or resistance of sports injuries is mainly conducted in developed countries in Europe and the United States, but the applicability of these genetic markers varies depending on the race or ethnic background of the study subjects. For example, the rs1800012 polymorphism of the COL1A1 gene, which is the most widely used for the prediction of sports injuries in Western populations, is monomorphic in Korea, which cannot help in the prediction of sports injuries. In order to discover genetic markers, it is necessary to discover useful genetic markers that can predict sports injuries in a group of Korean sports injuries, and currently no research has been conducted on this.

Korean Patent Publication No. 10-2005-0053670 (2005.06.08) United States Patent Publication No. 2013-0080182 (Mar. 28, 2013)

An object of the present invention is to discover useful genetic markers that can be applied to predict Korean sports injuries.

In addition, the present invention analyzes how much the genetic markers discovered can predict sports injury as a risk, and it can be applied to personalized exercise programs that can prevent sports injuries or elite prospects in the field of sports. It is to provide a useful genetic marker for predicting sports injury for application as a means and a method for preventing sports injury using the same.

The present invention predicts a sports injury including any one or more of the AA genotype constituting the rs970547 polymorphism (SNP) of the COL12A1 gene, the GG genotype constituting the rs679620 SNP of the MMP3 gene, and the CC genotype constituting the rs1800955 SNP of the DRD4 gene. It is a composition for.

In addition, the primer base pair for detecting rs970547 SNP of the COL12A1 gene is forward primer, 5 '-CTATTTGACAAGAGGGGAAA-3'

reverse primer, 5 '-TCTTCAAATCAGCCTTCCT-3'

genotyping primer, 5 '-TGGCAACAACGTACCTGGATAGC-3'

to be.

In addition, the primer base pair for detecting rs679620 SNP of the MMP3 gene is forward primer, 5 '-GCAAGCTAGAGAAATACTCC-3'

reverse primer, 5 '-CCTCTGAACCATTACCTG-3'

genotyping primer, 5 '-GAAATATCTAGAAAACTACTAHGACCTC-3'

to be.

In addition, the primer base pair for detecting rs1800955 SNP of the DRD4 gene is forward primer, 5 '-AGGATCAACTGTGCAACG-3'

reverse primer, 5 '-AAACCGACAAGGATGGAG-3'

genotyping primer, 5 '-CTCGCCTCGACCTCGTGCGC-3'

to be.

In addition, OR (95% Cl), a measure of the risk of the AA genotype constituting the rs970547 polymorphism (SNP) of the COL12A1 gene, is 2.44 (1.25 to 4.74).

In addition, OR (95% Cl), which is a measure of the risk of the GG genotype constituting the rs679620 SNP of the MMP3 gene, is 2.00 (1.04 to 3.83).

In addition, OR (95% Cl), which is a measure of the risk of CC genotype constituting the rs1800955 SNP of the DRD4 gene, is 2.69 (1.24 to 5.82).

* In addition, if the SOL of all three types, the AA genotype constituting the rs970547 polymorphism (SNP) of the COL12A1 gene, the GG genotype constituting the rs679620 SNP of the MMP3 gene, and the CC genotype constituting the rs1800955 SNP of the DRD4 gene, the risk of risk is included The measure OR (95% Cl) is 4.73 (0.54 to 41.52).

The present invention has an effect that a genetic predisposition useful to Koreans can provide useful information for developing personalized exercise programs that can prevent elite athletes from being discovered or prevented from being vulnerable to injury.

1 is 1 is a result of analyzing the rs970547 SNP of COL12A1 gene by SNaPshot method.
A) GG genotype; B) GA genotype; C) AA genotype.
2 is a result of analyzing the rs679620 SNP of the MMP3 gene by SNaPshot method.
B) GG genotype; B) GA genotype; C) AA genotype.
3 is a result of analyzing the rs1800955 SNP of the DRD4 gene by SNaPshot method.
A) CC genotype; B) CT genotype; C) TT genotype.

The present invention can be variously modified and have various embodiments, and specific embodiments are illustrated in the drawings and are described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood as including all modifications, equivalents, and substitutes included in the spirit and scope of the present invention.

In the present invention, a total of 8 types of genes including COL1A1, COL3A1, COL5A1, COL12A1, MMP3, MMP12, GDF5 and DRD4 genes were selected as candidate genes for the purpose of searching for useful genetic markers for Korean sports injuries. After preparing oligonucleotide primers for detecting SNPs present in genes by SNaPshot method that can be accurately and easily detected, a patient-control relationship study using them was conducted, and each step of the study was performed as follows.

1. Blood collection and DNA separation

About 3 ~ 5 mL of blood is collected from the sports injury patient group and the control group and transferred to the EDTA tube, and then total genomic DNA is separated from an automatic DNA isolator (Bionex, Co. Ltd. Korea).

2. Detection of SNP by SNaPshot method

For SNPs present in 8 types of candidate genes, forward primers, reverse primers, and genotyping primers were designed to detect them by SNaPshot method, and the base sequence for these is as follows.

[Base sequence]

For rs1800012 SNP in COL1A1 gene,

forward primer, 5 '-GGCTAAAAGTGACCTGGAG-3'

reverse primer, 5 '-CTTCCAACTCCAACCTCAG-3'

genotyping primer, 5 '-CCACCCCACMTGCCCAGGGAATG-3'

[Base sequence]

For rs1800 255 SNP in COL3A1 gene,

forward primer, '5 -TGTTTTTAGCTTTGGGTTG-3'

reverse primer, '5 -GACTTCCAAGACCTCCTCTT-3'

genotyping primer, '5 -AGGGGSCCCAGGACTTAGAGGTRGA-3'

[Base sequence]

For rs1800 255 SNP in COL5A1 gene,

forward primer, '5 -CACCTGACTTCATCTACGC-3'

reverse primer, '5 -GAAGGTCAGGGGAAATACA-3'

genotyping primer, '5 -CCCGCCCCACGCTCTGTCCACACCCA-3'

[Base sequence]

For rs970547 SNP in COL12A1 gene,

forward primer, '5 -CTATTTGACAAGAGGGGAAA-3'

reverse primer, '5 -TCTTCAAATCAGCCTTCCT-3'

genotyping primer, '5 -TGGCAACAACGTACCTGGATAGC-3'

[Base sequence]

For rs679620 SNP in MMP3 gene,

forward primer, '5 -GCAAGCTAGAGAAATACTCC-3'

reverse primer, '5 -CCTCTGAACCATTACCTG-3'

genotyping primer, '5 -GAAATATCTAGAAAAVTACTAHGACCTC-3'

[Base sequence]

For rs970547 SNP in MMP12 gene,

forward primer, '5 -AGGGCTTATTTTGTAGGAT-3'

reverse primer, '5 -AGCAGTATTAGAAGAAACTTCA-3'

genotyping primer, '5 -GAAATATCTAGAAAACTACTAHGACCTC-3'

[Base sequence]

For rs143383 SNP in GDF5 gene,

forward primer, '5 -TCAGTTGTGCAGGAGAAAG-3'

reverse primer, '5 -GAGTTTGGGGAGTCTCATC-3'

genotyping primer, '5 -TAACTCGTTCTTGAAAGGAKAAAGCC-3'

[Base sequence]

For rs1800955 SNP in DRD4 gene,

forward primer, '5 -AGGATCAACTGTGCAACG-3'

reverse primer, '5 -AAACCGACAAGGATGGAG-3'

genotyping primer, '5 -CTCGCCTCGACCTCGTGCGC-3'

SNaPshot analysis was performed using the primers described above, and the analysis process will be described in detail.

First, in order to perform amplification on the region on the DNA containing the SNP portion of the candidate gene, template DNA 10ng, forward and reverse primers 0.5pM, 10XPCR buffer 1uL, dNTP 250uM, Taq DNA polymerase 0.25 units each After performing the denaturation process of 10 cycles for 10 minutes at a temperature of 95 ° C for 10 minutes in a DUAL 384-Well GeneAmp®PCR System 9,700, the reaction solution of 10uL was 30 seconds at 55 ° C, 55 ° C (COL1A1, COL3A1, COL12A1, MMP12, GDF and DRD4 genes) or a total of 35 cycles of 1 minute at a temperature of 60 ° C (DRD4) and 1 minute at a temperature of 72 ° C were performed, and finally 1 cycle of 10 minutes at a temperature of 72 ° C A final elongation reaction is performed.

Primer extension reaction is carried out after the amplification reaction described above is completed. Put 1uL of purified PCR product into SNaPshot Ready Reaction mixture containing 0.15 pmol of genotyping primer, respectively, at a temperature of 96 ° C for 10 seconds and at a temperature of 50 ° C 5 Second, a total of 25 cycles of 30 seconds is performed at a temperature of 60 ° C.

In addition, in order to remove the excess fluorescent dye terminator, 1 unit of SAP (Shrimp Alkaline Phosphatase) was added to the reaction product, followed by reaction at 37 ° C for 75 minutes and 72 ° C for 15 minutes, followed by reaction product 1uL After adding 9uL of Hi-Di foramide, the mixture was placed at 95 ° C for 5 minutes, and then placed on ice for 5 minutes, then ABl Primer® 3730 xl Research results are calculated using DNA Analyzer (Applied Biosystems, USA), and analysis of research results is performed using GeneMapper 4.0 analysis software (Applied Biosystems, USA).

3. Data processing

If the genotype for the SNP present in each candidate gene is determined by the SNaPshot method described above, the allele frequency is inferred using this, and the genotype frequency constituting each SNP is Harqy- based on the inferred frequency. Calculate whether it deviates from the Wwinberg equilibrium.

In addition, in order to analyze whether the genotype and allele frequencies constituting each SNP represent a significant difference between the control group and the sports injury patient group, a χ²-test is performed, and a statistical analysis confirms a significant association with the sports injury. For the SNP, the 95% confidence interval was calculated by the logistic regression method along with the OR (odds ratio) value, which is a measure of the relative risk for the genotype, which showed a significantly higher frequency in the sports injury patient group. The level of risk is indicated.

[Example]

1. Subject

A total of 153 patients, including 73 patients with similar physical activity levels, as well as 80 patients who visited a hospital specializing in sports injuries due to sports injuries, were selected as subjects of this study.

2. Blood collection and DNA separation

For the sports injury patient group and the control group, about 3 to 5 mL of venous blood was collected from their main venous vein, transferred to an EDTA tube, and stored in a refrigerator at 4 ° C. until DNA analysis was performed. The separation of DNA from the venous blood was performed using total genomic DNA in an automatic DNA isolator (Bionex, Co. Ltd., Korea), an automated DNA separation device.

3. Detection of SNP by SNaPshot method

For SNPs present in a total of 8 types of candidate genes, forward primers, reverse primers, and genotyping primers for detecting them by SNaPshot method were designed with the above-described [base sequence], and thereafter SNPs of the candidate genes In order to perform amplification on the region on the DNA containing the part, dual 10uL of the reaction solution containing 10 ng of template DNA, 0.5 pM of forward primer and reverse primer, 1 uL of 10X PCR buffer, 250 uM of dNTP, and 0.25 unit of Taq DNA polymerase, respectively After performing a 1 cycle denaturation process at a temperature of 95 ° C for 10 minutes in a 384-Well GeneAmp®PCR System 9,700, 30 seconds at a temperature of 95 ° C, 55 ° C (COL1A1, COL3A1, COL5A1, COL12A1, MMP12, GDF5, and DRD4 Genes) or a total of 35 cycles with 1 cycle at 60 ° C (DRD4) and 1 minute at 72 ° C were performed, and finally the final elongation at 1 cycle over 10 minutes at 72 ° C. Carrying out the reaction It was.

After this process, in order to remove excess fluorescent dye terminators, 1 unit of SAP (Shrimp Alkaline Phophatase) was added to the reaction product and reacted for 15 minutes at 72 ° C and 75 minutes at 37 ° C. After 5 minutes on ice, ABl Primer® 3730 xl Research results were calculated using a DNA analyzer (Applied Biosystems, USA), and analysis of the results was performed using GeneMapper 4.0 analysis software (Applied Biosystems, USA).

4.Data processing

If the genotype for the SNP present in each candidate gene is determined by the SNaPshot method described above, the allele frequency is inferred using this, and the genotype frequency constituting each SNP is Harqy- based on the inferred frequency. Calculate whether we deviate from the Weinberg equilibrium.

In addition, in order to analyze whether the genotype and allele frequencies constituting each SNP represent a significant difference between the control group and the sports injury patient group, a χ²-test is performed, and a statistical analysis confirms a significant association with the sports injury. For the SNP, the 95% confidence interval was calculated by the logistic regression method along with the OR (odds ratio) value, which is a measure of the relative risk for the genotype, which showed a significantly higher frequency in the sports injury patient group. The degree of risk is indicated for.

5. Results

As a result of studying the patient-control relationship, in Koreans, with the rs970547SNP of the COL12A1 gene, the rs679620 SNP of the MMP3 gene and the rs1800955 SNP of the DRD4 gene showed a significantly higher frequency in the sports injury patient group, indicating the risk for sports injury. It could be selected as a predictable useful gene marker, and detailed study results for each SNP are described in <Table 1> to <Table 3>.

Rs970547 polymorphic distribution of COL12A1 gene in control group and sports injury patient group group Genotype No. (%) Allele No. (%) GG GA AA G A Control 11 (15.1) 40 (54.8) 22 (30.1) 62 (42.5) 84 (57.5) Sports shanghai 8 (10.0) 31 (38.8) 41 (51.2) 47 (29.4) 113 (70.6) all 19 (12.4) 71 (46.4) 63 (41.2) 109 (35.6) 197 (64.4) AA / GG + GA, OR (95% Cl) = 2.44 (1.25 ~ 4.74) AA / GG + GA, OR (95% Cl) = 2.44 (1.25 ~ 4.74) χ = 7.0390, df = 2, p = .0296. χ = 5.7050, df = 1, p = .0169.

As for the rs970547 polymorphism of the COL12A1 gene, SNaPshot analysis was performed using the primers described above. As a result, the GG, GA, and AA genotypes were 12.4%, 46.4%, and 41.2%, respectively, for the entire subjects, and observed genes The distribution of was observed to not deviate significantly from the Hardy-Weinberg equilibrium, and it was recognized that the subjects participating in this study were well representative of the Korean population (χ² = 0.0210, df = 1, p = .8841). In addition, in the results of comparing the genotype and allele frequencies constituting the above-described SNP for the control group and the sports injury patient group, in the genotype between two groups (χ² = 5.7050, df = 1, p = .0169), There was a statistically significant difference.

In addition, among the three types of genotypes, the AA genotype showed a higher frequency than in the sports injury patient group, and the 95% confidence interval (Cl) also appeared as 1.25 to 4.74, so the lower confidence limit exceeded 1.0. The relative risk of AA genotype to sports injury was found to be significant.

Rs679620 polymorphism distribution of MMP3 gene in control group and sports injury patient group group Genotype No. (%) Allele No. (%) GG GA AA G A Control 26 (35.6) 37 (50.7) 10 (13.7) 89 (61.0) 57 (39.0) Sports shanghai 42 (52.5) 34 (42.5) 4 (5.0) 118 (73.7) 42 (26.3) all 68 (44.4) 71 (46.4) 14 (9.2) 207 (67.6) 99 (32.4) AA / GG + GA, OR (95% Cl) = 2.00 (1.04 ~ 3.83) AA / GG + GA, OR (95% Cl) = 1.80 (1.11 ~ 2.92) χ = 6.1560, df = 2, p = .0460. χ = 5.7050, df = 1, p = .0169.

As a result of performing SNaPshot analysis on the rs679620 polymorphism of the MMP3 gene using the above-described primers, GG, GA and AA genotypes showed a frequency of 44.4%, 46.4% and 9.2%, respectively, for all subjects, and observed genes The distribution of was observed to not significantly deviate from the Hardy-Weinberg equilibrium, and it was recognized that the subjects participating in this study were representative of the Korean population (χ² = 0.5540, df = 1, p = .4568). In addition, in the results of comparing the genotypes and allele frequencies constituting the above-described SNPs for the control group and the sports injury patient group, in the genotype (χ² = 6.1560, df = 2, p = .0460) between the two groups, There was a statistically significant difference.

In addition, among the three genotypes, the relative risk (OR) of the GG genotype, which showed a higher frequency in the sports injury patient group, was about 2 times higher than the other two genotypes (GA and AA). In addition, the 95% confidence interval (Cl) was also found to be 1.04 to 3.83, so the lower confidence limit exceeded 1.0, and the relative risk of GG genotype to sports injury was found to be significant.

Rs1800955 polymorphic distribution of DRD4 gene in control and sports injury patients group Genotype No. (%) Allele No. (%) CC CT TT C T Control 12 (16.7) 40 (55.5) 20 (27.8) 64 (44.4) 80 (55.6) Sports shanghai 28 (35.0) 32 (40.0) 20 (25.0) 88 (55.0) 72 (45.0) all 40 (26.3) 72 (47.4) 40 (26.3) 152 (50.0) 152 (50.0) AA / GG + GA, OR (95% Cl) = 2.69 (1.24 ~ 5.82) AA / GG + GA, OR (95% Cl) = 1.53 (0.97 ~ 2.40) χ = 6.8870, df = 2, p = .0320. χ = 3.3773, df = 1, p = .0661.

As a result of performing SNaPshot analysis on the rs1800955 polymorphism of the DRD4 gene using the primers described above, CC, CT, and TT genotypes of 26.3%, 47.4%, and 26.3%, respectively, were observed for all subjects, and observed genotypes The distribution of was observed to not significantly deviate from the Hardy-Weinberg equilibrium, and it was recognized that the subjects participating in this study were representative of the Korean population (χ² = 0.4210, df = 1, p = .5164). In addition, in the results of comparing the genotypes and allele frequencies constituting the above-described SNPs for the control group and the sports injury patient group, in the genotypes between two groups (χ² = 3.3773, df = 1, p = .0661), There was a statistically significant difference. In addition, among the three types of genotypes, the CC genotype, which showed a higher frequency in the sports injury patient group, is about 2.69 times higher than the other two genotypes (CT and TT), which have a relative risk (OR) to cause sports injury. In addition, the 95% confidence interval (Cl) was also 1.24 to 5.82, so the lower confidence limit exceeded 1.0, and the relative risk of AA genotype to sports injury was found to be significant.

As a result of the combination of each SNP, the odds ratio (OR) and 95% confidence interval for sports injuries SNPs Combined genotype No. (%) Risk genotype (s) OR (95% Cl) COL12A1 (rs970547) AA 2.44 (1.25 ~ 4.74) MMP3 (rs679620) GG 2.00 (1.04 ~ 3.83) DRD4 (rs1800955) CC 2.69 (1.24 ~ 5.82) COL12A1 + MMP3 AA + GG 3.24 (1.21 ~ 8.69) COL12A1 + DRD4 AA + CC 2.54 (0.98 ~ 13.72) MMP3 + DRD4 GG + CC 6.67 (0.98 ~ 13.72) COL12A1 + MMP3 + DRD4 AA + GG + CC 4.73 (0.54 ~ 41.52)

Finally, when the three types of SNPs that showed significant relevance to sports injuries were analyzed together, the relative risks to sports injuries were analyzed, and the relative risk values when analyzing them individually were about 2.00 to 2.69. On the other hand, when two types of SNPs were analyzed together, the relative risk value increased from 2.54 to 3.67, and when all three types of SNPs were analyzed, the value increased to 4.73, constituting the rs970547 polymorphism of the COL12A1 gene. When the AA genotype and the CC genotype constituting the rs1800955 polymorphism of the MMP3 gene were combined together, the risk of developing sports injury increased by 4.73 times.

Although embodiments according to the present invention have been described above, it is impossible to be illustrative, and those skilled in the art will understand that various modifications and equivalent ranges of embodiments are possible therefrom. Therefore, the true technical protection scope of the present invention should be defined by the claims.

Claims (2)

A primer set for detecting rs1800955 SNP of the DRD4 gene,
Forward primer with the sequence '5 -AGGATCAACTGTGCAACG-3',
Reverse primer having the sequence '5 -AAACCGACAAGGATGGAG-3', and
Composition for predicting sports injury comprising a primer set, characterized in that consisting of a genotyping primer having the sequence '5 -CTCGCCTCGACCTCGTGCGC-3'. According to claim 1,
The composition for predicting sports injury, characterized in that OR (95% Cl), a measure of the risk of the CC genotype constituting the rs1800955 SNP of the DRD4 gene, is 2.69 (1.24 to 5.82).

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