Method for determining the risk of developing normal tension glaucoma in Koreans specific by polymorphism

Abstract

The present invention relates to a method for analyzing the risk of developing normal tension glaucoma in Koreans based on single nucleotide polymorphisms. Specifically, the invention relates to a method for identifying one or more single nucleotide polymorphisms (SNPs) selected from the group consisting of rs10483727 and rs33912345 from a biological sample; a biomarker composition for identifying normal tension glaucoma in Koreans containing the single nucleotide polymorphisms (SNPs); a composition for predicting the risk of developing normal tension glaucoma comprising a preparation capable of detecting the biomarker composition; and a kit for predicting the risk of developing normal tension glaucoma comprising the composition. According to the present invention, useful information can be provided for predicting the risk of developing normal tension glaucoma in Koreans based on single nucleotide polymorphisms. In particular, such information can be provided more easily when using the kit of the present invention.

Description

Technology Field

[0001] The present invention relates to a method for analyzing the risk of developing normal-tension glaucoma in Koreans based on single nucleotide polymorphisms. Background Technology

[0002] Glaucoma is a degenerative and progressive optic neuropathy characterized by glaucomatous optic nerve damage accompanied by visual field defects. Globally, glaucoma is one of the leading causes of visual field loss and blindness. Recent studies have revealed that open-angle glaucoma (OAG) is phenotypic and genetically heterogeneous and is virtually polygenic.

[0003] Increased intraocular pressure (IOP) is not an essential factor in the diagnosis of glaucoma. Normal tension glaucoma (NTG) is the most common subtype of glaucoma among Koreans. Studies on various racial groups suggest that while normal tension glaucoma and high tension glaucoma share some genetic risk factors, the association with glaucoma may differ depending on genetic variations.

[0004] Although several previous studies have reported that the SIX1 / SIX6 loci are associated with open-angle glaucoma (OAG) across various races, the definition of OAG patients has varied from study to study, and the association of the SIX1 / SIX6 loci with normal-tension glaucoma (NTG) in Koreans remains uncertain.

[0005] Accordingly, the inventors investigated the association between the incidence of normal-tension glaucoma and the SIX1 / SIX6 loci in a Korean cohort with the aim of utilizing this for the prediction or diagnosis of normal-tension glaucoma. By confirming that the OAG-related SNPs SIX1 / SIX6 are significantly associated with normal-tension glaucoma, the inventors completed the present invention. Prior art literature

[0006] Republic of Korea Published Patent No. 10-2023-0075213 (May 31, 2023) The problem to be solved

[0007] The objective of the present invention is to provide a method for identifying one or more single nucleotide polymorphisms (SNPs) selected from the group consisting of rs10483727 and rs33912345 from a biological sample in order to provide information necessary for predicting the risk of developing normal tension glaucoma in Koreans.

[0008] Another objective of the present invention is to provide a biomarker composition for identifying normal tension glaucoma in Koreans comprising one or more single nucleotide polymorphisms (SNPs) selected from the group consisting of rs10483727 and rs33912345.

[0009] Another objective of the present invention is to provide a composition for predicting the risk of developing normal tension glaucoma, comprising a preparation capable of detecting the biomarker composition.

[0010] Another objective of the present invention is to provide a kit for predicting the risk of developing normal tension glaucoma, comprising the above composition.

[0011] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings. means of solving the problem

[0012] The inventors investigated the relationship between normal-tension glaucoma (NTG) and the SIX1 / SIX6 locus in a Korean cohort and confirmed that SIX1 / SIX6 locus polymorphisms are significantly associated with genetic susceptibility to normal-tension glaucoma in Koreans. To this end, four polymorphisms of the SIX1 / SIX6 locus (rs33912345, rs12436579, rs2179970, and rs10483727) were genotyped in 327 subjects using TaqMan single nucleotide polymorphism genotyping analysis.

[0013] In addition, the rs33912345 polymorphism was significantly correlated with NTG in the recessive model (odds ratio (OR): 0.265; 95% confidence interval (CI): 0.078-0.898, P =0.033), but not in the allelic and dominant models (both P >0.05). The SNP rs10483727 was significantly associated with NTG in the allelic model (OR: 0.674; 95% CI: 0.464-0.979, P =0.038) and the recessive model (OR: 0.187; 95% CI: 0.058-0.602, P =0.005). However, genetic association analysis of SNPs rs12436579 and rs2179970 showed no significant difference in genotype distribution between controls in NTG cases, alleles, dominant or recessive models (all P > 0.05). Therefore, it was confirmed that SIX1-SIX6 locus rs10483727 and rs33912345 polymorphisms are significantly associated with the risk of developing NTG in Koreans.

[0014] The present invention provides a method for identifying one or more single nucleotide polymorphisms (SNPs) selected from the group consisting of rs10483727 and rs33912345 from a biological sample in order to provide information necessary for predicting the risk of developing normal tension glaucoma in Koreans.

[0015] In the present invention, polymorphism refers to the case where two or more alleles exist at a single locus, and among polymorphic regions, a single nucleotide polymorphism (SNP) is defined as a region where only a single nucleotide differs from person to person. A preferred polymorphic marker has two or more alleles that exhibit an occurrence frequency of 1% or more, more preferably 5% or 10% or more, in a selected population. Therefore, genetic association with a polymorphic marker means that there is an association with one or more specific alleles of a specific polymorphic marker. The marker may include alleles of any variant form found in the genome, including single nucleotide polymorphisms (SNPs), microsatellites, insertions, deletions, duplications, and translocations.

[0016] In the present invention, an allele or allelic trait refers to multiple types of a single gene located at the same locus on homologous chromosomes. Alleles are also used to express polymorphism; for example, an SNP has two types of alleles.

[0017] In the present invention, rs_id refers to the rs-ID, an independent marker assigned to all initially registered SNPs by NCBI, which began accumulating SNP information in 1998. In the present invention, it is described in the form of rs10483727. The rs_id listed in such a table refers to the SNP marker, which is a polymorphic marker of the present invention. A person skilled in the art will be able to easily identify the location and sequence of the SNP using the above rs_id.

[0018] rs10483727 and rs33912345, which are used in the present invention to predict the risk of developing normal-tension glaucoma in Koreans, are each registered in the single nucleotide polymorphism database of the National Center for Biotechnology Information (NCBI dbSNP, http: / / www.ncbi.nlm.nih.gov / snp / ).

[0019] Meanwhile, it was determined that the risk of normal tension glaucoma (NTG) is reduced in AA genotype carriers for rs33912345 or CC genotype carriers for rs10483727. Specifically, small alleles for two SNPs in SIX1 / SIX6 showed protective effects and were found to have a lower frequency in the NTG patient group than in the control group. According to one embodiment of the present invention, since AA genotype carriers for rs33912345 appear at a higher rate in the control group compared to the patient group, it is determined that the minor allele (A) has a protective effect, and since CC genotype carriers for rs10483727 appear at a higher rate in the control group compared to the patient group, it is determined that the minor allele (C) has a protective effect.

[0020] In the present invention, the single nucleotide polymorphism of rs33912345 is determined by a recessive model, and the single nucleotide polymorphism of rs10483727 may be determined by an allelic model or a dominant model.

[0021] In the present invention, the single nucleotide polymorphism (SNP) may be located in the intergene region between loci SIX1 and SIX6.

[0022] In the present invention, the biological sample may be blood, tissue, cell, serum, plasma, saliva, urine, etc., but is not limited thereto.

[0023] In the present invention, the subject of the biological sample may be a Korean.

[0024] In addition, the present invention provides a biomarker composition for identifying normal tension glaucoma in Koreans comprising one or more single nucleotide polymorphisms (SNPs) selected from the group consisting of rs10483727 and rs33912345.

[0025] In addition, the present invention provides a composition for predicting the risk of developing normal tension glaucoma, comprising a formulation capable of detecting the biomarker composition.

[0026] In the present invention, the agent capable of detecting the biomarker composition may be a primer or probe that specifically binds to a polynucleotide containing an SNP marker or a polynucleotide complementary thereof, but is not limited thereto.

[0027] In the present invention, the primer or probe that specifically binds to the polynucleotide or its complementary polynucleotide is allele-specific.

[0028] In the present invention, a primer refers to a short nucleic acid sequence having a short free hydroxyl group, capable of forming base pairs with a complementary template, and functioning as a starting point for template strand replication. The primer of the present invention can be chemically synthesized using methods known in the art, such as, for example, the phosphoramidite solid support method.

[0029] In the present invention, the probe refers to a nucleic acid fragment, such as RNA or DNA, composed of several to hundreds of bases that can specifically bind to mRNA, and is labeled so that the presence or absence of a specific mRNA can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc., and can be labeled with biotin, FITC, rhodamine, DIG, etc., or labeled with a radioisotope, etc.

[0030] In the present invention, the probe may be labeled with a detectable material, for example, a radioactive label having a suitable signal and a sufficient half-life. The labeled probe may hybridize to nucleic acids on a solid support as known in the literature (Sambook et al., Molecular Cloning, A Laboratory Manual, 1989).

[0031] In the present invention, the probe is an allele-specific probe in which a polymorphic region exists within the nucleic acid fragment, so that it may hybridize to a nucleic acid fragment containing one allele but not to a nucleic acid fragment containing another allele. In this case, the hybridization conditions must be sufficiently strict to ensure that hybridization occurs to only one of the alleles by showing a significant difference in hybridization intensity between the alleles. Preferably, the probe may be single-stranded for maximum efficiency in hybridization, but is not limited thereto.

[0032] In the present invention, the detection of a specific nucleic acid using the primer can be performed by amplifying the sequence of a target gene using an amplification method such as PCR, and then confirming whether the gene has been amplified by a method known in the art. Additionally, the detection of a specific nucleic acid using a probe can be performed by contacting a sample nucleic acid with the probe under suitable conditions and then confirming the presence of the nucleic acid that hybridizes.

[0033] Methods for detecting specific nucleic acids using the probe or primer in the present invention include, for example but are not limited thereto, polymerase chain reaction (PCR), DNA sequencing, RT-PCR, primer extension method (Nikiforeov et al., Nucl Acids Res 22, 4167-4175, 1994), oligonucleotide extension analysis (Nickerson et al., Pro Nat Acad Sci USA, 87, 8923-8927, 1990), allele-specific PCR method (Rust et al., Nucl Acids Res, 6, 3623-3629, 1993), RNase mismatch cleavage (RNase mismatch cleavage, Myers et al., Science, 230, 1242-1246, 1985), and single strand conformation lymorphism (single strand conformation lymorphism, Orita et al., Pro Nat Acad Sci These include USA, 86, 2766-2770, 1989), simultaneous analysis of SSCP and heteroduplex (Lee et al., Mol Cells, 5:668-672, 1995), denaturation gradient gel electrophoresis (DGGE, Cariello et al., Am J Hum Genet, 42, 726-734, 1988), denaturation high-pressure liquid chromatography (Underhill et al., Genome Res, 7, 996-1005, 1997), hybridization reactions, DNA chips, etc. Examples of the above hybridization reactions include Northern hybridization (Maniatis T. et al., Molecular Cloning, Cold Spring Harbor Laboratory, NY, 1982), in situ hybridization (Jacquemier et al., Bull Cancer, 90:31-8, 2003), and microarray (Macgregor, Expert, Rev Mol Diagn 3:185-200, 2003) methods.

[0034] In addition, the present invention provides a kit for predicting the risk of developing normal tension glaucoma, comprising the above composition.

[0035] In the present invention, the kit may include a polynucleotide, primer, or probe for identifying one or more of the polymorphic markers, as well as one or more other component compositions, solutions, or devices suitable for the analysis method.

[0036] In the present invention, the kit may be a kit containing essential elements necessary to perform PCR. In addition to polynucleotides, primers, or probes specific to the polymorphic marker, the PCR kit may include a test tube or other suitable container, a reaction buffer (with varying pH and magnesium concentration), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNAse inhibitors, DEPC-water, and sterile water. Effects of the invention

[0037] According to the present invention, there is an advantage in that it can be usefully employed to predict the risk of developing normal-tension glaucoma in Koreans based on single nucleotide polymorphisms. Specific details for implementing the invention

[0038] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0040] Example 1. Experimental Method

[0042] 1.1 Selection of Research Group

[0044] Unrelated NTG patients and healthy control subjects were recruited from the Department of Ophthalmology at Uijeongbu St. Mary's Hospital, South Korea. All subjects received written consent after being informed of the purpose of the genetic test and the blood collection process, and those who refused to participate were excluded. The study was approved by the Institutional Review Board of Uijeongbu St. Mary's Hospital, South Korea (Approval No.: UC16SISE0060) and conducted in accordance with the principles of the Declaration of Helsinki.

[0045] A thorough ophthalmic examination, including optic nerve head examination, was performed on all participants. Inclusion criteria were as follows: individuals with a characteristic glaucomatous optic nerve head exhibiting a VCDR ≥0.6; diffuse or focal neuroretinal rim thinning (e.g., notching or acquired notch of the optic nerve) and / or retinal nerve fiber layer (RNFL) defects; glaucomatous visual field defects measured by a Humphrey Automated Field Analyzer (Zeiss Humphrey Systems, Dublin, CA); open-angle structures; and an IO1 ≤21 mmHg without medication on the initial examination and repeated measurements on different days.

[0046] Glaucoma visual field defects are defined as at least one point with a probability of less than 1% in the pattern deviation map, or at least three point clusters with a probability of less than 5%, and the field result is the "normal limit in the external glaucoma visual field test" or a pattern standard deviation with a probability <5%.

[0047] A total of 210 NTG patients (80 men, 130 women) with an average age of 67.15 ± 12.8 years met the inclusion criteria. Patients with closed-angle glaucoma, pigmentary glaucoma, uveitis glaucoma, neovascular glaucoma, or a history of trauma were excluded. The control group (117 patients, 46 men, 71 women, average age 70.3 ± 10.9 years) met the following criteria at the time of recruitment: normal optic nerve head; normal IOP (≤21 mmHg); no history of elevated IOP; no family history of glaucoma.

[0049] 1.2 Sample Preparation and Allele Identification

[0051] Genomic DNA was isolated from blood using the MG Blood Genomic DNA Extraction SV kit (MGmed, Seoul, Korea). Four SNPs (rs33912345, rs12436579, rs2179970, and rs10483727) at the SIX1 / SIX6 loci were analyzed in this invention.

[0052] Genotyping analysis for rs33912345, rs12436579, rs2179970, and rs10483727 polymorphisms was performed using the QuantStudio real-time polymerase chain reaction system (Applied Biosystems) with the TaqMan SNP Genotyping Assay analysis IDs (Applied Biosystems, Foster City, CA) for C___2485201_10, C__11916555_30, C__15840320_10, and C___2485225_20, respectively.

[0054] 1.3 Statistical Analysis

[0056] Statistical analysis was performed to investigate the association between SIX1-SIX6 SNPs and susceptibility to NTG. The four major SNPs were tested for Hardy-Weinberg equilibrium using the χ² test. Frequency and odds ratio (OR) with 95% confidence intervals (CI) were calculated for all polymorphic SNPs. Comparisons between genotype and allelic frequency were performed using the χ² test or the Fisher exact test. Allelic association was evaluated by logistic regression to calculate the odds ratio (OR) and 95% confidence interval (CI).

[0057] SNP associations were tested using the following three genetic models: the dominant model [common allele homozygous (AA) vs. heterozygous (AB) + minority allele homozygous (BB)], the recessive model (AA+AB vs. BB), and the allele model (A vs. B). All statistical analyses were performed using R software (R Foundation for Statistical Computing, Vienna, Austria). A P-value of <0.05 was considered statistically significant in all association analyses.

[0059] Example 2. Experimental Results

[0061] To evaluate the possible association between NTG and the SIX1 / SIX6 loci, genotyping analysis was performed on four SNPs in a Korean cohort. The study included 327 subjects (210 NTG patients and 117 healthy controls). For each SNP, the genotypes were all in Hardy-Weinberg equilibrium in the controls (Table 1). Table 1 presents general information on the four SNPs.

[0063]

[0065] In addition, the association results of four SNPs in the allele model, dominant model, and recessive model were summarized and shown in Table 2.

[0067]

[0069] A significant association between rs33912345 and NTG was confirmed using the recessive model (OR: 0.265; 95% CI: 0.078-0.898, P=0.033), but no significant association between rs33912345 and NTG was found when using the dominant or allelic model (P= 0.204 or 0.064, Table 2).

[0070] Significant associations between rs10483727 and NTG were confirmed using recessive and allelic models (OR: 0.187; 95% CI: 0.058-0.602, P=0.005 and OR: 0.674; 95% CI: 0.464-0.979, P=0.038, respectively), but no significant association between rs10483727 and NTG was found when using the dominant model (P= 0.236, Table 2).

[0071] Genetic association analysis for rs12436579 and rs2179970 did not show statistically significant differences in genotype distribution between NTG patients and control subjects in allelic, dominant, or recessive models (all P > 0.05 Table 2).

[0072] In addition, carriers of the AA genotype for rs33912345 and the CC genotype for rs10483727 were found to have a reduced risk of NTG. We found that the rs10483727 polymorphism was significantly correlated with increased NTG risk in both recessive and allelic models.

[0074] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 A method for providing information necessary for predicting the risk of developing normal tension glaucoma in Koreans, comprising the step of identifying one or more single nucleotide polymorphisms (SNPs) selected from a group consisting of rs10483727 and rs33912345 from a biological sample, wherein the rs33912345 SNP is identified by a recessive model and the rs10483727 SNP is identified by an allelic model or a recessive model. Claim 2 delete Claim 3 A method according to claim 1, wherein the risk of normal tension glaucoma is determined to be reduced in the case of AA genotype carriers for rs33912345 or CC genotype carriers for rs10483727. Claim 4 A method according to claim 1, wherein the portion is located within the SIX1 gene or the SIX6 gene, or is located in the intergeneric region between the SIX1 and SIX6 genes. Claim 5 A method according to claim 1, characterized in that the biological sample is any one selected from the group consisting of blood, tissue, cell, serum, plasma, saliva, and urine. Claim 6 A method according to claim 1, characterized in that the subject of the biological sample is a Korean. Claim 7 A biomarker composition for determining normal tension glaucoma in Koreans comprising one or more single nucleotide polymorphisms (SNPs) selected from the group consisting of rs10483727 and rs33912345, wherein the rs33912345 single nucleotide polymorphism is used for determination according to a recessive model, and the rs10483727 single nucleotide polymorphism is used for determination according to an allelic model or a recessive model. Claim 8 A composition for predicting the risk of developing normal tension glaucoma, comprising a preparation capable of detecting the biomarker composition of claim 7. Claim 9 A kit for predicting the risk of developing normal tension glaucoma, comprising the composition of claim 8.

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